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ISSN: 0971-7196
Journal of
Volume 30 Ÿ Number 1 Ÿ June 2006
(Released August 2006)
Parasitic
Diseases
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The Official Organ of the Indian Society for Parasitology
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The Indian Society for Parasitology
Electronic version available on ISP Website
www.parasitologyindia.org
JOURNAL OF PARASITIC DISEASES
(ISSN: 0971-7196)
Editor-in-Chief
Professor Prati Pal Singh
National Institute of Pharmaceutical
Education and Research
Sector-67, Phase-X
S. A. S. Nagar-160 062, India
Managing Editor
Assistant Managing Editor
Dr. Varsha Gupta
Deptt. of Microbiology
Govt. Medical College & Hospital
Chandigarh-160 032
Dr. Savita Singh
National Institute of Pharmaceutical
Education and Research
S. A. S. Nagar-160 062
Advisory Board
Dr. A. B. Chaudhary, Kolkata
Prof. R. C. Mahajan, Chandigarh
Dr. G. P. Dutta, Lucknow
Dr. V. P. Sharma, New Delhi
Prof. N. K. Ganguly, New Delhi
Editorial Board
Prof. M. C. Agrawal, Jabalpur
Prof. Irfan Ahmed, Aligarh
Dr. H. K. Bajaj, Hissar
Prof. H. S. Banyal, Shimla
Prof. Neelima Gupta, Bareilley
Prof. B. C. Harinath, Sevagram
Dr. D. C. Kaushal, Lucknow
Dr. S. L. Hoti, Pondicherry
Prof. R. Madhubala, New Delhi
Prof. Sandeep Malhotra, Allahabad
Prof. Nancy Malla, Chandigarh
Dr. J. R. Rao, Izatnagar
Prof. M. L. Sood, Ludhiana
Dr. B. K. Tyagi, Mdurai
Journal of Parasitic Diseases is published biannually by the Indian Society for Parasitology in June and December
in each calender year. The subscription price for libraries and other multi-reader organizations for each number is
Rs. 400 in India and US $ 100 elsewhere. Subscription by Demand Draft in favour of 'The Indian Society for
Parasitology' should be sent to Dr. J. K. Saxena, Secretary, The Indian Society for Parasitology, Division of
Biochemistry, Central Drug Research Institute, Chattar Manzil, Lucknow - 226 001, India.
JOURNAL OF PARASITIC DISEASES
Volume 30, Number 1, June 2006
The official organ of
THE INDIAN SOCIETY FOR PARASITOLOGY
Central Drug Research Institute, Lucknow-226 001, India
Editorial Office: National Institute of Pharmaceutical Education and Research
Sector-67, Phase-X, S. A. S. Nagar-160 062, India
Phone: 0172-2214682-87; Fax: 0172-2214692; E-mail: [email protected]
Journal of Parasitic Diseases
Copyright © 2006 The Indian Society for Parasitology
All Rights Reserved
No part of this publication may be reproduced or utilized in any form or by any means, electronic or
mechanical including photocopying, recording or any other information storage and retrieval system
without the permission in writing from copyright owner.
JOURNAL OF PARASITIC DISEASES
Volume 30
Number 1
June 2006
CONTENTS
A Note from the New Editor-in-Chief
1-3
Reviews
Histochemical, biochemical and immunological studies in Haemonchus contortus
(Nematoda: Trichostrongyloidea) - an Indian perspective. M. L. Sood
Immunological perspectives and malaria vaccine. H. S. Banyal and N. Elangbam
4-15
16-29
Original papers
Isolation and characterization of the paraflagellar rod proteins of Leishmania
donovani. A. Lahiri and A. Bhattacharya
30-36
Onchocercosis in Benue state, Nigeria: comparative epidemiological
amongst the Etulo and Idoma ethnic groups. E. A. Omudu and B. O. Atu
studies
37-40
Distribution of iron in plasma, erythrocytes and tissues of calves with the
progression of Theileria annulata infection. N. Sangwan and A. K. Sangwan
41-44
Mosquito breeding in riceland agro-ecosystem near Chennai, Tamil Nadu, India. J.
Ravindran and J. Williams
45-52
Random amplified polymorphic DNA of Trichomonas vaginalis isolates from
Tarbiz, Iran. R. Jamali, B. Zareikar, A. Kazemi, M. Asgharzadeh, S. Yousefee, R.
Estakhri, S. Montazer and A. Ghazanchaei
53-57
Two new species of Trypanosoma from freshwater fish (Heteropneustes fossilis and
Channa punctatus) from Bareilley, India. D. K. Gupta, N. Gupta and R. Gangwar
58-63
Impact of anthelmintic therapy on live weight gain in gastrointestinal nematodeinfected goats. A. K. Jayraw and Y. V. Raote
64-67
Ultrastructure, differential density and distribution pattern of polymorphic
microtriches in tegument of Stilesia globipunctata infecting Ovis aries (sheep). C.
Venkatesh, K. Ramalingam and V. Vijayalakshmi
68-75
The protozoan fauna living in the digestive system of Periplaneta americana in
Kolkata, West Bengal, India. J. Ghosh and A. Gayen
76-80
Chelatrema neilgherriensis n. sp. (Trematoda: Gorgoderidae) infecting the
freshwater fishes from Noolpuzha river in Wynad district, Kerala, India. K. T.
Manjula and K. P. Janardanan
81-84
Short communications
Haemato-biochemical studies on fowl coccidiosis in layer birds. N. D. Hirani, J. J.
Hasnani, R. S. Joshi and K. S. Prajapati
85-88
Re-redescription of Dissurus farrukhabadi Verma, 1936
(Digenea Echinostomatidae) with a discussion of the genus Dissurus Verma, 1936. P. C.
Gupta and R. B. Singh
89-91
A case of vaginal bleeding due to leech bite. R. P. Ganguly, M. S. Mukhopadhyay
and K. K. Patra
92-93
Field evaluation of a rapid immunochromatographic test kit for the diagnosis of
Plasmodium falciparum and non-falciparum malaria parasites from Sonpur
district, Assam. C. Rajendran and S. N. Dube
94-97
Journal of Parasitic Diseases: Vol. 30, No. 1, June 2006, 1-3
J PD
A Note from the New Editor-in-Chief
“What the Author Expects from the Editors”. This has
been expressed by Dr. Earl H. Wood of Mayo Clinic as
“I expect the editor to accept all my papers, accept
them as they are submitted, and publish them
promptly. I also expect him or her to scrutinize all
other papers with utmost care, especially those of my
competitors”.
My association with Journal of Parasitic Diseases
(started as Indian Journal of Parasitology) is both
personal and somewhat historical, and goes nearly 30
years back when in 1976 the first issue of the journal
was released in the main conference hall of the Central
Drug Research Institute, Lucknow. Certainly, it must
have been born out of the hard work and
uncompromised dedication of several Members of the
Society, and the vision of its Founder Editor-in-Chief,
late Dr. B. N. Singh. And with this issue, in a new style
and format, my tenure as Editor-in-Chief has just
begun.
The Editor-in-Chief of a journal is ultimately
responsible for its quality standards and acceptability
by the audience it addresses. His or her singular goal
should be to deliver the readers a package of good
science in a good and simple language. Therefore, the
final decision to accept or not to accept a manuscript
for publication or to send it back for modification must
be the responsibility of the Editor-in-Chief only. For
arriving at such a decision, he or she should carefully
go through the manuscript and take into consideration
the recommendations, and the reports and comments
of the Editorial Board Members and Reviewers/
Referees/Consultants, respectively. However, for the
resolution of complicated and controversial matters,
the Editor-in-Chief should also be willing to take the
help of Advisory Board Members, should such a
situation arise.
The Members of Editorial Board, who must be experts
in their respective fields, in turn, have a very important
responsibility of sending the manuscripts to suitable
Reviewers/Referees/Consultants, determine the
quality of these manuscripts and then send their
recommendations to the Editor-in-Chief. Their
recommendations regarding the suitability of the
manuscripts for publication, revision or rejection are
crucial for the Editor-in-Chief in making a final
decision, and thus, in maintaining the quality
standards of the journal. Therefore, the Editor-inChief must decide upon an appropriate size of the
Editorial Board taking into consideration the expertise
of its Members which should be in consonance with
both the dimensions of the scope of the journal and the
number of manuscripts to be handled.
The Reviewers/Referees/Consultants read, evaluate
and return the manuscripts along with their comments
prepared in accordance with the instructions provided.
These comments must be specific and not abstract,
point errors related both to fact and interpretations,
indicate inaccuracies and ambiguities, and also must
clearly suggest as to how to condense, enlarge and
improve upon the style of writing. In biological
sciences, hardly 5% manuscripts are accepted without
any revision. “The Guidelines for Reviewers,
American Society for Microbiology”, formulated
based on the policies recommended by a committee of
the Council of Biology Editors is usually very helpful
for the Reviewers/Referees/Consultants. The help
provided by them is generally acknowledged in the
form of listing their names in the last issue of the year.
Because a manuscript is the intellectual property of its
authors, it must be treated as a privileged confidential
communication throughout, till published.
The Managing Editor of a journal is usually not
2
involved in decisions related to the acceptance or nonacceptance of a manuscript. Rather, he or she is
supposed to provide the Editor-in-Chief various
support services during the review process. However,
the main function of a Managing Editor starts only
after a manuscript has been accepted for publication,
and he or she takes the responsibility to convert it into a
printed product i. e. a published paper. The Copy
Editors and Production Editors, usually associated
with big journals are responsible for the final product
as related to grammar, spellings, syntax, style
polishing etc., and the quality of page layout and
image resolution, respectively. Therefore, in my
opinion, the entire Editorial Board must meet at least
once during the year, and our annual meetings can be
the most appropriate occasions for such meetings.
It is aptly said that the reputation of a journal primarily
depends upon the quality of research papers it
publishes. I shall, therefore, with the help of Advisors,
Members of the Editorial Board and Reviewers/
Referees/Consultants, strive hard to take the standards
of our journal to new heights. Towards this end, I shall
expand the current Editorial Board, both in terms of
the number of its Members and the diversity of their
fields of specialization to encompass various aspects
of parasitology. The speed of the editorial processing
of the manuscripts submitted for publication leading
to its final acceptance or non-acceptance is also
important. I shall try to strictly follow the 15-day
duration for the Reviewers/ Referees/Consultants to
submit their reports. To the extent possible, I shall
encourage the use of electronic mail for sending the
manuscripts for review and for receiving the review
reports. This can be expected to reduce both time and
cost.
The job of an Editor-in-Chief is a very responsible one.
I would, therefore, be a remiss if I did not acknowledge
the attendant challenges that I expect to face. First, I
believe, is to implement again the very vision with
which the journal was conceived and started. There is
no doubt that the primary constituency of Journal of
Parasitic Diseases is basic parasitology. During my
informal discussions with various members of the
Society, I gathered the impression that a large number
of them feel that the journal has seriously departed
from its primary constituency. Certainly, such wheels
must have turned slowly over a period of time. I would,
therefore, with the help of our authors/contributors
and Editorial Board Members, like to improve upon
this state and try to move the journal back to basic
Prati Pal Singh
parasitology. This certainly does not mean that the
newer cellular and molecular, and other important and
emerging dimensions of parasitology shall be
neglected. Second, the journal should orient itself in
such a way, that it must make an impact on the conduct
of the theory and practice (including clinical) of
parasiotology in our country. This may not turn out to
be an easy task. The third, I feel is that there are several
areas in parasitology which continue to remain
controversial, conflicting, confusing and
conjecturing. Therefore, Journal of Parasitic
Diseases must specifically publish comprehensive
and accessible reviews which should try to focus on
these areas of parasitology. Towards this end, I intend
to invite manuscripts from experts who would be
willing to specifically contribute such reviews.
However, this notwithstanding, the journal intends to
publish at least two invited reviews on various
important aspects of parasitology in its each issue.
Parasitology research is a very big field spanning
mainly into areas like agricultural, veterinary, medical
and general parasitology. These areas, in turn, have
their own sub-areas and allied areas. Put together, they
all constitute a big world of parasitology. In our
country Journal of Parasitic Diseases is probably the
only journal that is specifically devoted to parasites
and the diseases caused by them. Therefore, it should
be the aim of the journal to adequately address all such
areas and sub-areas effectively. Additionally, Journal
of Parasitic Diseases should also try to address to
areas related to parasitology and community/society,
parasitology and economics, and parasitology and
nation development.
One of the most important policies that I have already
introduced is the “Tutorial Editorial Policy”. This
policy aims towards working of the Editorial Board
with the authors, to improve the quality of their
submitted manuscripts so as to make them
publishable. Experience has shown that a greater and
intense interaction between the authors and Editorial
Board has always been beneficial in improving both
the quality of science and of writing. Scientific
writings are different from any other type of writings
and at times not easy, and thus require considerable
practice. Unfortunately, in our country, unlike in
several other countries, most of the universities do not
have a formal course related specifically to scientific
writings. It is now well known that in many reputed
journals, mostly the manuscripts are not turned down
because of bad science but bad writing. The effects of
Editor's Note
this policy may not become visible very soon, but
certainly in the long run it is going to be beneficial,
both to the authors and the journal. I would like to take
this opportunity to assure our authors that the entire
Editorial Board is on their side, to help them in
publishing good science. Here, I must bring to your
kind attention that our journal has a big problem of not
having a suitable number of quality manuscripts
available for publication. And, this often results in the
delayed release of a particular issue of the journal.
In an editorial titled “On the Future of Scholarly
Journals”, published in Science (17 April 1998, 279,
359), the author Alen M. Edelson has very
appropriately emphasized the potential impact of
digital/electronic publishing on print publishing.
Presently, most of our print journals are suffering from
financial crunch because of declining library budgets,
escalating costs, reduction/cancellation in
subscriptions and, of course, increasing manuscript
pressure. Digital/electronic publishing, on the other
hand, can be expected to be cost-effective and would
allow greater and relatively more convenient
accessibility to scientific information. Therefore, I
believe in the need that Journal of Parasitic Diseases
also must run with the times, and should evolve
strategies to digitalize it in a bigger way, including online submission and processing of manuscripts.
Nevertheless, it is becoming increasingly clear that
electronic journals will also be fraught with their own
3
sets of problems, and may not be able to make print
journals obsolete so soon.
In closing, I would like to sincerely thank Dr. V. P.
Sharma (Chairman) and Members of the committee to
constitute new Editorial Board Prof. M. S. Jairajpuri
(former President of the Society), Prof. Veena Tandon
(President of the Society), Prof. Nancy Malla (former
Editor-in-Chief), Dr. J. K. Saxena (Secretary of the
Society), Dr. L. M. Tripathi (Treasurer of the Society),
Dr. S. Dutta and Dr. S. L. Hoti, and to the Society at
large, for the honour of offering me this important
responsibility and for reposing confidence in me. I
should like to take this opportunity to especially thank
Prof. R. C. Mahajan and Dr. G. P. Dutta for their
valuable suggestions. I should also like to thank the
authors of this issue and to the future authors. I deem it
my greatest pleasure to thank Prof. P. Ramarao,
Director, National Institute of Pharmaceutical
Education and Research, for his help and
encouragement without which it would not have been
possible for me to undertake this responsibility.
Finally, I thank our printer Mr. Lalit Azad, for his keen
interest and excellent work in a timely manner. I am
confident that with our joint efforts, Journal of
Parasitic Diseases will continue to maintain its high
standards and surge to new heights. I shall continue to
interact with you all from time to time.
Prati Pal Singh, Ph. D., F. N. A. Sc.
Editor-in-Chief
Review
Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 4–15
J PD
Histochemical, biochemical and immunological studies
in Haemonchus contortus (Nematoda: Trichostrongyloidea) - an Indian perspective
M. L. Sood
Department of Zoology, Punjab Agricultural University, Ludhiana.
ABSTRACT. Haemonchus contortus is the most pathogenic nematode parasite of sheep/goats in
tropics/subtropics. Presently, the different aspects of H. contortus research in histochemistry
[absorptive surfaces-structure and composition (the body wall, the gut), anthelmintic effects;
spicules and gubernaculum], biochemistry [inorganic elements; carbohydrates (glucose and
glycogen utilization, glycolysis, TCA cycle, pentose phosphate pathway]; amino acids
(composition, metabolism, anthelmintic effects); lipids (composition, metabolism, enzymes,
anthelmintic effects), proteins; nucleic acids; nutrition (digestive enzymes), biochemistry in
taxonomy; miscellaneous] and immunology have been reviewed in Indian context.
Key words: biochemistry, Haemonchus contortus, histochemistry, immunology, India
INTRODUCTION
Heamonchus contortus
(Rud.,1803) commonly
known as the twisted stomach worm, is a bloodsucking nematode parasite, primarily occurring in the
abomasum (Fig.1) of small ruminants, notably sheep
and goats. It has been ranked as the most important
parasite of small ruminants in all regions across the
tropics/subtropics (Anonymous, 1992).
Haemonchosis, the disease caused by this nematode is
responsible for considerable economic losses
worldwide. In hyperacute disease, death of the host
may occur within one week without significant signs
(Fraser, 1991).
Over the years, attempts have been made to study
various aspects of Haemonchus spp. from different
parts of the world (see Sood and Kapur, 1982a).
Earlier, Haemonchus research in India was reviewed
Fig. 1. A part of goat abomasum cut open to show Haemonchus
contortus, in situ. Inset, adult female and male (From Sood and
Kapur, 1982; with permission from Shidha Publications,
Ludhiana, India).
Corresponding author: Prof. M. L. Sood, 500/4, Model Town
(Club lane), Ludhiana - 141 002, Punjab, India.
E-mail : [email protected]
(Sood, 1981) under the titles : morphology and
d i s t r i b u t i o n , b i o c h e m i s t r y, i m m u n o l o g y,
development and cytology, life-history, pathogenesis,
Haemonchus histochemistry, biochemistry and immunology
5
clinical symptoms, diagnosis, epidemiology,
treatment and prophylaxis. Subsequently, in a review
on haemonchosis in India, Sood (2003) covered
epidemiology, pathology, diagnosis and control,
including resistant strains. Presently, the upcoming
areas of Haemonchus research, and the ones not fully
covered in the above reviews have been taken up.
Hopefully, these three reviews, covering separately
the various aspects, would provide an up-dated
consolidated account of 'Haemonchus to
haemonchosis' in India.
the cuticle (Sood and Kaur, 1977), has been well
documented (Sood and Kaur, 1976).
HISTOCHEMISTRY
Histochemical studies have been made on the
absorptive surfaces (including in vitro anthelmintic
effects) and of the spicules and gubernaculum.
Absorptive surfaces
Parasitic nematodes have two absorptive surfaces- the
external surface or the body wall and the gut, though
latter is the main functional unit from nutrition point of
view. The body wall in nematodes is composed of the
cuticle, an underlying cell layer (hypodermis or
epidermis), and the longitudinally orientated somatic
musculature (Wright, 1987). Nematode cuticle is one
of the most complex acellular structures synthesized
by a living organism. In order to understand the
transport of solutes in the body wall, experimental
determination of diffusion coefficients of NaCl and
KCl in adult H. contortus at 298. 16 k has been made
(Sood et al., 1999).
The nematode gut consists of three parts, a muscular,
cuticular lined pharynx (oesophagus), a relatively
straight intestine, whose wall is one cell thick and a
rectum or cloaca, which like pharynx is lined by
cuticle (Bird, 1971; Chitwood and Chitwood, 1974).
Nematodes do not have a continuous muscle sheet
around the intestine and the pseudocoelomic fluid is in
direct contact with the basement membrane. The
nematode intestine is, therefore, an ideal cell
monolayer to study the transport mechanisms, the
only diffusion barrier being the basement membrane
(Barrett, 1981).
Absorptive surfaces in H. contortus have been
reviewed (Sood, 1999), under the titles: structure and
composition, immunology and anthelmintic effects.
Structure and composition
The body wall: The morphology of the vulvar
configurations in the female H. contortus, formed by
The histochemical studies on the body wall of H.
contortus (Sood and Kalra, 1977) have revealed that
the cuticle is mainly proteinaceous in nature. The
lipids and PAS-positive materials are only present in
cortical layers. In addition, haemoglobin and acid
phosphatase (AcPase) are also present. Glycogen,
lipids, RNA, AcPase and alkaline phosphatase
(AkPase) have been reported in the hypodermis. The
oval dense body is composed of keratinous and
collagenous proteins associated with acid
mucopolysaccharides. Muscles carry a greater
concentration of glycogen granules and
phospholipids. The functional significance of these
components has been fully discussed.
The gut: Singh and Johl (2001) studied the structure
of the fore-gut (stomodaeum). Histomorphology of
the intestine has also been studied (Johl, 2003). Sood
and Sehajpal (1978) made morphological,
histochemical and biochemical studies. The intestinal
epithelium (IE) is provided with a well-developed
brush border (BB) which contains periodic acidSchiff- positive mucoproteins. The IE stores glycogen
and lipids. It stains diffusely for phospholipids and
general proteins and also for terminal-NH2 group. The
presence of Fe2+ and Fe3+ containing pigments and
activities of AcPase and AkPase, glucose-6phosphatase (G-6-Pase) and 5'-nucleotidase have
been abserved in IE. Biochemically, pH optimum for
intestinal AcPase has been found to be 4.8. BB shows
positive reactions for Acpase and G-6-Pase, and
negative for AkPase and 5'-nucleotidase. Presence of
enzymes in the BB is related to extracellular digestion
and absorption of nutrients.
Anthelmintic effects
Study of alterations induced in the absorptive surfaces
(and neuromuscular system) of the parasites by the in
vitro incubations with the anthelmintic drugs is of
prime importance, particularly in view of the
repeatedly reported resistant strains of H. contortus.
The mechanisms by which the drugs act on these
absorptive surfaces are quite obscure. During their
absorption, anthelmintics are expected to induce some
cellular and chemical changes in the absorptive
surfaces. Anthelmintics during their absorption
affect/and/or modify the enzyme activity and may also
alter the normal metabolism of the absorptive
surfaces.
6
Sood
Anthelmintic effects (and other aspects) on the
absorptive surfaces have been reviewed (Kaur and
Sood, 1986).
well as neuromuscular system) and hypodermis
emphasize that both absorption and motility of the
parasite are effected.
The morphological and histochemical effects of in
vitro incubations with thiabendazole, morantel
tartrate, tetramisole hydrochloride and piperazine
hexahydrate on the body wall and intestine have been
investigated (Sood and Kaur, 1982). Similar effects of
dl-tetramisole (TMS) and rafoxanide (RFX) have
been observed for AkPase, AcPase adenosine
triphosphatase (ATPase) and G-6-Pase (Kaur and
Sood, 1982 d). Also, histological study on the effects
of these two anthelmintics has been made (Kaur and
Sood, 1983a). In vitro alterations induced by Nilzan
(NLZ) and albendazole (ABZ) have also been studied
in AcPase, AkPase, ATPase, G-6-Pase, cytochrome
oxidase, monoamine oxidase, non-specific esterases,
acetylcholine esterase (AChE), and in succinic, lactic,
glutamate and glucose-6-phosphate dehydrogenases,
reduced nicotinamide, adenine dinucleotide
diaphorase and reduced nicotinamide adenine
dinucleotide diaphorase (NADH-D) and reduced
nicotinamide adenine dinucleotide phosphate
diaphorase (Kaur and Sood, 1990). Also, the effects of
Nilverm (NLV) and Nilverm forte (NLF) (Kaur and
Sood, 1992a) and thiophenate (TP) and fenbendazole
(FBZ) (Kaur and Sood, 1992b) have been abserved for
these enzymes except NADH-D. Also, the
morphological and histochemical effects of ALB,
FBZ, TP, TMS, dl-tetramisole and oxyclozanide (TO),
and levamisole HCl (LMS) have been studied on the
absorptive surfaces (and neuromuscular system) of
adult H. contortus. ABZ, FBZ, TP, TO, HCl and LMS
reduced the quantity of neutral mucopolysaccharides
in the intestine. TO and ABZ caused the loss of acid
mucopolysaccharides in the microvilli of intestine.
Loss of lipids from the intestine was evident after TO,
ABZ, FBZ and TP treatments. LMS caused
accumulation of very large lipid droplets in the
intestine (Kaur and Sood, 1996).
Spicules and gubernaculum
In general, morphological changes are most evident in
the intestine and muscles. Enzymatic intensities and
alterations induced by the in vitro incubations of the
drugs in H. contortus are stronger in the intestine than
in any structure, indicating it to be the main structure
involved in the absorption and also in the action of
various anthelmintics. Different structures of the
parasite respond differently to a particular drug,
indicating the response and function correlation. The
alterations induced by the drugs in the intestine (as
Morphological and histochemical studies on the
spicules and gubernaculum (Sood and Kaur, 1983),
have revealed that these are mainly proteinaceous. The
sclerotized part consists mainly of keratin and
collagen. The presence of traces of AcPase in the
spicules may represent some metabolic activity. The
protoplasmic part is composed of proteins,
carbohydrates and lipids.
BIOCHEMISTRY
Much of the interest in parasite biochemistry comes
from the ways in which the metabolic pathways have
been modified to suit the highly specialized parasitic
mode of life. In addition to intrinsic interest, parasite
biochemistry has great practical importance through
chemotherapy and vaccine production, and in
understanding of the complex association involved in
the host-parasite relationship. However, information
in parasite biochemistry is patchy, and mainly the
large-sized worms have been exploited (Barrett,
1981).
Of the various biochemical parameters, in H.
contortus, carbohydrates and lipids have been studied
more extensively compared to others. Further, most of
these studies are restricted to adult form, though in L3
carbohydrate catabolism has been studied in detail.
Moreover, variation in metabolic pathways due to
strains poses a great problem. Biochemistry of
Haemonchus has been reviewed (Kapur and Sood,
1987a) under the titles: chemical composition
(carbohydrates, lipids, proteins, amino acids, nucleic
acids, hormones, inorganic elements, pigments),
absorption/transport, biosynthesis (carbohydrates,
lipids, proteins, amino acids, nucleic acids),
catabolism/utilization (carbohydrates-glycolysis,
TCA cycle), carbon dioxide fixation, electron
transport system, pentose phosphate pathway,
glyoxylate cycle, strain variations in energy
metabolism; lipids; proteins; biochemistry in
taxonomy and miscellaneous.
Inorganic elements
These play a significant role in the physiology of
parasites. Their impotance is also demonstrated by the
influence of mineral deficiencies in host's diet.
Haemonchus histochemistry, biochemistry and immunology
7
Various elements detected in H. contortus (Sood and
Kapur, 1980) include phosphorous (P), zinc (Zn),
calcium (Ca), iron (Fe), magnesium (Mg), copper
(Cu), manganese (Mn), boron (B) and potassium (K),
in decreasing concentrations in female. In the male
also, same elements are present, with the exception of
B. The relative order of occurrence in the male is
P>Ca>Zn>Fe>Mn>Mg>Cu>K. Presence of more Ca,
P and Fe in male has been discussed.
of glycogen utilization is similar both in male and
female (Premvati and Chopra, 1979). Under in vitro
conditions, the worms readily utilize glucose from the
medium (Chopra and Premvati, 1977; Kaur and Sood,
1982a). At the same time, glucose is excreted and this
excretion is possibly at the expense of endogenous
glycogen reserves, which falls rapidly with time. An
outstanding feature of carbohydrate catabolism in
nematodes is the production of reduced organic end
products, even under aerobic conditions.
Carbohydrates
Carbohydrates form the chief energy source in
parasitic nematodes. In view of the importance of
carbohydrates in helminths, any difference in their
carbohydrate metabolism and that of their hosts might
be usefully exploited in helminth control. The
outstanding feature of carbohydrate breakdown in
nematodes is the production of reduced organic end
products and this occurs even under aerobic
conditions.
Carbohydrate metabolism in H. contortus has been
reviewed (Kaur and Sood, 1983b) under the titles :
glucose and glycogen utilization, carbohydrate
metabolism, including the glycolytic pathway, the
pathway of CO2 fixation, the TCA cycle and the
pentose phosphate pathway.
Glucose and glycogen utilization: Glucose in very
important energy source for many helminths
inhabiting the gut of vertebrates. It is generally
believed that helminths absorb glucose against a
concentration gradient and use their endogenous
carbohydrates as an energy source only when it is
unobtainable from outside. Similarly, glycogen in
most of the nematodes provides a significant reserve
store of energy, particularly in forms which are
parasitic in animals and which exist in environments
of low oxygen tension.
Amount of glucose and glycogen contents in H.
contortus has been determined. It contains
considerable amount(8-12% fresh tissue of glycogen;
Chopra and Premvati, 1977; Premvati and Chopra,
1979; Kaur and Sood, 1982a). Famale has more
glycogen than male (Premvati and Chopra, 1979).
This is possibly related to the reproductive role of
female rather than with general metabolism. Adult
worm utilizes glycogen reserves rapidly. In male, it
decreases less rapidly with time, while lin female, it
decreases exponentially with time. Thus, in female,
rate of glycogen utilization at any time is directly
proportional to glycogen content. However, initial rate
The in vitro effects of TMS and RFX on these
constituents have also been studied (Kaur and Sood,
1982a).
The pathway of carbohydrate breakdown in H.
contortus has been worked out as follows:
Glycolysis: Adult worm utilizes glucose both
aerobically and anaerobically. Under both aerobic and
anaerobic conditions, end products of glucose
metabolism include CO2, propanol, acetate, and npropionate as the major and ethonol, lactate and
succinate as the minor components. It is unique among
nematodes that propanol is a major excretory product.
This is possibly produced by reduction of propionate
and hence glucose catabolism in H. contortus does not
indicate a major departure from the known pathways
of anaerobic glucose utilization. Production of lactate
from glucose in H. contortus has been demonstrated
by Chopra and Premvati (1977). In female 90-100% of
glucose and in male 80-90% is catabolized to lactate.
Both glucose consumption and lactate production
decrease with the progression of incubation.
Enzymes of glycolysis have been detected in adult H.
contortus (Kaur and Sood, 1982b). These include
hexokinase, phosphoglucomutase, phosphoglucoisomerase, aldolase, glycerladehyde-3phosphate dehydrogenase, phosphoglycerate kinase,
phosphoglyceromutase-enolase-pyruvate kinase,
pyruvate kinase (PK), and lactate dehydrogenase
(LDH). Low PK and LDH activities suggested an
alternate pathway from phosphoenolpyruvate. LDH
exhibits optimum activity (about 180 nmoles/min/mg
protein at a pH of 6.6 and a temperature of 37° C
(Kapur et al., 1985). Thermostability of LDH has also
been studied (Harpreet et al., 1991).
Varying degrees of inhibition of glycolytic enzymes
have been observed with 50 µg/ml of TMS and RFX
treatments, former being more effective. These effects
may block the glycolytic pathway and deprive the
8
parasite of its ATP production (Kaur and Sood,
1982b).
TCA cycle: Studies on aerobic catabolism of
carbohydrates in helminthology is an important area
of research. In small nematodes, the TCA cycle plays
an important but not exclusive part in their energy
economy (Ward, 1982). The occurrence of TCA cycle
in parasites has not been demonstrated with the same
precision as in vertebrate tissue or bacteria. A
functional TCA cycle may exist in the developing eggs
and larvae of H. contortus. The presence of aerobic
enzyme systems in the larvae may be a preparation for
the next stage of development, since development of
larvae to adults requires oxygen. Various enzymes of
the TCA cycle viz. aconitase (ACO), isocitrate
dehydrogenase (ICDH), succinate dehydrogenase
(SDH), fumerate reductase (FR), fumarase and malate
dehydrogenase (MDH) have been detected in adult H.
contortus (Kaur and Sood, 1983c). Low activities of
ACO and ICDH suggest that TCA cycle has a minor
function and the pathway of CO2 fixation is the major
pathway in the energy metabolism of the parasite.
Incorporation of carbon into proteins and
carbohydrates (Kapur and Sood, 1986c). and of C14
into various amino acids (Kapur and Sood, 1984b),
also give evidence for the TCA cycle operation in H.
contortus.
In a study on the effects of pH and temperature, Kapur
et al. (1984), found that SDH exhibits maximum
activity (about 120 nmoles/ min/mg protein) at an
optimum pH of 8.2 and temperature 32° C. pH
optimum for ICDH (about140nmoles/min/mg
protein) and MDH (about 30 nmoles/min/mg protein)
is 9.0 and 7.8 respectively; optimum temperature
being 32º C for both the enzymes (Kapur et al., 1985).
The in vitro effects of TMS and RFX on various
enzymes at 50 µg/ml, have shown varying degrees of
inhibition of SDH and FR. At the same concentration,
the activities of other enzymes remained unaltered
(Kaur and Sood, 1983c). Also, the in vitro effects of
NLZ, ABZ, NLV, NLF, TP and FBZ have been studied
on LDH (Kaur and Sood, 1993).
Pentose phosphate pathway: This is an alternative
pathway of glucose catabolism. There is, however, no
definite evidence that this pathway is involved in
energy metabolism in parasitic nematodes. Its main
function may be to provide NADPH and C5 and C7
sugars for synthetic reactions (Barrett, 1976). In H.
contortus, key enzymes of this pathway, viz. glucose-
Sood
6-phosphate dehydrogenase (GDPH) and 6phosphogluconate dehydrogenase, as also the in vitro
effects of TMS and RFX on these enzymes have been
demonstrated (Kaur and Sood, 1985). GDPH exhibits
activity (4 nmoles/min/mg protein) at pH 7.4 and 37º C
(Kapur et al., 1984). Thermostability of GPDH,
separately for male and female worms has been
studied (Harpreet et al., 1991).
Synthesis of nucleic acids in adult H. contortus, is also
indicative of the operation of pentose phosphate
pathway (Kapur and Sood, 1986c).
Amino acids
Amino acids commonly found in proteins, also occur
as free acids. There are number of amino acids which
are never found as constituents of proteins, but play
important metabolic roles. Therefore, it is essential to
study both free and bound amino acids.
Composition: In adult H. contortus, both free and
bound acids are present (Kapur and Sood, 1984b). The
level of free amino acids is 191 mg in male and 523 mg
in female per 100 g on fresh weight basis. Various
acids in free form include aspartic acid (Asp),
glutamic acid (Glu), leucine + isoleucine (Leu+Ile),
tryosine (Try), lysine+histidine (Lys+His),
glycine+serine (Gly+Ser), arginine (Arg),
valine+methionine (Val+Met), cystine+cysteine
(Cys-Cys+Cys), β-alanine (Ala) and α−Ala in
decreasing order of concentration in female, and in
male, the order of occurrence is: Asp>Glu>Tyr>βAla>Lys+His>Gly+Ser>Leu+Ile>Val+Met>Agr.
Thus, in male Cys-Cys+Cys and α−Ala are missing. In
female, concentration of Leu, Ile and Asp is
significantly higher than in male, and in male it is of
Tyr, β−Ala, Lys+His and Glu. However, concentration
of Val, Met, Gly, Ser and Arg is similar in both the
sexes. Asp is present in the highest concentration in
both the sexes and the acid, in least concentration is
Arg in male and α-Ala in female. Like other
invertebrates, in H. contortus too, free amino acid
pools are dominated by one or two amino acids, i.e.,
Asp in female and Asp, Glu in male. Proline (Pro) not
reported has been earlier demonstrated by Nigam
(1979). Higher amount of Met in female than in male
indicates the extent of transaminase reactions. More
of Lys in male in indicative of its role in maintaining
sperm viability (having a histone-like function) as in
humans. As in birds, more of Glu in male may play a
role in the maintenance of osmolarity and pH of
seminal plasma.
9
Haemonchus histochemistry, biochemistry and immunology
In both the sexes, bound acids detected are almost
similar to those of the free fraction. The differences
include the absence of β-Ala and presence of α-Ala,
Cys-Cys+Cys, and Pro in both the sexes.
Concentration of both total and individual bound acids
is far more than that in male. Relative order of
occurrence in female is Tyr>Lys+His>Glu>Gly+
Ser>Leu+Ile>Val+Met>Asp>Arg>Cys-Cys+Cys>
α-Ala, and in male it isTyr>Gly+Ser>Lys+His>Glu>
Leu+Ile> Val+Met>Asp>Cys-Cys+Cys>Arg>α-Ala.
However, concentration of Pro is not known.
GLUCOSE
GLUCOSE -6- PHOSPHATE
SERINE
TRIOSE PHOSPHATE
TRYPTOPHAN
PHOSPHOENOL PYRUVATE
Metabolism: No significant variation in the
composition of amino acids of nematodes inhabiting
different environments has been found. This indicates
that in nematodes, the composition of the amino acid
pool remains largely unaffected, not being dependent
only on their absorption form external environment.
Thus, the nematodes must be capable of amino acid
biosynthesis. This has been demonstrated in many
nematodes. Further, in parasitic nematodes, where the
major metabolic activities are directed towards egg
production, the emphasis on protein and hence amino
acid biosynthesis must be considerable. If we are able
to find some differences in the enzymes involved in
amino acid biosynthesis by the parasite and the host,
we can selectively check the synthesis of amino acids
in the parasites. Thus, these would not be available for
incorporation into egg proteins, and hence the
propagation of a species could be checked.
Adult H. contortus has been investigated for its ability
to utilize various C14-labelled precursors, i.e., glucose,
acetate, CO2 and palmitic acid for amino acid
biosynthesis (Fig. 2) (Kapur and Sood, 1984c). It has
been demonstrated that H. contortus is capable of
synthesizing essential as well as non-essential amino
acids. Possible mechanisms for the involvement of
various precursors in amino acids have been
examined. It is not possible to predict whether or not
the synthesis of amino acids is at a level commensurate
with reproduction. Therefore, further studies need to
be carried out on these lines. Also, it would be
interesting to elucidate the pathways of amino acid
biosynthesis. These studies can give a deeper insight
into the metabolism of the worms.
ACETATE
PHENYL ALANINE
TRYOSINE
TRYPTOPHAN
ALANINE
PYRUVATE
ACETYL CoA
LEUCINE
VALINE
LEUCINE
ISOLEUCINE
ALANINE
LYSINE
ASPARTATE
THREONINE
SERINE
OXALOACETATE
GLYCINE
CO 2
METHIONINE
FUMARATE
ARGININE
Concentration of α-Ala, Lys+His and Tyr is higher in
female and that of Gly, Ser, Cys-Cys+Cys in male.
Levels of Leu, Ile, Val, Met, Asp, Glu and Arg are
similar in both the sexes. Thus, Tyr is present in the
highest concentration and α-Ala the least, in both the
sexes.
PALMITIC ACID
ERYTHROSE-4-PHOSPHATE
CITRATE
CYSTEINE
SERINE
CYSTINE
TCA CYCLE
α - KETOGLUTARATE
SUCCINYL CoA
ARGININE
TRYPTOPHAN
GLUTAMATE
PROLINE
PHENYL ALANNE
Fig. 2. Proposed scheme for the involvement of C14-glucose,
acetate, carbon dioxide and palmitic acid in amino acid
biosynthesis in adults of H. contortus (After Kapur and Sood,
1984; with permission from Elsevier).
Absorption/incorporation studies of C14-labelled
amino acids in H. contortus by Kocher et al. (2000)
have revealed significantly higher absorption of
amino acids in females than the males. The absorbed
amino acids first get incorporated into cystosolic
proteins and after periods of time (30 min), the
incorporation in the deoxycholate and sodium dodecyl
sulphate extractable membrane bound and nuclear
proteins increased significantly. It is postulated that H.
contortus absorbs amino acids through cuticle or
possibly by ingestion. Although there is no definite
proof for the mechanism of ingestion of soluble
nutrients, yet it can be similar to the way the parasites
ingest blood after attachment to the abomasal mucosa.
Anthelmintic effects: The two transaminases- alanine
(ALT) and asparate (AST) are involved in
transamination, i.e., interconvert a pair of amino acid
and a pair of keto acid. ALT is involved in the
interconversion of L-alanine to pyruvate, whilst AST
converts asparate to oxaloacetate (Rodwell, 1990).
Anthelmintic effects of NLZ, TP, NLV, NLF, ALZ and
FBZ on these transaminases (Kaur and Sood, 1993),
have revealed the increase of these transaminases
except with NLZ.
Lipids
Carbohydrates form the major and possibly the sole
energy source of parasitic nematodes. However, the
importance of lipids cannot be overlooked, these the
10
being structural and functional constituents. These are
important components of membranes, which are in
constant stage of dynamic equilibrium. Also, these are
incorporated into eggs and are important energy
reserves in the free-living stages of animal parasitic
nematodes. Thus, it we are able to selectively inhibit
lipid biosynthesis in parasites, these would not be
available for incorporation into eggs, and we can
check the propagation, if not eliminate the parasite.
Further, lipid biochemistry (and nutritional
requirement) studies indicate the basis of host
specificity, which can be exploited for in vitro growth
of the parasite. Also, study of lipid biochemistry of
different groups of nematodes may reveal
phylogenetic relationship or adaptations of parasites.
Lipid composition and metabolism in nematodes has
been reviewed (Kapur and Sood, 1995) under the
titles: composition, absorption/transport, nutritional
requirements, biosynthesis, catabolism, lipids in
intermediary metabolism, enzymes involved in lipid
metabolism and anthelmintic effects. In H. contortus,
lipid composition and metabolism has been reviewed
(Sood and Kapur, 1989) under the titles: composition,
distribution, biosynthesis, catabolism, enzymes
involved in lipid metablism, lipids in intermediary
metabolism and anthelmintic effects.
Composition: Qualitative and quantitative aspects of
lipid composition have been studied (Kapur and Sood,
1985). The total lipids constitute 44mg/g of fresh
tissue and 200 mg/g of dry tissue. Possibly, higher
amount of lipids in H. contortus accounts for the daily
production of its own weight of eggs. The non-polar
(NP) lipids are present in higher concentration than
polar (P) lipids, the NP/P ratio being 1:4. NP lipids
include monoacylglycerols (MG), diacylglycerols
(DG), sterols, free fatty acids (FFA), hydrocarbons
and pigments (Hyd+Pig), triacylglycerols (TG), and
sterol esters (S. esters). Of these, TG are present in the
highest concentration, followed by FFA, the sterols
being the least. Among P lipids, the following
components are present in the decreasing order of their
occurrence: phosphatidyl choline (PC), phosphatidyl
ethanolamine (PE), lyso PE, sphingomyelin+lyso PC,
cerebrosides (Cereb.), phosphatidyl inositol (PI) and
phosphatidyl serine (PS). Fatty acid analysis of total, P
and NP lipids revealed 28, 13 and 15 fatty acids
respectively. Fatty acids detected range from C12 to C24,
with 1-4 double bonds. C16 and C18 fatty acids
constitute more than 50% and C21, C22 about 25% of
total fatty acids. An unidentified acid with carbon
Sood
number 22 or 23 is also present in traces.
Metabolism: Nematodes usually contain lipids in
considerable amounts, and these are used for energy
production, usually under aerobic conditions. It is
reasonable to assume that some lipid catabolism could
occur in H. contortus because of its aerobic
conditions.
Adult H. contortus has been investigated for its ability
to utilize lipids with regard to total lipids, sterols, FFA,
acylglycerols and phospholipids produced during
incubation in vitro (Kapur ans Sood, 1987c). All these
components exhibit extensive fluctuations,
decreasing at some times and increasing at others, thus
indicating both biosynthesis and utilization. Also,
changes in fatty acid components of total lipids have
been analyzed by GLC.
Investigations on adult H. contortus for its ability to
synthesize various lipids form C14-glycerol (Kapur
and Sood, 1987b), have revealed that the worm is
capable of utilizing it for lipid biosynthesis. There was
significantly more incorporation into P than into NP
lipids (P/NP ratio > 1.89). Further the worm is capable
of synthesizing all the classes of complex lipids
present, viz., acylglycerols, FFA, sterols and S.esters
among NP lipids, and PC, PE, PI, PS,
sphingomyelin+lyso PC, lyso PE and Cereb among P
lipids. Time-course incorporation studies revealed the
operation of the MG-pathway for the synthesis of TG.
Information has also been obtained indicating which
pathways are utilized by the worm for phospholipid
synthesis. The results are indicative of de novo
synthesis of lyso PC and lyso PE.
Incorporation studies with labelled substrates,
sodium-1-C14-acetate and U-C14-D-glucose
demonstrated that adult H. contortus has extremely
active mechainisms for synthesizing all classes of
complex lipids including free cholestrol. More of the
label from acetate than glucose is incorporated into
total lipids. With both the precursors, there is more P
than NP synthesis, the P/NP ratio being 3.991 in case
of acetate and 1.223 in case of glucose. Among NP
components, most of the label from acetate is
incorporated into FFA, TG, Hyd+Pig, and label from
glucose into TG and FFA only. Among P lipid classes,
most of the label from acetate is incorporated into PC
and from glucose into PC and PE (Kapur and Sood,
1984a). It is suggested that in order to establish a
particular metabolic pathway for lipid biosynthesis,
the pool size and turnover lipids must be determined.
Haemonchus histochemistry, biochemistry and immunology
11
Accordingly, the presence and functions of the
enzymes involved in lipid metabolism must be
considered.
concentrations greater than 4mm. It was stable at 4ºC
for several weeks, but lost 60% of its activity when
liated at 60°C for 5 min. Physostigmine and
neostigmine inhibited enzyme activity at low (µm)
concentrations, whereas phenylmethyl sulfonyl
fluoride and sodium fluoride were inhibitory only at
high concentrations (Joshi and Singh, 2000). Specific
activity of ChE was 33.64± 0.2 (µ moles substrate
formed/h/g tissue). After treatment with TMS and
RFX, ChE activity was reduced to 31% and 30%
respectively (Kaur and Sood, 1982c).
H. contortus has also been investigated for its ability to
synthesize lipids form simple 14C-precursors (Kapur
and Sood, 1986a). It has been shown to have
extensively active machanisms for synthesizing all
classes of complex lipids present, including free
cholestrol from HCO3 and 14C-palmitic acid. With
both the precursors, there is more of NP than P lipid
synthesis, the NP/P ratio being 1.258 in case of HCO3
and 1.917 in case of palmitic acid. Thus, H. contortus
has extremely active mechanisms for the synthesis of
complex lipids from exogenously supplied fatty acids
from CO2 fixation.
Results of the incorporation of carbon from 14Cglycerol into total P and NP lipids (Kapur and Sood,
1991) reveal that radioactivity lost into the medium as
lipids decreased during the first 3 h of incubation. This
was followed by a drastic increase during the 4th h.
Again, there was decline by 75% during the 5th h.
However, there was a slight increase in the NP lipids
during the 5th h.
Enzymes: Lipases hydrolyse esters of long chain fatty
acids. In H. contortus, lipases and phospholipases
have been detected in adult worms (Kapur and Sood,
1986e). Lipase exhibits an optimum activity at pH 7.4
and temperature of 42° C, and phospholipase at pH 5.0
and temperature of 37° C. An indirect evidence of
lipase is also obtained from time-course incorporation
studies with glycerol, as decrease in the amount of
label in triacylglycerols has been observed (Kapur and
Sood, 1987b).
Among esterases AChE is involved in nervous
transmission in nematodes and choline esterase (ChE)
serves to destroy the transmitter, as it does in other
groups of animals (Lee, 1965). Both AChE and ChE
have been demonstrated in a variety of nematodes,
trematodes and cestodes (Barrett. 1981). AChE was
purified from extract of adult H. contortus by gel
filtration and ion-exchange of ConA-Sepharose
chromatography. The enzyme was also secreted by the
parasite during in vitro cultivation, and it was partially
purified from the excretory-secretory products. The
presence of enzyme-specific antibodies was observed
in animals infected with H. contortus. The MW of the
enzyme by SDS-PAGE was 144 KDa. It showed
typical Michaelis-Menten kinetics at low substrate
concentrations, but was inhibited by substrate
Anthelmintic effects: The in vitro effects of TMS on
lipid biosynthesis from simple precursors such as C14acetate and glucose have been studied in adult H.
contortus (Kapur and Sood, 1986d). It has a
considerable inhibitory effect on lipid biosynthesis,
percentage inhibition being 26 in case of acetate and
34 in glucose.
Proteins
Parasitic helminths are capable of efficient protein
synthesis and incorporation of labelled amino acids
into proteins has been demonstrated in several species.
Adult H. contortius is capable of efficient protein
synthesis from acetate, glucose, palmitic acid and CO2
(Kapur and Sood, 1986c). Of these, palmitic acid is the
most efficient precursor and CO2, the least. Extent of
synthesis from acetate and glucose is intermediate.
Nucleic acids
Adult H. contortus is capable of efficient nucleic acid
biosynthesis from acetate, glucose, palmitic acid and
CO2 (Kapur and Sood, 1986d). Synthesis from acetate
and CO2 is negligible, compared to that from glucose
and palmitic acid. It is suggested that glucose is
partially decomposed via HMP-pathway into ribose
and hence incorporated into RNA.
Nutrition
Digestive enzymes: The nutritive functioning of H.
contortus depends on the activity of the digestive
enzymes, especially protease (s), which play a number
of critical roles such as digestion of host proteins,
retardation of blood coagulation, evasion of immune
response, invasion of host tissues and blood feeding
and its digestion (see Kocher et al., 2002a). In a study
on the characterization and purification of cystosolic
and membrane-bound protease (s) in adult H.
contortus, different fractions showed optimum
protease activity at 37° C, pH 8.5 and 8.0 mg casein of
12
concentration (Kocher et al., 2002a). The female
fractions had a particularly high activity of protease (s)
in comparision with the male fractions, especially of
membrane-bound enzymes in the anterior half.
Inhibition/activation studies revealed the presence of
four kinds of protease (s) in the cystosolic and
membrane-bound fractions. Protease (s) in different
fractions are purified to a greater extent by higher
concentrations of saturated ammonium sulphate
solutions, i.e., ranging from 50 to 65%. The
purification study revealed the presence of multiple
forms of protease (s) in cystosolic and membranebound extracts of H. contortus.
Studies have also been made to elucidate the inhibition
of protease (and lipase activity) from excretorysecretory products, cystosolic and membrane bound
fractions of male and female H. contortus by
immunoglobulins raised in rabbits against cytosolic
fraction (Kocher et al.,1996).
Biochemistry in taxonomy
Possibility of the use of electrophoresis in
polyacrylamide gel for taxonomy of helminths is well
established. Electrophoretic analysis of proteins of
knobbed and linguiform morphs of female H.
contortus revealed marked differences (Sood and
Kapur, 1982a). Proteins of knobbed form have 13
fractions, and those of linguiform 14. Further, in
knobbed form, there are 3 bands at cathode and 2 at
anode, while in linguiform morph, at cathode, there
are 5 bands and at anode only one. This observation
(along with other evidences) tends to support the view
of subspeciation in H. contortus.
Miscellaneous
Glutamate dehydrogenase (GDH) plays a central role
in amino acid deamination and in the formation of αamino nitrogen groups form ammonia. Its optimum
activity (162 nmoles/min/mg protein) is at pH 7.0 and
temperature of 27 ° C (Kapur et al., 1984).
Thermostability of GDH has also been studied
(Harpreet et al., 1991. The in vitro effects of NLZ,
ABZ, NLV, NLF, TP and FBZ have been studied in
AkPase and AcPase. All the six drugs caused decrease
in the enzyme levels of AkPase, the maximum effect
being exerted by NLZ. In AcPase, marked decrease in
the level was observed, maximum effect being caused
by NLF (Kaur and Sood, 1993).
Malic enzyme (ME) is one of the enzymes involved in
CO2 fixation and its presence suggests an alternate fate
Sood
of phosphoenolpyruvate and ATP is generated, when
the latter is reduced to pyruvate. Activity of ME has
been studied in adult H. contortus (Kaur and Sood,
1982b). The in vitro treatments with TMS had no
significant effect. However, after RFX treatment, ME
activity was reduced to 38%.
Kaur and Sood (1982a) studied the effect of TMS and
RFX on total volatile fatty acids under in vitro
conditions.
IMMUNOLOGY
Chemotherapy is an inadequate means of controlling
haemonchosis because of the development of
resistance to anthelmintics. In view of the severe
pathogenicity and economic losses due to
haemonchosis, various other strategies, including
breed resistance, biological control and
immunological control through vaccine are being
studied. Immunology is an exciting area for
formulating new products for therapeutic and
diagnostic purposes. In immunological control
methods, with vaccines as targets, specific
immunodominant antigens play a significant role.
Immunoparasitology of Haemonchus infestation has
been reviewed (Kapur et al., 2001). The topics
discussed are: vaccination, genetic control, immune
responese, transfer of immunity, immunodiagnostics
and host effects.
The various aspects of H. contortus immunology,
reported from India are as follows:
Kaur et al. (2002a) used Dot-ELISA to study kinetics
of hyperimmune sera of rabbits immunized with adult
H. contortus. A high titre sera were obtained in
immunized rabbits and observed to be maintained for
180 days, in response to adult H. contortus soluble
extract which can be of significant value in the identification of immunodominant antigens and their
further characterization using natural host sera.
Earlier studies of Sood et al. (1996) have shown that
competitive-inhibition Dot-ELISA using adult H.
contortus antigen is able to detect as low as 10 pg of the
antigen and can be suitably applied at the field leval for
mass screening of infected animals. To enhance this
sensitivity Kapur-Ghai et al. (2004) have developed
avidin-biotin ELISA for detection of H. contortus
antigens.
Immunodominant antigens of adult H. contortus that
can evoke a protective immune response in the host
have been identified (Kaur et al., 2002b). Since the
Haemonchus histochemistry, biochemistry and immunology
13
antigenic component with MW 91.2 KDa was
recognized in the immune sera of all the rabbits and
also in sera collected at intervals, it is suggested as the
immunodominant component of adult H. contortus.
and smooth gave three precipitin lines with rabbit
antisera. Also, lines unite in a manner indicating
marked antigenic differences between the three types.
Since serological techniques are considered to be
sensitive tools in taxonomy, the present work has
indicated that taxonomic importance should be
attached to the vulvar configurations in female H.
contortus.
Two low molecular weight proteins of 15 and 22 KDa
were isolated from the extract of adult H. contortus by
gel filtration conA-sepharose and affinity
chromatography on antibody-sepharose. These
proteins were also indentified in the excretorysecretory products of adult parasites. Antibodies
against these proteins were identified in the sera of
animals infected with H. contortus. Upon
immunization, these proteins protected animals
against challenge H. contortus infection, as egg
shedding and worm burden significantly reduced. The
importance of these antigens in the host-parasite
relationship has been discussed (Joshi and Singh,
1999).
Preliminary studies carried out by Kocher et al.
(2002b) to determine the relationship between level of
circulating immune complex (CIC) and the possibility
of ascerting mortality caused by H. contortus
infestation in sheep and goat, have revealed that
determination of CIC level may prove to be important
parameter for early diagnosis of the state of the host
immunized with H. contortus.
To detect anthelmintic resistance using thiabendazole,
a larval development assay (LDA) has been
standardized. A linear dose-response relationship was
observed between the probit of larval development of
the logarithms of anthelmintic concentration of both
egg hatch assay and LDA (Singh et al., 2003).
Sood and Kapur (1981) observed immunological
changes in the spleen of rabbits in response to antigens
of H. contortus female. The study of sections showed
hyperplasia of reticuloendothelial cells and
duplication of red pulp. Haemosidrin pigment was
also observed. A few eosinophils and neutrophils were
also seen at the cortical region. Antibodies being
proteins, their synthesis is similar to that of other
proteins and hyperplasia of reticuloendothelial cells
indicates increased protein synthetic activity.
In support of the biochemical techniques in taxonomy
in H. contortus (Sood and Kapur, 1982b),
immunodiffusion patterns of antigens from
phenotypically different females have been studied
(Sood and Kapur, 1982c). The studies indicate that the
three phenotypes---smooth, linguiform, and knobbed
differ from each other serologically. Linguiform
antigens gave five precipitin lines, knobbed four lines
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Review
Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 16–29
J PD
Immunological perspectives and malaria vaccine
H. S. Banyal and N. Elangbam
Department of Biosciences, Himachal Pradesh University, Shimla.
ABSTRACT. Even after more than a century of efforts to either eradicate or control malaria, it still
remains a major vector borne parasitic disease that affects nearly a third of world's population,
mainly in poor developing countries. Increasing incidences of parasite resistance to available antimalarial drugs and of mosquitoes to commonly used insecticides necessitates an alternative combat
strategy for the control of malaria. The development of an effective malaria vaccine is one such
strategy. Herein, we focus on various immunological aspects of malaria including target antigens
that can serve as potential candidate vaccines, and on the status and prospects of the development of
a malaria vaccine.
Keywords: immune response, malaria, Plasmodium, target antigens, vaccine
INTRODUCTION
Malaria, derived from the Italian word for bad air was
attributed to fevers in populations living in the vicinity
of marshes. Edwin Smith Surgical Papyrus 1600 B. C.,
indicated about the disease while the Greek physician,
Hippocrates gave the first accurate clinical description
of malarial fever in 400 B. C. (Boyd, 1949). Malarial
fevers were known in ancient China and Arabian
countries, and have also been mentioned in ancient
Indian literature like 'Charaka Samhita'.
Meckel in 1847 was the first person to observe black
granules embedded in protoplasmic masses in the
blood of a severely ill malarious patient. In 1880, a
French army physician, Charles-Louis-Alphonse
Laveran observed exflagellation of a parasite and
described it as Laverania falcipara in 1884. Manson
suggested mosquitoes to be the host for malaria
parasite's extrinsic development which encouraged
Sir Ronald Ross to investigate the fate of malaria
parasite in various mosquito species in India. In 1898,
Corresponding author: Prof. H. S. Banyal, Laboratory of
Parasitology and Immunology, Department of Biosciences,
Himachal Pradesh University, Shimla-171 005, H. P., India.
E-mail: [email protected]
Ross succeeded in completely elucidating the
sporogony of Plasmodium relictum in Culex pipens
fatigans. Meanwhile, Italian worker Bignami in 1899
succeeded in infecting a healthy volunteer with
Plasmodium falciparum through the bites of
mosquito. At the same time, sporogony in P.
falciparum and P. vivax was elucidated in anopheline
mosquitoes (Bastianelli and Bignami, 1899; Grassi et
al., 1899a), and also the development of sporogonic
stages of P. malariae in Anopheles claviger (Grassi et
al., 1899b). Differential descriptions of P. vivax and P.
malariae were given by Grassi and Feletti (1892) and
for P. falciparum by Welch (1897). The fourth human
malaria parasite species, P. ovale was described in
1922 (Stephens, 1922).
Even after more than a century of efforts to either
eradicate or control malaria, it still remains a major
global health hazard and one of the most important
vector borne human diseases prevalent in more
than107 countries that affects nearly a third of the
world's population. Malaria is prevalent mainly in
poor developing countries and causes more deaths
than any other parasitic disease with sub-Saharan
Africa accounting for nearly 90% of the cases. Other
places like South-East Asia, Oceania, Middle East and
Malaria vaccine development
Latin America also face serious malaria problems. In
Africa, every 30 seconds, a child dies of malaria.
Various strategies have been adopted to control the
spread of malaria through vector elimination and
chemotherapy. Taking into account the increasing
resistance of Plasmodium to chemotherapeutic
agents, and of Anopheles to conventional insecticides,
there is a critical need for an effective malaria vaccine
to combat malaria. Great advances have been made in
understanding the immunological perspectives of
malaria which should help in the fight against the
disease.
LIFE CYCLE OF MALARIA PARASITE
Malaria is caused by a protozoan parasite,
Plasmodium, four species of which infect humans:
Plasmodium falciparum, P. vivax, P. ovale and P.
malariae. Mortality and morbidity are mainly due to P.
falciparum, although P. vivax is more widespread
geographically. Malaria parasite has a complex life
cycle alternating between a vertebrate, ranging from a
reptiles to mammals, and an arthropod host female
anopheline mosquitoe. Each bite of an infected female
Anopheles inoculates 5-20 sporozoites which, within
30 min, find their way into hepatocytes. Each
sporozoite multiplies and differentiates intracellularly
into a liver stage trophozoite and ultimately a schizont.
Rupture of hepatocytes releases merozoites (Mzs) into
circulation, which continue a cycle of red cell invasion
and multiplication causing clinical manifestations of
the disease. Within erythrocyte, each Mz develops into
a trophozoite that matures and divides, generating a
schizont that gives rise to up to 32 Mzs within 48 or 72
h depending upon the species. The Mzs upon invading
new erythrocytes either maintain the blood
schizogony or some of them differentiate into male or
female gametocytes which are ingested by the blood
feeding mosquito. In the mosquito gut, the
gametocytes emerge as gametes and fertilize to
produce motile ookinetes which burrow into the gut
wall of mosquito to form oocysts. Finally, sporozoites
released into the body cavity ultimately find their way
to the salivary glands, and are injected to a new host
during the next blood-meal.
IMMUNOLOGY OF MALARIA
Malaria infection gives rise to immune responses by
the host which are regulated both by the innate and
adaptive immune systems. Immunity to malaria
involves both cell-mediated and humoral immune
mechanisms through both T-cells and B-cells. The
17
cellular and humoral arms of immunity are tightly
bound through cytokines which control the immune
response with both antibody and cellular immunity
playing critical roles in protective immunity. The
mechanisms of non-specific innate defense are poorly
defined. Neutrophils, mononuclear phagocytes and
natural killer (NK) cells appear to play an important
role(s) in innate immunity to malaria infections.
Humoral immunity: B-cells are primarily concerned
with production of antibodies, which form humoral
immune response. In malaria endemic areas, induction
of strong humoral immune responses involving
predominantly IgM and IgG have been reported
(Chelimo et al., 2005; Couper et al., 2005). Antibodies
act against different stages of parasites, and are
predominantly effective against erythrocytic stages.
IgG 1 and IgG 3, the two cytophilic isotypes in
humans, predominate in malaria protected individuals
and IgG2a in P. yoelii has been reported to protect
rodents (Druilhe et al., 2005).
The possible mechanisms of action of antibodies
include inhibition of Mz invasion of erythrocytes and
inhibition of intraerythrocytic development of
parasites or both. Antibodies can also cause
neutralization and agglutination of Mzs. Protective
antibodies have very limited direct effect on parasite
growth and invasion but act in co-operation with blood
monocytes, known as antibody-dependent cellular
inhibition (ADCI). In ADCI, soluble mediators are
released at the time of schizont rupture triggered by the
contact between Mz surface components and
cytophilic antibodies bound to monocytes which
diffuse in the serum and block multiplication of
surrounding parasites at the uninucleate stage
(Bouharoun-Tayoun et al., 1995). Tumor necrosis
factor-α (TNF-α) has been implicated in ADCI and is
thought to inhibit the ring stages of parasite
development.
Cell-mediated immunity: The importance of cellmediated response in parasitaemia suppression in
animal models was suggested by the resolution of
acute malaria infection by several Plasmodium sp. in
B-cell deficient but not athymic mice (Grun and
Weidanz, 1981; Cavacini et al., 1990). Acquisition
and maintenance of protective immunity to malaria is
T-cell dependent as T-cells are essential both in
regulating antibody formation and in inducing
antibody independent immunity (Webster et al.,
2005). T-cells act as helpers for antibody response but
also as effector cells as they can inhibit parasite growth
18
in vitro. The cell-mediated immune effector
mechanisms include macrophage activation by
interferon-γ (IFN-γ ) derived from γ T cells, NK cells
or T helper1 (Th 1) and inhibition of parasite growth
and development inside hepatocytes mediated by
CD8+ cytotoxic and IFN-γ producing T cells (Tsuji
and Zavala, 2003). Cell-mediated immune responses
may protect against both pre-erythrocytic and
erythrocytic stage parasites.
Immunity against pre-erythrocytic stages: In preerythrocytic stage immunity, infected hepatocytes
expressing major histocompatibility complex (MHC)
molecules are the primary target of cell-mediated
immune responses. Both CD 4+ T-cells and CD8+ Tcells recognize parasite derived peptides presented by
class I and II MHC molecules, respectively, present on
the surface of infected hepatocytes. CD8+ T-cells are
thought to be the primary mediators against preerythrocytic stages (Meraldi et al., 2005). It has been
suggested that CD 8+ T-cells interact with MHCpeptide complex on the surface of infected
hepatocytes and secrete IFN-γ, which in turn induces
the infected hepatocytes to produce nitric oxide (NO)
that renders the parasite non-infectious. CD8+ T-cells
rather than CD8+ cytotoxic T lymphocytes (CTLs) per
se, are the critical effector cells of pre-erythrocytic
stage immunity.
Immunity against erythrocytic stages: Erythrocytic
stage immunity is thought to involve both antibodies
and T-cell. Antibodies are presumed to work against
parasite proteins on the surface of erythrocytes and
prevent the sequestration of parasites in
microcirculation resulting in their destruction by
spleen. Anti-Mz antibodies may also act in other
protective mechanisms like complement-mediated
lysis and act through co-operation with Fc bearing
cells. Parasite proteins present intracellularly or
expressed on the surface of Mzs are also target of
antibodies that prevent invasion of erythrocytes by
parasites (Banyal and Inselburg, 1985). In humans,
cytophilic antibodies IgG1 and IgG3 may bind to Mzs
and thus facilitate their uptake by phagocytes or
mediate antibody-dependent cellular cytotoxicity
(ADCC) or ADCI (Tebo et al., 2001). Pouvelle et al.
(1991) reported that antibodies can penetrate the
infected RBC through the parasitophorous ducts and
bind to the intracellular parasite.
Studies in animal models have implicated T-cell
mediated, antibody-independent mechanism(s) in
Banyal and Elangbam
immunity against blood-stage malaria parasites.
CD4+ T-cells are essential for immune protection
against asexual blood stages in both murine and
human malaria infections. Adoptive transfer of CD4+
T-cells from immune donor mice to nude mice
suppresses acute infection. The antibody-independent
mechanisms of action of CD4+ T-cells have been
suggested to involve cytokines and NO and T-cells
(Seixas et al., 2002). In P. chabaudi infection T-cells
are stimulated during primary infection and their
expansion is observed during recovery from acute
infection. CD4+ T-cells act through the release of
cytokines that may exert parasiticidal or parasitostatic
effect, activate macrophages and provide help for
antibody production by B-cells. Activated T-cells but
not T-cells from malaria naïve donors inhibit parasite
replication in erythrocytes in vitro, suggesting their
protective function (Perlmann and Troye-Blomberg,
2002).
In humans, T-cells act not only as helpers for an
antibody response but also act as effector cells as they
can inhibit parasite growth in vitro. As human
erythrocytes do not express MHC antigens, lysis of
infected-erythrocytes by CD8+ cytotoxic T
lymphocytes (CTLs) plays no role in protection
against blood-stage parasites. Sterile immunity
induced by repeated low grade infection of red cells
without any detectable antibodies suggests that T-cell
mediated protection operates in humans (Pombo et al.,
2002).
Role(s) of cytokines: In malaria, parasite killing
requires the production of inflammatory cytokines
like IFN-γ, interleukin (IL)-1 and IL-6 and others that
can have deleterious systemic effects and have been
correlated with malaria pathology. The cytokines
TNF-α, IL-1 and IL-6 have been connected with
severe malaria (Artavanis-Tsakonas et al., 2003). In
murine malaria infection, TNF-α and IFN-γ activate
macrophages to phagocytose parasitized-erythrocytes
and release NO that causes destruction of parasite
which results in protection from severe disease (Good
and Doolan, 1999). In humans, TNF-α, IFN-γ and NO
are associated with the resolution of fever and parasite
clearance (Kremsner et al., 1996). However, high
levels of circulating TNF-α are associated with severe
P. falciparum malaria and high levels of IFN-γ with
fever. IL-12, produced by mononuclear phagocytes is
involved in protection against pre-erythrocytic and
blood-stage infection by a Th1 anti-malaria response
(Doolan and Hoffman, 1999). In contrast, anti-
19
Malaria vaccine development
inflammatory cytokines like IL-10 and transforming
growth factor-β (TGF-β) counteract the production
and cytopathic effects of the pro-inflammatory
cytokines (Omer et al., 2000). Lower than normal
levels of circulating TGF-β are seen in patients with
symptomatic P. falciparum infection. Also risk of
febrile illness is associated with high ratios of IFN-γ,
TNF-α or IL-12 to TGF-β (Dodoo et al., 2002) which
emphasize the importance of the balance between proand anti-inflammatory cytokines. TGF-β plays an
essential role in down regulating the production of
potentially pathogenic pro-inflammatory cytokines
and may be a novel mechanism of pathogen- mediated
TGF-β activation. TNF plays a central role both in
protection and pathogenesis of malaria, whereas
elevated levels of serum IFN-γ enhance production of
TNF, NK and T-cells and have been identified as
potential sources of IFN-γ in malaria infection.
NO can modulate anti-microbial activity, smooth
muscle contraction, cytokine production and has been
correlated with malaria immunity as well as
pathogenesis (MacMicking et al., 1997). NO has also
been implied in protection from blood-stage malarial
parasites in humans as plasma NO levels increased in
patients with P. falciparum and P. vivax.
Target antigens: Problems of the resistance to
chemotherapeutic agents and insectides emphasize
the urgency of a suitable malaria vaccine. The
complex life cycle of malaria parasite with distinct
developmental stages expressing multiple antigens
could provide targets of immune responses. There are
essentially six targets for a malaria vaccine in the
whole life cycle of the parasite namely, sporozoites,
liver stages, Mzs, infected-erythrocytes, parasite
toxins and sexual stages.
Sporozoite: In 1967, Nussenzweig and co-workers
showed that mice immunized with radiation
attenuated sporozoites of P. berghei were protected
against challenge with infectious sporozoites. This
immunization confers sterile protective immunity
which is species-specific but not strain specific
(Nussenzweig et al., 1967). Immunization with heat
killed, formalin treated or lysed sporozoites could not
induce protection emphasizing the requirement for
live sporozoites in the hepatocytes for protective
immunity. Intrahepatocytic parasites are a major
target of protective immunity induced by
immunization with irradiated sporozoites and CD8+
T-cells specific for epitopes of parasite proteins
expressed in the hepatocytes are considered primary
effectors with IFN-γ playing a major role (Plebanski
and Hill, 2000).
Circumsporozoite proteins: Protective antibodies
against sporozoites are mainly directed against
circumsporozoite proteins (CSP). A 44K CSP
exhibited protection against P. berghei (Potocnjack et
al., 1980). Passive protection by polyclonal antibodies
against central repeat has also been reported for P.
berghei and P. yoelii (Egan et al., 1987; Wang et al.,
1995). P. vivax CS-derived synthetic peptides have
shown good antigenicity and immunogenicity
eliciting both humoral and cellular responses (Herrera
et al., 2004; 2005).
Liver stage antigens (LSA): Immunity during this
stage is mostly mediated by cellular-dependent
mechanisms involving CD8+ T-cells, CD4+ T-cells,
NK cells and T-cells. Studies in mice indicate that
IFN-γ produced by activated CD8+ T-cells induce
infected- hepatocytes to synthesize NO which has
potent anti-parasitic activity (Doolan and Hoffman,
2000). Some of the identified liver stage antigens
include LSA-1, LSA-2 and LSA-3. LSA-1 is
expressed specifically in liver stage parasites and no
homologue has been identified in mouse or nonhuman primate malarias. It is a 200 kDa protein with
conserved sequence across strains and involved in
naturally acquired responses in human protection.
LSA-1 specific protective immune responses includes
CD8+ T-cells, IFN-γ, IL-10 and antibodies in natural
transmission. Daubersies et al. (2000) reported
protection of chimpanzees against P. falciparum by
immunization with LSA-3, 200 kDa protein expressed
both in sporozoites and liver stages and is highly
conserved. Pf LSA-3 DNA immunization induced
potent Th 1 response with protective properties and
conferred protection against P. yoelii challenge in
mice.
Sporozoite surface protein 2 (SSP-2): SSP-2 is also
known as thrombospondin-related anonymous protein
(TRAP) involved in sporozoite invasion and is carried
into hepatocytes along with CSP ( Muller et al., 1993).
SSP-2 contains a sulphated glycoconjugated binding
peptide sequence needed for parasite invasion.
Antibodies to SSP-2 prevent sporozoites from
invading hepatocytes in vitro.
Merozoite
The Mz is the only extracellular stage of the malaria
20
parasite in the human host other than the sporozoite
making it a visible target for antibodies. Antibodies
targeting Mz proteins, mainly its surface proteins,
interfere in the invasion process through agglutination
followed by phagocytosis or blocking of Mzerythrocyte interaction. Several Mz surface proteins
have been identified and some are targets for leading
malaria candidate vaccines.
Mz surface proteins (MSP): MSP-1 is the most
abundant protein on the surface of Mzs (Polley et al.,
2003). It is a 190230 kDa protein on the surface of Mz
that is processed into smaller fragments at the time of
invasion of erythrocytes and exists as a non-covalently
linked complex of four fragments (83, 28, 34 and 42
kDa). The C-terminal 42 kDa fragment (MSP-142)
undergoes further processing to form MSP-133, which
is shed, and MSP-119, which remains on the Mz surface
and is taken into the newly invaded erythrocytes.
Monoclonal antibodies to MSP-119 inhibit Mz
invasion in vitro, and sera from P. falciparum immune
adult humans and P. chabaudi immune mice revealed a
major role for MSP-119 specific antibodies in
mediating the invasion-inhibition (O'Donnell et al.,
2001). The 19 kDa fragment is reported to be highly
conserved in P. falciparum and contains a series of
cysteine residues that are conserved among species of
plasmodia infecting humans, primates and rodents
(Daly et al., 1992).
Different effector mechanisms of antibodies against
MSP-1 are being suggested which primarily involve
inhibition of erythrocyte invasion by Mzs (Tolle et al.,
1993; Locher et al., 1996; O'Donnell et al., 2001;
Vukovic et al., 2003), blocking the processing of
larger mature MSP-1 protein on the Mz surface
(Blackman et al., 1994) and through macrophage Fc
receptors to induce ADCC (Bouharoun-Tayoun et al.,
1995; Ravetch and Clynes, 1998). MSP-119-specific
immunoglobulin IgG 3 monoclonal antibody can
passively transfer protection to mice deficient in FcãRI receptors whose macrophages cannot bind IgG 3
(Vukovic et al., 2000). Studies in P. yoelii model by
Wipasa et al. (2002) show that antibodies specific to
MSP-119 alone can induce protective immunity and
that effector T-cells specific to MSP-119 play no role in
immunity. However, such antibodies must be
produced during challenge. Specific cellular immune
responses induced by MSP-1 can be protective against
exoerythrocytic forms of P. yoelii (Kawabata et al.,
2002).
Banyal and Elangbam
MSP-2 is encoded by a single gene and is a 4552 kDa
integral membrane glycoprotein anchored on the
surface of Mz by a glycosylphosphatidyl inositol
(GPI) moeity (Weisman et al., 2001) and a target of
host protective immune responses as monoclonal
antibodies specific to MSP-2 have inhibited parasites
growth in vitro. Mice immunized with conserved
regions of P. falciparum MSP-2 have been protected
against challenge with P. chabaudi. Antibodies to
MSP-2, mainly IgG 3, have been detected in sera of
people living in endemic areas. Human trials of multi
subunit vaccines containing MSP-2 have been
undertaken both in non-exposed and malaria-exposed
individuals (Genton et al., 2000).
MSP-3 is a secreted polymorphic antigen associated
with erythrocytic schizonts and Mzs. P. falciparum
MSP-3 has been shown to range from 40-76 kDa
depending on the isolate and has been implicated in
induction of ADCI (Audran et al., 2005; Druilhe et al.,
2005). P. vivax MSP-3 is associated with but not
anchored in the Mz membrane and is structurally
related to P. falciparum MSP-3 and 140 kDa MSP of P.
knowlesi (Galinski et al., 1999).
P. falciparum MSP-4 is a 40 kDa protein containing a
single epidermal growth factor (EGF)-like domain at
the C-terminus that is synthesized at the late ring stage
and transported to the parasite surface, anchored to the
Mz membrane by a GPI moiety. Studies analyzed a
region of chromosome 2 in P. falciparum and
identified 3 clustered genes that encode GPI-anchored
Mz surface proteins in tandem MSP-2, MSP-5 and
MSP-4 and MSP-5 encoding a 40 kDa protein located
on the Mz surface (Marshall et al., 1998). A
homologue of P. falciparum MSP-4 and MSP-5 in P.
chabaudi designated Pc MSP 4/5 encoding a protein of
apparently 36 kDa contains a single EGF-like domain
near the C-terminus. Murine homologue of MSP-4
induces protective immunity in mice against lethal
challenge with P. yoelii. Anti-MSP-4 antibodies are
highly prevalent and present at high level in
individuals in malaria endemic area and are mainly
IgG 1 and IgG 3.
Proteolytic processing of MSP-1 precursor produces
two components p 36 (MSP-636) and p 22 (MSP-722)
which are associated with the shed MSP-1 complex.
The 36 kDa protein is derived from a larger precursor
MSP-6 and so designated as MPS-636. Antibodies
against recombinant protein containing the C-terminal
of MSP-6 36 bound to parasite surface or the
parasitophorous vacuole within schizonts (Trucco et
21
Malaria vaccine development
al., 2001). MSP-6 reactive antibodies are generated in
a natural human infection and antibodies on an MSP-3
peptide cross reacted with MSP-6 which suggest it to
be a target of ADCI. The 22 kDa protein designated
MSP-722 is the result of protease cleavage of precursor
MSP-7 expressed in mature schizonts (Pachebat et al.,
2001). Another asexual stage parasite protein of P.
falciparum containing two EGF-like domains near the
C-termini is designated as MSP-8 (Black et al. 2001).
Apical membrane antigen (AMA)-1: Apical
membrane antigen is a trans-membrane protein
present on the surface of Mzs and involved in the
parasite invasion of erythrocytes. AMA-1 has been
identified in all Plasmodia as a relatively conserved
sequence and synthesized de novo as a 66 kDa protein
except in P. falciparum and P. reichenowi. In P.
falciparum and P. reichenowi, AMA-1 is a 83 kDa
protein processed by proteolytic cleavage between
different domains into a 66 kDa form (Pf AMA-166)
which is further proteolytically cleaved and shed as
soluble ectodomain (Howell et al., 2001). P.
falciparum Pf 83/AMA-1 is the analogue of 66 kDa P.
knowlesi protective Mz protein, Pk 66/AMA-1 that is
expressed in late schizonts and localized in the Mz
apex. The full length 83-kDa remains apically
restricted while the processed 66-kDa becomes
circumferentially associated with the Mz surface. An
Escherichia coli expressed recombinant P. falciparum
AMA-1 induced in vitro growth inhibitory anti-AMA1 antibodies which recognize both strain specific and
conserved epitopes and show AMA-1 to be a natural
target of protective antibody responses (Hodder et al.,
2001). Recombinant AMA-1 proteins either alone or
in combination with MSP-1 have also been evaluated
for its efficacy against blood-stage malaria in animal
models (Burns et al., 2003).
Several of the potential malaria blood-stage vaccine
candidate antigens are expressed on the rhoptries,
apical organelles involved in erythrocyte invasion.
Rhopty Asociated Protein (RAP-1) is synthesized as a
86 kDa precursor N-terminally cleaved to generate 82
kDa molecule, P 82, that is further processed to give a
68 kDa molecule, P 68 (Howard et al., 1998). Purified
complexes of RAP-1 and RAP-2 used in experimental
immunization of Saimiri monkeys have shown partial
protection against P. falciparum infection, whereas
anti-RAP monoclonal antibodies have shown
inhibition of parasite replication in vitro (Ridley et al.,
1990).
Proteins involved in the targeting of particular
erythrocyte subpopulation for Mz invasion have been
intensely studied as a possible means of blocking Mz
attachment to erythrocytes. In P. falciparum,
glycophorin A is the erythrocyte receptor for Mzs
whose major ligand is the erythrocyte-binding
antigen-175 (EBA-175) located in the microneme.
Blocking an EBA-175 binding site inhibits parasite
multiplication in vitro and immunization of Aotus
monkeys with recombinant EBA-175 region II
induced anti-parasite effect (Jones et al., 2001). In P.
vivax, Duffy antigen receptor for chemokines (DARC)
is the receptor in erythrocyte for Mz which expresses
Duffy-binding protein (DBP; Yazdani et al., 2005). P.
vivax and P. knowlesi DBPs and their orthologue EBA175 bind well-defined glycoprotein motifs on
erythrocyte membrane. P. vivax reticulocyte binding
proteins (PvRBP-1 and PvRBP-2) attach to
reticulocyte enriched erythrocytes. PvRBP-1 and
PvRBP-2 have molecular masses of 325 kDa and 330
kDa, respectively, and share similar structures
(Galinski et al., 2000). PvRBP-2 appears to be
distantly related to a 235 kDa rhoptry protein of P.
yoelii. P. falciparum genes homologous to P. vivax
RBP-1 and -2 encoding high molecular mass proteins
of > 300 kDa are expressed in late schizonts. Two
PvRBP-2 orthologues of P. falciparum termed P.
falciparum RBP-2 homologues a and b (Pf RBP 2-Ha
and Pf RBP 2-Hb) along with RBP-2 of P. vivax and P.
yoelii 235 kDa protein, constitute an important
Plasmodium family important for Mz invasion.
Parasitized erythrocytes
Parasite antigens expressed on the infectederythrocytes are targets for antibodies to act on, and
several such molecules have been identified. Ringinfected erythrocyte surface antigen (RESA) is one of
the most studied with molecular weights 155 and 130
kDa, and anti-RESA antibodies raised in mice inhibit
parasite growth in vitro (Chopra et al., 2000). RESA
has also been used as a component in multiantigen
blood-stage vaccine together with MSP-1 and-2 in
Phase I clinical trials. The serine-rich protein
(SERP/SERA) is a 120 kDa soluble protein expressed
in the schizont stage, whereas glutamate rich protein
(GLURP) is expressed during all stages of the parasite
development in human host as a 220 kDa protein.
Several immuno-epidemiological studies have
identified high anti-Ro-GLURP IgG levels as
predictors of protection against high parasitaemia and
febrile malaria episodes (Soe et al., 2004). There is an
association between protection against febrile malaria
22
and presence of anti Ro-GLURP antibodies in intense
malaria transmission areas, and further that increasing
levels of IgG 1 and IgG 3 antibodies are associated
with reducing P. falciparum parasite densities
(Lusingu et al., 2005). Erythrocyte membrane
proteins (EMP) e. g. EMP 1, 2 and 3 are also located on
erythrocyte membrane; however, Pf EMP-3 is
expressed not only on erythrocyte surface but also by
liver stage parasites and sporozoites (Gruner et al.,
2001).
Parasite toxins
The toxic basis of malarial pathogenesis was first
conjectured by Camillo Golgi in 1886 (Golgi, 1886).
Malaria GPI is considered a candidate toxin as it
induces cytokine like TNF-α and adhesion expression
in macrophages and vascular endothelium which are
associated with malaria pathogenesis. Antibodies to
GPI lipid domains have been associated with
protection against malaria. GPI is a highly conserved
pro-inflammatory endotoxin of parasite origin and
synthetic anti-GPI can be used as a plausible anti-toxic
vaccine (Schofield et al. 2002).
Sexual stage antigens
Various studies have demonstrated that antibodies
directed against the sexual-stage antigens can prevent
fertilization in the mosquito thereby interrupting the
transmission of malaria. Transmission blocking
vaccines (TBVs) would reduce or interrupt malaria
transmission in human and mosquito populations
within a community as a whole but confer no
protection to an individual recipient. Such a
transmission blocking vaccine when given in
combination with a pre-erythrocytic or blood-stage
vaccine would prevent or reduce the spread of
parasites which become resistant to such vaccines and
would thus prolong the effective life of other malaria
vaccines. Antigens expressed on the surface of sexual
stages i.e. gametocyte, gamete, zygote and ookinete of
malaria parasite are being considered as promising
targets for developing a transmission blocking
vaccine. These vaccines induce antibodies in human
host that inhibit parasite development within
mosquito vector, thus, blocking parasite transmission.
Cloning of genes and subsequent recombinant
proteins have shown to induce transmission-blocking
antibodies in animal models. Two groups of antigens
have been explored to block the propagation of sexual
parasites. (i) Pfs 48/45 and Pfs 230 antigens. These
proteins belong to a family unique to Plasmodium with
Banyal and Elangbam
characteristic arrangement of cysteine containing
domains within the proteins. Monoclonal antibodies
against these proteins have been effective in blocking
the infectivity of the parasites to mosquitoes
(Templeton and Kaslow, 1999). (ii)
Ps 25 and
Ps 28 antigens. These are surface proteins expressed
on the zygotes and mature ookinetes of malaria
parasite. The ookinete surface proteins of P.
falciparum, Pfs 25 and Pfs 28, are target antigens for a
possible vaccine and their homologous proteins have
been cloned from P. vivax and other species. P.
falciparum Pfs 25 has been tested in Phase I clinical
trials in human volunteers (Kaslow and Shiloach,
1994). Yeast produced recombinant Pvs 25 and Pvs 28
are highly immunogenic and antisera recognized
corresponding molecules expressed by field-isolated
parasites in Thailand (Sattabongkot et al., 2003).
CURRENT STATUS AND PROSPECTS OF
MALARIA VACCINES
The development of a malaria vaccine remains an
urgent need for most people living in malaria endemic
regions. Two lines of evidence suggest the feasibility
of a malaria vaccine: firstly, immunity can be acquired
as a result of natural exposure to infection, and
secondly, numerous experimental malaria infections
in animal models and human volunteers can be
protected through various immunization strategies.
The distinct developmental stages of malaria parasite
provide numerous targets for vaccine development.
Because the production of live, attenuated or killedinactivated malaria vaccine is not practical, the aim
has been to develop sub-unit vaccines. In this, part or
complete antigens are identified from the pathogen's
proteomic complement, which can induce protective
immunity to the whole pathogen on vaccination. The
general target for a subunit vaccine has been to
identify critical target antigens at each stage of the lifecycle of malaria parasite. Another strategy is to
assemble peptide sequences from a range of parasite
antigens into different combinations that are then
tested for immunogenicity in animal models and
human volunteers. In the new generation of DNAbased subunit vaccines, DNA sequences from P.
falciparum parasites have been inserted into plasmid
DNA molecules (DNA vaccines) or various
recombinant attenuated DNA viruses (recombinant
viral vaccines) to generate candidate vaccines (Wang
et al., 1998). DNA vaccines are taken up by host cells,
protein expressed and T-cells are primed to form
memory T-cell populations while in recombinant viral
Malaria vaccine development
vaccines, cells are actively infected and express the
recombinant malaria proteins. Both vaccines induce
high levels of effector T-cell immune responses which
in combination with antibody responses are both
critical in protection against malaria. Three stages in
the life-cycle of malaria parasite are targeted for
vaccine development (Table I).
Pre-erythrocytic vaccines: These would prevent the
disease by targeting sporozoites or schizont infected
liver cells and by preventing the release of primary
Mzs from infected hepatocytes. Such a vaccine that
successfully interferes with pre-erythrocytic
development would terminate infections before they
had any chance of causing clinical illness and would
be beneficial to travellers and residents of malaria
endemic countries. Numerous vaccine constructs
have been developed with CS protein being the prime
target.
RTS, S/ASO 2A: This vaccine candidate is the most
advanced in clinical development and is based on the
CS protein of P. falciparum comprising of two
polypeptides RTS and S expressed in yeast. RTS is a
single polypeptide chain comprising of CS protein
fused to hepatitis B surface antigen (HBsAg), while S
is a polypeptide of HBsAg. The two polypeptides
assemble to form composite particulate structure
(RTS, S) that constitute the vaccine antigen. This
vaccine when administrated with the adjuvant
ASO2A, an oil in water emulsion consisting of the
immunostimulants monophosphoryl lipid A and
saponin derivative QS 21, has shown good
immunogenicity and efficacy in clinical trials. Phase
II safety and immunogenicity trials in malaria naïve
and malaria-immune subjects have shown it to be safe
and immunogenic and conferred 50% sterile
immunity in volunteers but immunity waned with time
lasting only up to six months in a few cases. Phase I
field evaluation in malaria experienced adults was
done in Gambia (Doherty et al., 1999). Later a
randomized, double-blind controlled Phase IIb
efficacy study was carried out in malaria-experienced
adult Gambian men that gave overall efficacy of 34%
(Bojang et al., 2001). Efficacy trial in children in
Mozambique and Phase I trials in children in The
Gambia gave encouraging results. A Phase IIb proof of
concept efficacy study was done in children in
Southern Mozambique which showed that the vaccine
was highly immunogenic and had an efficacy of 58%
against severe malaria (Alonso et al., 2004).
ICC/-1132 CS/hepatitis B core particle: This is a
23
CS-based particle vaccine that uses the highly
immunogenic hepatitis B core antigen (HBcAg) as a
delivery platform. It includes T-cell epitopes and Bcell epitopes and is engineered so that the B-cell
epitopes are exposed on the surface of virus-like
particles (VLPs), whereas the T-cell epitopes are
within the interior. Three Phase I clinical trials in
healthy, malaria-naïve adults have been undertaken.
Phase I trials in UK and Germany evaluated aluminum
hydroxide and Montanide ISA 720 formulations safe
and well tolerated (Walther et al., 2005).
Multi-epitope TRAP: This vaccine construct
consists of plasmid DNA or recombinant attenuated
live viral vector like adenovirus, fowl pox and
modified vaccinia Ankara (MVA) in a prime-boost
strategy, the most advanced of which is a multiepitope (ME) string fused to TRAP. The ME portion
contains two B-cell, 14 CD8+ and three CD4+
epitopes from six sporozoite and/or liver stage
antigens, including CS, LSA-1 and LSA-3. Several
phase I and Phase IIb sporozoite challenge studies
have been conducted in malaria-naïve adults in UK
which is reported to induce high T-cell responses
(McConkey et al., 2003). Phase I safety trials were
investigated in adults and children in The Gambia also
(Moorthy et al., 2003) and a further Phase IIb proof of
concept trial is being conducted in The Gambia.
Plasmid DNA vaccines: Intense efforts have been
made to develop effective DNA-based vaccines to the
liver and blood stages with combinations of plasmid
DNA vaccines targeting one or more P. falciparum
antigens. Several single and combination vaccines,
including CS alone and CS with SSP2/TRAP, LSA-1,
EXP-1 and LSA-3 (MuStDO-5) have undergone
Phase I and IIa clinical trials (Doolan and Hoffman,
2001). DNA vaccines require viral boosting to induce
strong T-cell immunogenicity.
Asexual blood-stage vaccines: A second strategy for
vaccine development is to target immune responses
against the asexual blood stages with the aim to
prevent or contain the disease process by suppressing
parasite replication either by preventing Mz invasion
of erythrocytes or by attacking parasite inside host
erythrocytes. A blood stage vaccine would be effective
in reducing mortality in endemic countries. The
principal target of asexual stage vaccine is the Mz with
the major target proteins like MSP-1, 2, 3 and AMA-1.
A vaccine based on the C-terminal of P. falciparum
MSP-1 consisting of a 42 kDa protein produced as a
lyophilized recombinant antigen expressed in E. coli
24
Banyal and Elangbam
Table I: Malaria vaccines in clinical trials
Stages
Vaccine/antigen
Research group
Trial phase
Pre-erythrocytic
Stage
RTS,S: Hybrid P. falciparum
CSPHBsAg particles + AS02
adjuvant
CSP C-ter peptide + Montanide
ISA 720
ICC-1132: Hybrid CSP
multiepitope-HBc VLPs
DNA vaccines (including
MuStDO-5: CSP/LSA-1/
LSA-3/EXP1/TRAP)
Live recombinant FPV- or
MVA-CSP + LSA-1 epitope
Live recombinant MVAmultiepitope string + TRAP
Live recombinant Ad-CSP
Other live recombinant vaccines
(MVA, cold-adapted influenza
virus)-CSP
LSA-3 (long peptides;
lipopeptide;recombinant)
LSA-1, SALSA, other
liver-stage antigens
GSK / WRAIR/MVI
Phase IIb
Dictagen / Lausanne Univ
Phase Ib
Apovia/MVI
Phase II
US Navy/ Vical
Phase I
Oxford Univ/ Oxxon/MVI
Phase Ib
Oxford Univ/Oxxon
Phase Ib
Crucell/GSK/WRAIR/NIAID
Oxford Univ; NYU
Phase Ia
Preclinical
Pasteur Institute/WRAIR/ GSK
Phase Ia
Hawaii Biotech; Epimmune
Preclinical
GSK/WRAIR/MVI
MVDU; NIAID
Sec Military Med Univ Shanghai/
Wanxing harmaceuticals/WHO
Phase Ib/ II
Phase Ib
Phase I
NIAID; Hawaii Biotech; AECOM;
Univ Maryland
Preclinical to
Phase I
Pasteur Institute/ AMANET/EMVI
EMVI/SSI
EMVI/SSI
Phase Ib
Phase I
Phase I
Monash
Queensland Med Res Institute/
WEHRI
Osaka Univ/Biken
Various groups
Preclinical
Phase II
Phase I
Preclinical
NIH
NIH
Phase I
Preclinical
Asexual
erythrocytic
stage
Sexual stage
MSP-1 42 kD + AS02
AMA-1
PfCP 2.9: MSP-1-AMA-1 fusion
protein (yeast) + Montanide
ISA 720
Other MSP-1 derivatives
(peptides, Salmonella or
BCG recombinants)
MSP-3 long peptides
GLURP long peptide
MSP-3-GLURP hybrid long
peptide + Montanide ISA 720
MSP-4, -5
Combination B: MSP-1, -2,
RESA + Montanide
SE36
Other blood-stage antigens
(EBA-175,EBP2, MAEBL,
RAP-2, EMP-1, DBL-á..)
PfS25 (yeast)
PvS25 and other sexual-stage
antigens
Source: www.who.int/vaccine_research/documents/en/status_table.pdf.
25
Malaria vaccine development
has shown great promise (Angov et al., 2003). This
vaccine reconstituted with ASO2A, falciparum Mz
protein 1 (FMP-1)/ASO2A was found to be safe and
immunogenic in mice, rabbits and rhesus macaques.
The safety and immunogenicity trial has been
conducted in malaria-naïve individuals. A Phase I
safety study in malaria-experienced Kenyan adults
has been completed and is scheduled to begin a Phase I
study in children in malaria-endemic region of
western Kenya.
A chimeric fusion of domain III of AMA-1 and the 19kDa portion of MSP-1 called P. falciparum chimeric
protein 2 (Pf CP-2.9) containing conserved portions of
both proteins is also being developed (Genton and
Corradin, 2002). Pf CP-2.9 expressed in Pichia
pastoris has demonstrated good immunogenicity to
both portions of antigen in mice, rabbits and nonhuman primates. A preclinical trial showed its
effectiveness, and a Phase I trial in malaria-naïve
healthy adults with Montanide ISA720 formulation is
under way that began in February 2006.
The idea of a multi-component vaccine against bloodstage parasites was approached by Patarroyo and
colleagues with a synthetic peptide vaccine SPf66,
containing sequence from MSP-1 and two other
blood-stage antigens combined with CS protein
(Patarroyo et al., 1987). The vaccine underwent four
Phase III clinical trials in different locations. Studies
in Colombian adults and children (Valero et al., 1993)
and in Tanzanian children (Alonso et al., 1994) gave
encouraging results but no significant protection was
observed in studies in Gambian infants (D'Alessandro
et al., 1995) and in Thailand (Nosten et al., 1996).
Sexual-stage vaccines: The third vaccine strategy is
the development of a sexual-stage vaccine. Antigens
of gametocyte 48/45 kDa and 230 KDa and the 25 kDa
and 28 kDa post-fertilization antigens are leading
candidates for transmission blocking vaccine (TBV).
Pvs 25 adjuvanted with aluminum hydroxide is
currently in Phase I clinical trial. Pvs 25 adjuvanted
with cholera toxin (CT) when used to immunize intranasally induced significant Th-2 type immune
response in mice and the antisera completely blocked
parasite transmission (Arakawa et al., 2003).
CONCLUSIONS
Immunity to malaria is complex and not clearly
defined with the parasite exhibiting various
immunoevasive mechanisms like antigenic variation,
polymorphism and polyclonal B-cell activation.
Protection in endemic areas takes years to develop and
there is no clearly defined immune response that can
effectively protect against the disease. There is also
the need to develop novel adjuvants that can enhance
the cell-mediated immunity, and also for the
development of novel vectors and vaccine
formulations that will produce optimal protective
immune responses. Some of the recent vaccine trials
like RTS, S which showed 58% efficacy against severe
disease have given encouraging results. Malaria
vaccine development is at an exciting stage with an
optimistic outlook. Recent advances in understanding
the immune mechanisms and the volume of
information generated from malaria parasite genome
would help in better understanding and identification
of critical antigens for vaccine development. Perhaps,
given the complexity of the parasite, vaccine based on
all or many antigens would be more effective than the
one based on a single antigen.
ACKNOWLEDGMENTS
Ms. N. Elangbam is grateful to University Grants
Commission, New Delhi, for financial assistance in
the form of a Junior Research Fellowship (National
Eligibility Test).
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Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 30–36
J PD
Isolation and characterization of the paraflagellar rod
proteins of Leishmania donovani
A. Lahiri and A. Bhattacharya
Immunoparasitology Research Unit, Department of Zoology, University of Calcutta, Kolkata.
ABSTRACT. The paraflagellar rod (PFR) is a unique cytoskeletal structure present in the
kinetoplastid protozoans but absent from their mammalian hosts. It is a massive network of woven
cytoskeletal filaments attached to one face of the common 9+2 axoneme of the flagellum. The PFR is
necessary for proper parasite motility, viability and successful infection. Biochemical studies
carried out on the PFR have revealed that it is composed of both major and minor protein
components. In this paper, purification of the PFR proteins of Leishmania donovani has been
described, which involves a combination of flagella isolation, non-ionic detergent treatment and
restricted proteolysis. Scanning electron microscopy was done to monitor the process of isolation of
flagella from intact cell bodies. The major PFR proteins have been identified as two distinct bands
of mol wt 76 kDa and 68 kDa, by using sodium dodecyl sulphate polyacrylamide gel electrophoresis
analysis. The ultrastructure of the flagellum was studied by using a transmission electron
microscope.
Keywords: axoneme, flagellum, kinetoplastid, paraflagellar rod
INTRODUCTION
Leishmania donovani, an intracellular protozoan
parasite, causes Kala-azar (visceral leishmaniasis) in
humans. The parasite is transmitted by various species
of female sandflies (Phlebotomus argentipes). It
exists in two morphological forms: the promastigote,
residing in the gut of female sandflies, and the
amastigote, living in the reticuloendothelial system of
the mammalian hosts. The promastigotes possess a
full-length, free-flagellum whereas it is rudimentary
in amastigotes.
The flagellum of promastigotes helps in motility and is
involved in hemidesmosomal attachment to the
chitinous itima, and maintenance of the parasite
within the sandfly gut (Killick-Kendrick, 1979;
Killick-Kendrick et al., 1974; Walters et al., 1987).
Corresponding author: Dr. A. Bhattacharya, Immuno-parasitology
Research Unit, Department of Zoology, University of Calcutta, 35
Ballygunge Circular Road, Kolkata-700 019, W.B., India.
The flagellar modes of attachment have been observed
with other kinetoplastids also and appear to be
essential for their survival within the insect vector
(Killick-Kendrick, 1979; Rowton et al., 1981;
Vickerman, 1973; Vickerman and Preston, 1976). The
flagellum of Leishmania is also involved in
chemotactic responses (Bray, 1983). Structurally, the
flagellum of Leishmania has a typical 9+2
microtubular axoneme and also possesses a
filamentous, lattice-like structure called the
paraflagellar rod (PFR) or paraxial rod. The
microtubules of the axoneme are arranged in a precise
pattern of nine outer double microtubules and two
separate central ones.
The PFR is a unique cytoskeletal structure found in
kinetoplastids, euglenoids and some dinoflagellates
(Bastin et al., 1996; Hyams, 1982; Cachon et al.,
1988). The paraxial rod of kinetoplastida is similar to
that of euglenoids, although apparently, they appear to
be morphologically distinct from each other (Farina et
31
The PFR proteins of Leishmania donovani
al., 1980; Fuge, 1969; Gallo and Schrevel, 1985). The
PFR is a massive network of woven cytoskeletal
filaments, running alongside the typical eukaryotic
9+2 axoneme of the flagellum (Russell et al., 1983).
Recent molecular studies have demonstrated that the
PFR is necessary for proper promastigote motility and
viability (Santrich et al., 1997; Bastin et al., 1998).
Ablation of a specific molecule of the PFR resulted in
mutant cells that were paralyzed, indicating the
essential role of PFR in cell motility (Deflorin et al.,
1994). Also, ATPase activity was detected in the PFR
of euglenoids (Moreira-Leite et al., 1999). The
ultrastructure of kinetoplastid PFR appears largely
similar in all species of the group (De Souza and
Souto-Padron, 1980; Beard et al., 1992). The structure
has been divided in three distinct zones in relation to
the axoneme namely, proximal, intermediate and
distal (Freymüller and Camargo, 1981). The proximal
and distal regions each contain filaments of 7-10 nm
that intersect at an angle of 100°. The intermediate
region contains thin (5 nm) filaments that intersect at
an angle of 45° and connect the proximal and distal
regions (Maga and LeBowitz, 1999). Attachment
filaments extending from axoneme microtubule
doublets 4-7 connect the proximal region to the
axoneme (Ismach et al., 1989). In Leishmania, the
promastigotes possess a full-length flagellum with a
PFR, whereas amastigotes contain only an attenuated,
non-emergent flagellum completely lacking a PFR.
The biochemical composition of the PFR is quite
complex and includes both major and minor PFR
proteins. Among them, the major PFR proteins have
been extensively studied in the parasitic
haemoflagellates Trypanosoma cruzi, T. brucei, L.
mexicana and L. amazonensis. In these organisms, the
major PFR proteins migrate in two bands on sodium
dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) with mol wt of about 68-75 kDa, and
appear to be present in approximately equimolar
amounts (Deflorin et al., 1994; Schlaeppi et al., 1989).
The major PFR components are conserved in the
trypanosomatidae family and form a doublet of
homologous proteins in most species of the family
(Araujo and Morein, 1991; Saborio et al., 1989). By
SDS-PAGE analysis, PFR1 of L. amazonensis
migrates at 74 kDa and PFR2 at 69 kDa (Bastin et al.,
1996). The PFR1 and PFR2 genes from T. cruzi, T.
brucei and L. mexicana are highly conserved across
species (over 80% amino acid homology; Maga and
Lebowitz, 1999). More complex patterns have been
described in T. cruzi in which four major PFR proteins
have been identified (Fouts et al., 1998). No
significant homology has been reported between
major PFR proteins and other known proteins
(Imbodem et al., 1995).
The ultrastructural study of the flagellum including
the PFR and the purification of the PFR proteins
provides an important guideline to the researchers for
understanding the complete biology of this structure.
The parasite L. donovani represents a standard model
for studying unique structures such as the PFR
because it is one of the most common human
pathogens in tropical countries such as India.
MATERIALS AND METHODS
Parasite culture: The promastigotes were cultured at
22-25° C in liquid M-199 medium supplemented with
10% heat-inactivated fetal bovine serum (FBS, Sigma
Aldrich, St. Louis, MO, USA; Morgan et al., 1950).
The culture medium was filtered under sterile
conditions in a culture room with laminar flow. The
medium was kept for 48 h at room temperature to
check for any contamination, and then stored at -20° C.
Aliquots of the above culture of L. donovani were used
in all the experiments.
Isolation of flagella: The culture medium containing
promastigotes of L. donovani was centrifuged at 2100
x g for 20 min (Cunha et al., 1984). The pellet
containing the cells was washed twice in phosphate
buffered saline (PBS; 10 mM, pH 7.2; Sigma) and then
in buffer A (25 mM TRIS-HCl , 0.2 mM EDTA, 5 mM
MgCl2, 12 mM β-mercaptoethanol, 0.32 M sucrose
(all from Sigma; pH 7.4) and resuspended in buffer A
supplemented with 1% bovine serum albumin
(Sigma), 0.1 mM CaCl2 (Sigma), 0.5 mM phenyl
methyl sulfonyl fluoride (Sigma) and 5 µg/ml
leupeptin (Sigma; Moreira-Leite et al., 1999). The
cells were subjected to three different centrifugation
speeds: 2600 x g, 3200 x g and 3700 x g, and the
flagella were isolated from the cell bodies by
centrifugation at an optimum speed of 3200 x g for 30
min. The supernatant containing the flagella was
separated from the pelleted and deflagellated cell
bodies. This supernatant was then concentrated by
centrifugation at 6780 x g for 20 min. The above
procedure was carried out at 0-4° C. The pellets
containing the flagella were finally suspended in
buffer A.
Scanning electron microscopy: A scanning electron
microscope (SEM) uses a fine beam of electrons to
scan back and forth across the metal-coated surface.
The principal application of SEM is in the study of
32
surfaces such as those of cells. The aliquots of the
treated cells were fixed with 2.5% glutaraldehyde
(Sigma) in 0.1M phosphate buffer (pH 7.2-7.4; Sigma)
for 24 h and washed in the same buffer for 15 min x 4.
The samples were rinsed thrice with double distilled
water, each time for 5 min. Then they were dehydrated
in different grades of alcohol: 50, 70, 80, 90, 95 and
100% ethanol (20 min each). Gold coating of 200Å
was done at 5 mA using Giko Engineering-IB2 ion
coater. The cells were dried using vacuum pump and
observed under a SEM (model HITACHI S-530), and
the photographs were taken by MAMIYA 6 x 7 camera
using NOVA 120 ASA films.
PFR protein purification: To purify the protein
component of PFR, it was necessary to remove the
flagellar membrane. The flagellar membranes were
removed by non-ionic detergent treatment. Flagellar
fractions of L. donovani promastigotes, obtained as
described above, were subjected to three rounds of
treatment with 2% Nonidet P-40 (Sigma) in PBS
(Sigma) at 0-4° C under constant shaking. Each 15 min
round of detergent treatment was followed by
centrifugation at 17,300 x g for 20 min at 4° C
(Moreira-Leite et al., 1999). The pellet of the final
centrifugation step was dissolved in PBS (Sigma) and
subjected to a brief treatment with 0.0015% trypsin
(type XIII, TPCK-treated, Sigma) for 90 s at 28° C.
The trypsin treatment was stopped by adding 20-fold
excess soyabean trypsin inhibitor (Sigma). The
resultant protein fractions were subjected to SDSPAGE.
SDS-PAGE: This method is based on the separation of
proteins according to mol wt and is particularly useful
for monitoring protein purification. The sample to be
run on SDS-PAGE was mixed with loading dye
(protein:loading dye, 1:1) and boiled for 5 min in a
water bath. The stock loading dye had the following
composition: double distilled water, 4.8 ml; TRIS (pH
6.8), 1.2 ml; 10% SDS (Sigma), 2 ml; glycerol
(Sigma), 1 ml and bromophenol blue (Sigma), 0.5 ml.
Before use, 950 µl of stock solution was mixed with 50
µl of β-mercaptoethanol. The sample was run
simultaneously with protein markers (Broad Range,
Bangalore GENEI, Bangalore, India) in different
wells at a constant voltage of 100 V in 10% resolving
gel and 4% stacking gel. The gel was then fixed in
methanol and stained with Coomassie Brilliant Blue
R-250 (Sigma) for a few h, and then washed in
destaining solution until clear bands were visible
(Laemmli, 1970).
Lahiri and Bhattacharya
Ultrastructural study of the flagellum by
transmission electron microscopy: Cells were fixed
in a suitably buffered aldehyde fixative (2.5%
glutaraldehyde grade I; Sigma) in 0.1 M sodium
cacodylate buffer (pH 7.4; Sigma) at 4° C for 1-4 h.
Then the cells were washed for 2 h or overnight at 4° C
in three changes of 0.1M sodium cacodylate buffer
(pH 7.4). Cells were post-fixed in 1% OsO4 (Sigma) in
0.8% potassium ferricyanide (Sigma) for 1-2 h at room
temperature and protected from light (Nakano et al.,
2001). The above cells were washed for 5 min x 2 with
distilled water. Dehydration was done using the
following grades of alcohol: 50, 70, 90 and 95%
ethanol, each for 15 min, and 100% ethanol for 15 min
x 4. Finally, the treated cells were embedded in Epon
polybed 820 epoxy resin. Ultrathin sections were cut
and stained with 5% aqueous uranyl acetate (Sigma)
and lead citrate (Sigma) and observed under a
transmission electron microscope.
RESULTS
When observed under a SEM, it was revealed that the
cells centrifuged at 2600 x g retained their flagella
(Fig. 1). The flagella were detached from the intact cell
bodies when centrifugation was carried out at 3200 x g
(Fig. 2). However, the cells were ruptured when
centrifuged at 3700 x g (Fig. 3).
SDS-PAGE analysis showed two protein bands of mol
wt 76 kDa and 68 kDa that are presumed to be two
fractions of the PFR proteins (Fig. 4).
Transmission electron microscopy study revealed the
ultrastructural details of PFR. The longitudinal
section of a flagellum shows that the lattice-like PFR
runs parallel to the axoneme even before the
emergence of the flagellum from the flagellar pocket
0002
15KV
5um
Fig. 1: SEM of a L. donovani cell centrifuged at 2600 x g.
33
The PFR proteins of Leishmania donovani
0016
15KV
5um
Fig. 2: SEM of a L. donovani cell centrifuged at 3200 x g.
0021
15KV
5um
Flagella
Marker
Fig. 3: SEM of a L. donovani cell centrifuged at 3700 x g.
kD
F
PFR
A
BB
K
205
97
N
68
29
14
Fig. 4: SDS-PAGE of PFR proteins of L. donovani. The marker
proteins are shown on the left side.
Fig. 5: Transmission electron microscope photograph of a L.
donovani promastigote. [F, flagellum; PFR, paraflagellar rod; A,
axoneme; BB, basal body; K, kinetoplast; N, nucleus]
(Fig. 5). It also shows the attachment of the flagellum
to the basal body at the anterior end of the parasite. The
axoneme of the flagellum showed typical eukaryotic
34
9+2 microtubular arrangement. The basal body as well
as the base of the flagellum remains ensheathed by a
membrane, and the central rod of the flagellum
emerges freely. Centrifugation at 2600 x g detached
the flagellum partially from the basal body. Complete
detachment was obtained at 3200 x g but the flagellum
was still surrounded by a membrane. Non-ionic
detergent treatment removed the flagellar
membranes.
DISCUSSION
Among protozoans, the kinetoplastid trypanosomes
have always held an important position in the
scientific research scenario, not only for their medical
importance but also for possessing diverse cellular
processes. One such unique cytoskeletal structure is
PFR. The canonical 9+2 axoneme of the flagellum
initiates beating of the latter in most eukaryotes, but
the elaborate PFR structure has been observed in only
kinetoplastids, euglenoids and some dinoflagellates.
Till date, the PFR and its proteins, namely PFR1 and
PFR2 have been studied in T. cruzi, T. brucei, L.
mexicana and L. amazonensis. These studies revealed
that PFR is essential for proper parasite motility and
viability, and both these functions are directly
attributable to the PFR proteins – PFR1 and PFR2
(Santrich et al., 1997; Bastin et al., 1998). Using new
molecular-genetic techniques, PFR1, PFR2 and
PFR1/PFR2 null mutants of T. brucei and L. mexicana
have been generated. The mutant cells lacked a native
PFR structure, showing that despite sharing over 60%
amino acid homology and similar physical properties,
PFR1 and PFR2 are essential and significantly
functional components of PFR. Mutants of L.
mexicana having a PFR2-phenotype were unable to
colonize in the vector's gut whereas PFR1 and/or
PFR2 mutants were viable in axenic culture. This
indicates that the PFR plays a key role in the viability
of Leishmania in a natural habitat (Hunger-Glaser and
Seebeck, 1997). Leishmania parasites lacking PFR
were found to display severe disturbances in flagellar
waveforms e.g. reduced wavelength and amplitude
and decrease in frequency as compared to the beating
patterns of normal parasites (Santrich et al., 1997). So,
it is of prime importance to isolate and study the
ultrastructure of the flagellum and PFR of L.
donovani, and to determine its role in the life cycle of
the parasite with special reference to PFR proteins.
The present studies were carried out with an aim to
fulfill the above objectives.
Lahiri and Bhattacharya
To isolate and purify the PFR proteins, the first
essential step was to successfully separate the flagella
from intact cell bodies. The process of flagella
isolation was monitored by using SEM to make sure
that most of the cells remained intact and only lost
their flagella. Because the promastigotes were
obtained by centrifuging the culture medium at 2000 x
g, the cells were subjected to three different higher
centrifugation speeds starting from 2600 x g and
increasing gradually to 3700 x g to isolate the flagella.
When observed under a SEM, it was observed that
most of the cells centrifuged at 3700 x g were ruptured
(Fig. 3), while those centrifuged at 2600 x g retained
their flagella (Fig. 1). Thus, the optimum
centrifugation speed for successful isolation of
flagella was chosen to be 3200 x g (Fig. 2).
The flagellar membranes were removed by treatment
with the non-ionic detergent Nonidet P-40. It latter
appears to have no effect on the integrity of the
flagellar cytoskeleton and is, therefore, unable to
break the attachment between the PFR and axoneme
(Fig. 5). Beacuse it has been determined that the links
between PFR and axoneme are highly sensitive to
trypsin (Moreira-Leite et al., 1999), the detachment of
PFR from axoneme was accomplished by proteolytic
treatment.
SDS-PAGE analysis showed two protein bands of mol
wt 76 kDa and 68 kDa that are presumed to be two
fractions of the PFR proteins (Fig. 4). Extensive
studies carried out till date on the major PFR proteins
in the parasitic haemoflagellates T. cruzi, T. brucei, L.
mexicana and L. amazonensis have shown that the
PFR proteins migrate in SDS-PAGE as two bands with
mol wt of 70-75 kDa and 68-72 kDa, respectively, and
appear to be present in approximately equimolar
amounts. So, this result is in conformity with the
results reported in previous studies.
Transmission electron microscopy study revealed the
ultrastructural details of PFR. The longitudinal
section of a flagellum shows that the lattice-like PFR
runs parallel to the axoneme even before the
emergence of the flagellum from the flagellar pocket
(Fig. 5). It also shows the attachment of the flagellum
to the basal body at the anterior end of the parasite. The
axoneme of the flagellum showed typical eukaryotic
9+2 microtubular arrangement (Fig. 5). The basal
body as well as the base of the flagellum remains
ensheathed by a membrane and the central rod of the
flagellum emerges freely. Centrifugation at 2600 x g
detached the flagellum partially from the basal body.
The PFR proteins of Leishmania donovani
Complete detachment was obtained at 3200 x g but the
flagellum was still surrounded by a membrane. Nonionic detergent treatment removed the flagellar
membranes.
The alternation of flagellate and nonflagellate forms
of the parasite is attributable to their needs for
infection and survival within the host cells. The
flagellum is essential for attachment to macrophages,
and subsequent penetration and stabilization within
macrophages. Apart from attachment, penetration and
stabilization within host cells, the main function of
flagella is to confer motility and viability to cells so
that they can infect host cells. Once the infection
process is completed, the presence of flagella is no
longer required. So, the flagella become rudimentary
and non-functional in the intracellular forms.
The present study shows the successful isolation of
flagella through scanning electron microscopy. It
reports that PFR is composed of two major proteins
(mol wt 76 kDa and 68 kDa) in L. donovani, which are
unique to that structure and bear no significant
homology to other known proteins. Transmission
electron microscopy study of the flagellum and PFR
provides a relatively complete picture of the biology
of these structures. It shows the position of the
axoneme and PFR within the flagellum, how they are
connected to each other and their structural
peculiarities. This study is an important guideline for
those who are carrying out researches on the flagellum
and PFR of L. donovani.
ACKNOWLEDGEMENTS
The financial assistance from the Council of Scientific
and Industrial Research, New Delhi, [scheme No. 60
(0042)/01/EMR-II] is gratefully acknowledged.
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Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 37–40
J PD
Onchocercosis in Benue state, Nigeria: comparative
epidemiological studies amongst the Etulo and Idoma
ethnic groups
E. A. Omudu and B. O. Atu
Department of Biological Sciences, Benue State University, Makurdi.
ABSTRACT. The prevalence of onchocercosis caused by Onchocerca volvulus amongst two distinct
ethnic groups in Benue State was investigated using Rapid Assessment Methods (RAMs) and skin
snip technique. Results showed that of the 2126 individuals examined in the Idoma-speaking
Okpokwu Local Government Area, 16.6% were positive either for Leopard skin (LS) or palpable
nodules. While of the 1005 individuals examined in the Etulo area, 5.6% had either LS or palpable
nodules. The prevalence rate varied significantly (p < 0.05). Correlation analysis showed significant
association between LS, palpable nodules and aging (r=0.43, p < 0.001). Skin snip results showed
communities with higher LS and nodules had higher community microfilarial load. Correlation
coefficient r=0.64 was obtained when skin snip prevalence was compared with RAMs in the Etulo
area. The implications of these results were discussed especially in terms of prioritizing the
implementation of community-directed treatment with ivermectin.
Keywords: microfilariae, Nigeria, onchocercosis, Rapid Assessment Methods
INTRODUCTION
Onchocercosis (river blindness) is a parasitic disease
caused by the nematode Onchocerca volvulus and
transmitted by the bites of blackflies belonging to
Simulium damnosum complex. It is estimated that over
81 million people are at risk of infection, some 18
million already infected and one million people
visually impaired of whom about 340,000 are blind
(WHO 1995). Apart from ophthalmological
degeneration leading to blindness, the disease also
presents bizarre dermatological, lymphatic and
systemic manifestations (Edungbola et al., 1990).
Onchocercosis has been reported in some parts of
Benue State, (Gemade and Dipeolu, 1983; Amuta and
Olusi, 2000), where it is affecting socio-economy of
Corresponding author: Dr. Edward Agbo Omudu, Department
of Biological Sciences, Benue State University, P. M. B.
102119, Makurdi, Nigeria. E-mail: eddieomudu@yahoo. com
human population. The development and use of Rapid
Assessment Methods (RAMs) using prevalence of
Leopard skin (LS) and palpable nodules for diagnosis
of onchocercosis and free distribution of ivermectin
for treatment has heightened expectation that the
disease can be contained. For this to happen, there is a
need to know the extent of the disease especially in
communities without baseline information. This
study, using RAM, was undertaken to provide pretreatment prevalence and identify communities
eligible for mass distribution of ivermectin.
MATERIALS AND METHODS
Study areas:
The study areas comprised of
nineteen Idoma speaking villages in Okpokwu Local
Government Area (LGA) and twelve Etulo speaking
villages in Buruku and Katsina-Ala LGA. There are
distinct cultural and occupational differences that may
influence exposure to biting blackflies and perception
of disease manifestation. The vegetation and
38
topographical features of the study areas have been
described by Nwoke et al. (1998) as being favourable
and suitable for onchocercosis transmission.
Okpokwu: The local population of this area (8°00 and
8° 30 E and 6° 58 and 7° 25 N) practice peasant
agriculture, fishing and hunting are secondary
occupations. With numerous breeding sites on the
Okpokwu river system that drain these villages and
outdoor activities performed by the population, they
are continually exposed to bites of the vectors.
Buruku and Katsina-Ala: The Etulo community
inhabits twelve villages which are bisected by river
Katsina-Ala, a major tributary of Benue River.
Majority of the villages are located along the bank of
the river with fishing and farming constituting the
major occupation.
Apart from river Katsina-Ala, another perennial river
known as Ogaturu drains most of Etulo land. The two
predominant rivers are fast flowing and harbour larvae
and pupae of S. damnosum complex (Nwoke et al.,
1998; Gemade and Dipeolu, 1983).
RAMs for diagnosis: Based on the analysis of large
volumes of epidemiological data on onchocercosis,
depigmentation of skin LS and palpable onchocercal
nodules were recommended as rapid assessment
procedures to determine communities eligible for
treatment with ivermectin. The merits and limitations
of these methodologies are summarized by Edungbola
et al. (1993).
Community: Only adults 18 years and above were
examined for presence of nodules and LS. All persons
sampled were engaged in one or more rural occupation
and have resided in the community for at least five
years (Gemade, 1993). Communities were visited a
day prior to the fixed date for examination in order to
mobilize people for the survey and free treatment.
Search for nodules: For search of nodules, the
patients were requested to strip down to the loin. As the
person stretched his arms above the head, the whole
chest region was examined both visually and by
palpation. Special attention was given to iliac crest
around knees, ankles, ribs (from and rear), chest wall,
shoulders, elbows, wrist and head.
Search for LS: The search for LS on patients was
more specific in location and faster as typical
onchocercal depigmentation commonly occurs on
lower limb of adults above 30 years. Patients were
asked to pull up trousers and/or wrappers to the thigh
region.
Skin snip examination: Skin snip parasitological
procedure for identification of microfilariae of O.
Edward and Bernard
volvulus was carried out in whole population of the
Etulo area; however, this was not possible in Okpokwu
LGA as a result of immunological and parasitological
studies just concluded in the same area by Amuta and
Olusi (2000). Two snips, one each from the right and
left iliac crest were taken from each individual with the
aid of a 2 mm bite corneoscleral punch (E-2802, Holt
Storz, Germany). Each snip was incubated in a well of
microtitre plate containing normal saline. The plates
were then examined within 30 min to one hour by
microscopy for the presence of O. volvulus
microfilariae (Wentworth, 1988).
RESULTS AND DISCUSSION
Out of 31 villages examined, all were found to have
individuals with LS, whereas 27 (87%) villages were
carrying nodules of onchocercal origin. Out of 2126
individuals examined from the Idoma speaking
villages in Okpokwu LGA, 248 (11.6%) had LS, 105
(4.9%) had nodules and 35 (1.6 %) had both nodules
and LS while 353 (16.6%) had either LS or nodules
(Table 1). In 1005 Etulo-speaking individuals, 45
(4.5%) had LS, 12 (1.2%) had nodules and 2 (0.2 %)
had both LS and nodules while 57 (5.6%) had either LS
or nodules (Table 2). The number of cases with LS,
visible or palpable nodules observed in the Okpokwu
area varied significantly (p < 0.05) from that of Etulo
area.
Whereas LS in Okpokwu area ranged from
7.3–28.8%, in Etulo area it ranged from 0.8–14.0%.
Only one village (Ashitenaku) had 14% LS, whereas
all the other Etulo villages had LS below 7.3%. In
Okpokwu, the prevalence of onchocercal nodules
ranged from 2.2%–10.9% and in Etulo area it ranged
from 0.8–3.1% only one village (Ugye) in Etulo had
nodule prevalence of 3.1%, whereas all other villages
had the prevalence below 2.3%. LS and onchocercal
nodules were mostly noticed in individuals within
40–69 years of age, highest (48.7%) being in between
50–59 years. This same age group accounted for
20.5% of onchocercal nodules. Correlation analysis to
determine association between these onchocercal
manifestations with age showed significant
correlation (r = 0.45, p < 0.001). The patients
complained varying degrees of visual impairment but,
only five cases of total blindness were recorded. One
classical blind case was seen in a 61 years old man in
Effa (Okpokwu), who had Leopard skin on both shins
and a nodule on the knee. The Etulo area on the other
hand had four cases of blindness (two in Ashitenaku,
one each in Oglazi and Agbou respectively).
Skin snip examination in the Etulo area showed 536
(53.3%) persons were positive for O. volvulus
microfilariae. The prevalence by villages varied with
39
Epidemiology of onchocercosis in Nigeria
Table I: Determination of the prevalence of onchocercosis in Okpokwu villages by using LS and palpable nodules as
diagnostic methods
Villages
No. of persons
examined
No. positive
LS (%)
No. positive for
nodules (%)
No. positive for both
nodules and LS (%)
Ojoga
Ai-Ebiega
Ipole
Iwewe
Obotu
Opialu
Ollo
Ai-Ohida
Ai-Okpe
Ogene
Ijege
Ogege
Effa
Idobe
Oto-Oklenyi
Odokpo
Ai-Akpa
Aidogodo
Ede
82
51
49
45
85
132
72
72
80
102
90
250
92
60
52
68
58
418
268
23(28.8)
11(21.5)
10(20.4)
3(6.6)
12(14.1)
13(9.8)
8(11.1)
6(8.3)
10(12.5)
15(14.7)
9(10.0)
22(14.6)
21(14.6)
6(10.0)
6(11.5)
5(7.3)
10(17.2)
36(8.6)
22(8.2)
9(10.9)
3(5.8)
2(4.0)
2(4.4)
8(9.4)
10(7.5)
5(6.9)
2(2.7)
3(3.7)
3(2.9)
2(2.2)
8(3.2)
10(10.8)
6(10.0)
3(5.7)
2(2.9)
2(3.8)
15(3.5)
10(3.7)
5 ( 6.1 )
2 ( 3.9 )
3 ( 3.5 )
4 ( 3.0 )
1 (1.3 )
1 (1.3 )
1 (0.9 )
1 (0.4 )
5 (5.4 )
2 (3.3 )
1 (1.4 )
3 (5.1 )
4 (0.9 )
2 (0.7 )
Total
2126
248 (11.6)
105 (4.9)
35 (1.6)
Table II: Determination of the prevalence of onchocercosis in Etulo villages by using LS, palpable nodules and
skin snip as diagnostic methods
Villages
No. of persons
examined
No. of positive
LS (%)
No. of positive
nodules (%)
No. positive for
both LS and
nodules (%)
No. of positive
skin snip (%)
Agbou
Agbatala
Agia
Angwauje
Ashitenaku
Oglazi
Otsaazi
Otsafu
Ugye
Ogurube
Otanga
Okpashila
64
55
87
92
100
68
99
47
95
94
84
120
2(3.1)
2(3.6)
1(1.1)
2(2.1)
14(14.0)
5(7.3)
9(9.0)
3(6.3)
2(2.1)
2(2.1)
2(2.3)
1(0.8)
1(1.8)
2(2.3)
1(1.0)
1(1.4)
2(2.0)
1(2.1)
3(3.1)
1(0.8)
1(1.0)
1(1.05)
-
40(62.5)
44(80.0)
39(44.8)
44(47.8)
74(74.0)
46(67.6)
59(59.5)
31(65.9)
31(34.0)
38(40.4)
29(34.5)
61(50.8)
Total
1005
45 (4.5)
12 (1.2)
2 (0.2)
536 (53.3)
40
four villages being meso-endemic (60–89%) while the
other eight were hypo-endemic (< 60%; Table 2).
Communities with higher prevalence of LS and
onchocercal nodule also have proportionately higher
prevalence of skin microfilariae. Correlation
coefficient (r) = 0.64 was obtained when skin snip
prevalence was compared with RAMs; there was,
however, no correlation between microfilarial
prevalence and visual impairment (r) = 0.017 in the
Etulo area.
The prevalence of onchocercosis varied significantly
(p < 0.05) in the different villages. Others have also
reported varying endemicity within same
biogeographical zones (Nwoke et al., 1994; Nocks et
al., 1998). It may be due to differences in duration and
degree of exposure of members from different
communities to bites of infected vectors (Nwoke et al.,
1994) and immunological factors (Murdock, 1992).
Another reason might be topographical differences,
whereas Okpokwu area is characterized by hilly
terrain with many fast flowing streams, the Etulo area
is predominantly flat plain with few fast flowing
streams.
A total of 479 (89.4%) individuals who had
microfilariae in their skin biopsy showed no clinical
sign of nodules or LS. Many other studies in Nigeria
where skin snip, LS and palpable nodules were
combined to determine onchocercosis prevalence
revealed similar findings where LS and visible
nodules varied significantly (p < 0.05) with the
community microfilarial rate (Nwoke et al., 1994;
Nock et al., 1998). Amuta and Olusi (2000) also
reported high prevalence rate using skin snip in some
villages where we recorded lower prevalence using LS
and onchocercal nodules.
The very low incidence of blindness recorded in both
study sites agreed with Nwoke et al. (1994) and has a
common observation because rainforest region of
Nigeria is endemic for O. volvulus strains that rarely
cause blindness unlike to savanna types which are
more invasive and more pathogenic to the eyes
(Nwoke et al., 1994).
The use of LS and palpable nodules as diagnostic
procedures for onchocercosis are highly desirable as
they are cheap, fast, convenient and highly efficient
(Carme et al., 1993; Edungbola et al., 1993; Nwoke et
al., 1998; Nock et al., 1998). A combination of these
two methods as done in this study is of exceptional
advantage since it identifies community's endemicity.
There is a need to commence treatment with
ivermectin in some of the villages investigated,
especially those with LS prevalence of more than
Edward and Bernard
10%. It is also important to train communities on how
to conduct rapid assessment procedures in order to
prioritize community eligibility.
ACKNOWLEDGEMENTS
We acknowledge with thanks the assistance and
support of National Onchocercosis Control
Programme Coordinators for Buruku, Katsina-Ala
and Okpokwu LGAs in carrying out the present
studies.
REFERENCES
Amuta EO and Olusi TA. 2000. Sero-epidemiological study of
Onchocerca volvulus using eluate of blood collected on
filter paper. Nigerian Journal of Parasitology 21:33-38.
Carme B, Samba Y, Ntsoumou MV and Yebakima A. 1993.
Prevalence of depigmentation of skin: a simple and cheap
way to screen for severe endemic onchocercosis in Africa.
Bulletin of the World Health Organization 70: 755-758.
Edungbola LD, Babata AL, Asaolu SO, Duke BOI and Connor
DH. 1990. Leopard skin and onchocercosis. Nigerian
Journal of Parasitology 9:77-82.
Edungbola LD, Nwoke BEB, Onwurili COE, Akpa AUC and
Tayo-Mafe M. 1993. Selection of rapid assessment
methods for community diagnosis of onchocercosis.
Nigerian Journal of Parasitology 12:45-50.
Gemade EI and Dipeolu OO. 1983. Onchocerciasis in Benue
State of Nigeria. II. Prevalence of the disease among the Tivs
living in the Kwande local government area. Annals of
Tropical Medicine and Parasitology 77:513-516.
Gemade EI. 1993. Training manual in Rapid Assessment
Methods using nodules and Leopard skin, treatment
schedule based on weight and height and health education
for large-scale distribution of Mectizan. Revised and
updated by sight savers international (Nigeria). pp 42.
Murdock ME. 1992. The skin and the immune response in
onchocercosis. Tropical Doctor (Supplementary) 1:44-62.
Nock IH, Ripiye P and Galadima M. 1998. Diagnostic value of
nodules and Leopard skin in community assessment of
human onchocercal endemicity. Nigerian Journal of
Parasitology 19:19-24.
Nwoke BEB, Edungbola LD, Mencias BS, Njoku AJ, Abanob
OC, Nwogu, FU et al. 1994. Human onchocercosis in rain
forest zone of southern-eastern Nigeria. 1: Rapid
assessment methods for community diagnosis in Imo river
basin. Nigerian Journal of Parasitology 15:7-18.
Nwoke BEB, Dozie INS, Gemade EI and Jiya JY. 1998. The
present status of human onchocercosis in southeastern
Nigeria using rapid epidemiological mapping (REMO).
Nigerian Journal of Parasitology 19:11-18.
Wentworth BB. 1988. Diagnostic Procedures for Mitotic and
parasitic infections. American Public Health Association
Publication Inc. Washington DC. 637 pp.
WHO. 1995. Expert Committee Report on Onchocercosis.
Fourth Report, Technical Report Series. 253 pp. Geneva.
Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 41–44
J PD
Distribution of iron in plasma, erythrocytes and tissues
of calves with the progression of Theileria annulata
infection
1
N. Sangwan and A. K. Sangwan
1
2
2
Department of Veterinary Biochemistry, CCS Haryana Agricultural University, Hisar.
Department of Veterinary Parasitology, CCS Haryana Agricultural University, Hisar.
ABSTRACT. The distribution of iron in plasma, erythrocytes and tissues was studied in relation to
the progression of Theileria annulata infection in cross-bred, 4-6 months old male calves. Group-I
animals were experimentally infected with Theileria annulata by attaching ten Theileria positive
ticks on the ear of each calf and Group-II was kept as a healthy uninfected control. The blood and
plasma samples were collected at 0, 10, 15, 20 and 25 days post-infection. The tissue samples of liver
and spleen were collected immediately after the death of the infected calves. The iron
concentrations were estimated by using atomic absorption spectrophotometer. The infected calves
had significantly (p < 0.05) low levels of iron in whole blood and erythrocytes, whereas the plasma
iron levels did not change much. The liver and spleen iron concentrations increased significantly (p
< 0.05) as compared to the normal range reported in the literature. This study suggests that care
should be taken while treating theileriosis cases by not supplementing with iron, but by
administering agents which minimize oxidative damage and help in the better utilization of iron for
erythropoiesis.
Keywords: cattle, haematology, iron, liver, spleen, Theileria annulata
INTRODUCTION
Tropical theileriosis is a tick-borne disease of cattle,
which causes morbidity and loss of productivity in
zebu cattle and lethal disease in European and crossbred stock (Gill et al., 1977; Purnell, 1978).
Theileriosis is characterized by fever and
lymphoproliferative disorders, which may be
associated with leucopenia, wasting, anaemia and
jaundice in susceptible cattle (Preston et al., 1992).
The damage to the host is caused by both schizonts in
lymphocytes/monocytes and piroplasms in
erythrocytes, resulting in lymphadenopathy and
Corresponding author: Dr. Nirmal Sangwan, Department of
Veterinary Biochemistry, CCS Haryana Agricultural University
Hisar-125 004, Haryana, India. E-mail:[email protected]
haemolytic anaemia with icterus. Serum iron has been
found to be reduced significantly in Friesian cattle
naturally infected with Theileria annulata (Omer et
al., 2003), whereas, on the contrary, it increased in
Holstein calves infected with T. sergenti;
concentrations of non-haem iron and ferritin in liver
and spleen were also significantly higher in infected
calves (Watanabe et al., 1998). Therefore, to know
whether the animals suffering from theileriosis should
be supplemented with iron or not, the present studies
were conducted to estimate the status of iron in blood,
plasma, erythrocytes and tissues of T. annulatainfected calves.
MATERIAL AND METHODS
Two groups of six cross-bred, 4–6 months old male
calves were de-wormed, sprayed with acaricides and
42
vaccinated against Foot and Mouth Disease. The
animals were fed concentrate as per National Research
Council (1988). Wheat bhoosa (roughage) and green
fodder were provided ad libitum. After 15 days,
Group-I animals were experimentally infected with T.
annulata by attaching ten Theileria positive ticks (five
pairs of females and males) on the ear of each calf, and
the other group was kept as a healthy uninfected
control. Infection was monitored by daily clinical
examination (rectal temperature and lymph node
palpation) and at 2-day intervals, Giemsa-stained thin
blood and lymph node biopsy smears were examined
after the appearance of lymphadenopathy. The day of
the death of infected calves was recorded. The blood
and plasma samples were collected in heparinised and
sterilized glass tubes on 0, 10, 15, 20 and 25 days postinfection. The tissue samples of liver and spleen were
collected immediately after the death of infected
calves (four animals). The iron concentrations in
different samples were estimated by using atomic
absorption spectrophotometery (Dunkan, 1976),
following acid digestion of organic matter and by
using calibration standards made in 0.1N HCl. The
samples of blood (1 ml) and plasma ( 2 ml) were
digested separately with 10 ml of digest acid (4:1;
nitric:perchloric acid ), dried, washed x 2 with 5 ml of
deionised water, dried each time and reconstituted
with 10 ml and 5 ml of 0.1 N HCl, respectively. The
tissue samples of liver and spleen ( 0.25 g , dried and
ground ) were also digested as for plasma and then
reconstituted in 10 ml of 0.1 N HCl. Simultaneously,
blanks were also run. The iron concentrations in
erythrocytes were calculated by using the following
formula: trace-element in erythrocytes/ml of blood =
whole blood-plasma (1-packed cell volume/100).
Haemoglobin and haematocrit were estimated by
using cyanmethaemoglobin and microhaematocrit
methods, respectively (Schalm et al., 1975). The data
were subjected to standard error of means (SE) and
Analysis of Variance (ANOVA) for statistical
significance (Snedecor and Cochran, 1967).
RESULTS AND DISCUSSION
During the course of Theileria infection, the calves
showed fever, anaemia, anorexia, cachexia, diarrhoea,
respiratory distress and recumbancy. The clinical and
parasitological findings are recorded in Table I. The
death of infected calves occurred between 18–27 days
post-infection. The haematological responses of
calves are given in Table II. As the disease progressed,
marked fall in hemoglobin and packed cell volume
(PCV) were observed. The values for hemoglobin and
PCV were reduced significantly (p < 0.05) from the
Sangwan and Sangwan
initial mean value of 9.88 ± 0.14 (g%) and 30.71 ± 0.63
(%) to 2.9 ± 0.5 (g%) and 9.0 ± 1.41(%), respectively,
on day 25 post-infection. The clinical and
haematological findings of bovine tropical theileriosis
reported here resembled those documented by other
authors (Preston et al., 1992; Sahu et al., 1996; Forsyth
et al., 1999).
The infected calves had significantly (p < 0.05) low
levels of iron in whole blood and erythrocytes,
whereas in plasma, the levels of iron did not change
much (Table II). The levels of iron in blood and
erythrocytes reduced significantly (p < 0.05) from
266.9 ± 7.38 to 67.9 ± 8.37 µg/ml and 384.3 ± 12.46 to
72.7 ± 10.7 µg/ml, respectively, on day 25 postinfection. Generally, the risk of iron inadequacy is
assessed by measuring blood hemoglobin and/or the
highly correlated PCV. However, in the study under
report, the low levels of hemoglobin and PCV did not
correspond to the low levels of iron in the body
reserves. The liver and spleen iron concentrations
were found to be 628.8 ± 78.23 [µg/g dry matter (DM)]
and 1647.7 ± 182.53 (µg/g DM), respectively. These
values in infected group are much higher than the
range reported by Georgievskii et al., 1982 i.e.
180–376 µg/g DM for liver of adult cattle and
200–400 µg/g on fresh basis for spleen, respectively
(Georgievskii, 1982). Therefore, it is important to
distinguish anaemia associated with Theileria
infection from that caused by iron deficiency. Iron
deficiency is indicated by low levels of iron in the
liver, and a marginal band of 150–250 µg/g DM is
tentatively proposed to separate deficient from normal
claves (Green et al., 1993).
The high levels in liver and spleen could be attributed
either to the increased haemolysis or animals' limited
capacity to excrete iron (Kreutzer and Kirchgessner,
1991). If there would have been haemolysis that would
have resulted in high plasma iron concentration. But in
the present study, the plasma iron levels did not
increase, which point towards the elimination of
infected erythrocytes by phagocytosis. Also, while
studying the pathogenesis of anaemia in T. annulata
infection, Hooshmand-Rad (1976) suggested that an
autoimmune reaction was largely responsible for the
development of anaemia and postulated that the
production of antibodies was triggered by the
development of schizonts; erythrocytic forms
apparently were not involved. This contention has
been supported by the finding that autohaemagglutinin antibodies were detected only in cases
of theileriosis due to a field or an agamogenous strain
(lacking erythrocytic forms) but not in premune
43
Iron distribution in Theileriosis
Table I. Host responses (clinical and parasitological) in calves infected with Theileria annulata
Clinical observations
Parasitological observations
Thermal reaction
Group Calf No.
Maximum
(°F)
Appearance
(day)
Piroplasms
Max. Appearance
(%)
(day)
(died on
day)
Max.
(%)
1
2
3
4
5
6
4
5
4
4
5
4
9
9
9
8
9
8
105.5
104.8
105.8
106.0
104.4
106.0
10
10
10
9
10
10
15
10
20
10
25
10
13
13
13
11
13
13
85
14
25
20
15
20
7-12
-
-
-
-
-
-
-
I
II
Lymph- Commencement
adenopathy
(day)
(day)
Macroscizonts
Result
23rd
29th
27th
18th
19th
20th
-
Group-I: calves were experimentally infected with Theileria annulata by attaching ten positive ticks on the ear of each
calf. Group-II:healthy uninfected control.
Table II. Estimated values for haemoglobin, packed cell volume and iron in the blood, plasma and erythrocytes of
cross-bred calves infected with Theileria annulata1
Parameters
Days post-infection
0
10
15
20
25
Hb
(g/dl)
9.88a ± 0.14
6.03b ± 0.48
5.88b ± 0.41
5.10c ± 0.42
2.90d ± 0.5
PCV
(%)
30.71a ± 0.63
21.42b ± 0.69
20.60b ± 0.36
20.00b ± 1.88
9.00c ± 1.41
Blood
(µg/ml)
266.9a ± 7.38
188.5b ± 12.7
165.9c ± 10.31
151.1cd ± 11.77
67.9e ± 8.37
Plasma
(µg/ml)
2.00a ± 0.05
1.52ab ± 0.21
1.33b ±0.18
1.64ab ± 0.05
2.03ab ± 0.35
Erythrocytes
(µg/ml of blood)
265.5a ± 7.39
187.3b ± 12.81
164.8cb ± 10.27
149.8c ± 11.83
66.1d ± 8.72
1
Calves were experimentally infected with Theileria annulata by attaching ten positive ticks on the ear of each calf. Hb,
hemoglobin; PCV, packed cell volume.
2
Values with common superscripts do not differ significantly (p < 0.05).
splenectomised calves. Only mild anaemia and
bilirubinaemia occurred in the premune
splenectomised calves in spite of the high level of
parasite load and it was suggested that the infected
erythrocytes may be eliminated by augmented
phagocytosis. During infection, iron is redistributed
by the host in an attempt to deplete the pathogen of iron
(Weinberg, 1984). The redistribution of iron by the
host may cause the secondary anaemia by depriving
iron to erythropoietic tissues. At a time when the liver
and spleen iron levels are already high, additional iron
supplementation in the Theileria-infected animals
could result in increase in the liver iron concentration
to toxic levels. The liver iron concentration of 1000
µg/g DM is considered hepatotoxic (Underwood and
Suttle, 1999) and sufficient reactive iron may be
available to cause peroxidative damage to liver (Kent
and Bahu, 1979), and this may be the underlying
44
pathogenic mechanism of peroxidative damage to
lipid membranes (Gordeuk et al., 1987). The extent of
injury depends on the antioxidant status of the animal,
particularly its vitamin E status (Omara and Blakeley,
1993; Ibrahim et al., 1997). Caeruloplasmin, a copper
containing protein, has also been reported to provide
antioxidant defences by scavenging free-iron and
free-radicals (Saneko et al., 1994).
In summation, clinical signs in terms of anaemia, low
levels of iron in whole blood and erythrocytes and the
presence of high concentrations of iron in liver and
spleen suggest that T. annulata can harm its host by
disrupting the normal functioning of liver and spleen,
upon which effective protective immune responses
and biochemical mechanisms depend. So, it is
important not to supplement Theileria-infected
animals with iron, but rather give them other nutrients
which help in preventing the oxidative damage (such
as vitamin E) and better utilization of iron for
erythropoiesis.
ACKNOWLEDGEMENTS
This work was carried out at CCS Haryana
Agricultural University, Regional Research Station,
Uchani, Karnal, and was financially supported by the
National Agricultural Research Project of the Indian
Council of Agricultural Research.
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Underwood EJ and Suttle NF. 1999. The Mineral Nutrition of
Livestock. Third Ed., CAB International Publishing Co. p
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Watanabe K, Ozawa M, Ochiai H, Kamohara H, Lijima N,
Negita H, Orino K and Yamamoto S. 1998. Changes in iron
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Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 45–52
J PD
Mosquito breeding in riceland agro-ecosystem near
Chennai, Tamil Nadu, India
1
J. Ravindran and J. Williams
2
1
Integrated Disease Vector Control Project (National Malaria Research Institute), Field Station, Chennai.
P. G. and Research Department of Zoology, Loyola College, Chennai.
2
ABSTRACT. Breeding ecology of mosquitoes was studied in riceland area near Chennai during the
period 1992-1999. The study area was a transition zone with withdrawal of intense agricultural
activities to facilitate urbanization. A myriad of mosquitoes including potential vectors of malaria
and Japanese encephalitis (JE) were observed breeding in various habitats present. A longitudinal
survey showed no malaria vector breeding in rice fields. Extensive breeding of Culex
tritaeniorhynchus (JE vector) was observed. Natural breeding pattern indicated peak anopheline
and culicine density during first two weeks after transplantation. Pre- and post-monsoon surveys
in irrigation wells indicated breeding of 16 species of mosquitoes. Both Anopheles stephensi and An.
culicifacies, the urban and rural malaria vectors, respectively, were encountered breeding in the
wells; An. stephensi breeding was predominant. Observations on species-specific habitat
preferences showed An. stephensi to breed extensively in open wells and An. culicifacies in
puddles/bed pools. Cx. tritaeniorhynchus was found breeding in almost all types of habitats
surveyed. In retrospective, the study revealed information on mosquito species breeding in various
habitats, which may be helpful for optimizing species sanitation.
Keywords: breeding habitats, immature stages, vectors
INTRODUCTION
Rice is the staple food in India, and its cultivation has
been traditionally carried out under different
geographical and climatic conditions (Singh et al.,
1989). Modern irrigation facilities and plant breeding
technologies have resulted in increased acreage and
cultivation of rice crops throughout the year leading to
its increased production. Unfortunately, along with
increased production it has also led to widespread
outbreaks of mosquito borne diseases like malaria and
Japanese encephalitis (JE; Lacey and Lacey, 1990).
Corresponding author: Dr. K. John Ravindran, Integrated
Disease Vector Control Project (NMRI), 332-A, Spartan
School Road, Paneer Nagar, Mogappair, Chennai-600 037,
Tamil Nadu, India. E-mail: [email protected]
For their breeding, mosquitoes are known to exploit
aquatic habitats present in riceland agro-ecosystem.
Any change in this ecosystem, either naturally or man
made, tend to alter the biotic community including
mosquitoes, which may be advantageous or
disadvantageous for mankind. Therefore, information
on mosquito fauna and breeding ecology in a
transitory environment is imperative for successful
intervention. Ongoing urbanization in agricultural
area in the outskirts of Chennai, Tamil Nadu, India,
resulted in an ecologically transitional region, which
received our attention. Information on breeding
ecology of mosquitoes in this environment may be
helpful to ensure preparedness for effective vector and
disease control.
46
MATERIALS AND METHODS
Chennai (longitude: 80° 15'E, latitude: 13° 05'N) isthe
capital city of the state of Tamil Nadu, India, and is
situated in the northeastern part of the state. Minimum
mean and maximum mean temperatures recorded
during 1990–2000 were 19.8° C and 38.5° C,
respectively. Maximum rainfall is during Northeast
monsoon (October–December). The study was
undertaken in Chennai metropolitan area in western
outskirts of the city during the period 1992–1999.
Agricultural activities have been totally withdrawn in
areas bordering the city due to ongoing urbanization
and partially withdrawn in areas away from the
outskirts of the city. The area represents an ecological
transition zone.
Studies on mosquito breeding in rice fields were
carried out in agricultural fields located at
Poonamallee and Thirukandalam areas, which are 10
km and 20 km away from the outskirts of Chennai.
Observations were carried out from September 1992
to September 1993, covering one long-duration and
two short-duration crops. Five fields were selected
and regularly observed for mosquito breeding and
population structure on a weekly basis during each
crop. A quadrat with a dimension of 33 x 33 x 20 cm
enclosing an area of 0.1m2 was used as a sampler
(Chandrahas, 1990). Ten samples were taken/plot.
Sampling was confined to previously earmarked
sampling sites, which included four corner, four
central and two random sites within 0.5 m from the
bund. During each sampling occasion, height of rice
plant, depth of standing water and peak noon water
temperature were recorded with other information
such as fertilizer used.
In Irrigation wells, study period covered a premonsoon (August–October) and post-monsoon
(November–February) period during 1998–1999.
Observations were carried out in irrigation wells in
riceland areas in Vanagaram area near Poonamallee,
where irrigation wells suitable for the study were
found. The conditions for selection of a well were
absence of fishes, no floating vegetation and
accessibility for sampling from all sides. Weekly
observations were carried out in ten irrigation wells. A
well net with a 20 cm diameter was used as a sampler.
Immature samples were collected by pulling well nets
through a distance of 1 m along the edges of the wall.
Repeated sampling in the same area was avoided.
Depth of water table and peak noon water temperature
was recorded during each visit. Chi square analysis for
Ravindran and Williams
test of independence was performed to establish
preferences towards breeding at various well depths.
Habitat specific preferences were studied in few
selected habitats namely open wells, bed pools,
puddles and hoof-prints present in outskirts of periurban localities with or without ongoing agricultural
activity. Any water stagnation with a surface area of
less than 1 m2 was classified as a puddle and those with
more than 1 m2 but less than 50 m2 as bed pool.
Random surveys were undertaken and immatures
were collected using well nets (wells), ladles (bed
pools and puddles) and spoons (hoof-prints).
Interspecific association and index of association in
relation to breeding habitats of various mosquito
vector species were calculated using the method of
Cole (1949) and Whittaker and Fairbanks (1958).
During sampling, immatures collected were counted
instar-wise. Fourth instar larvae and pupae were
brought to laboratory and reared until emergence for
identification.
RESULTS AND DISCUSSION
A total of 11 Anopheles, 10 Culex and four Aedes
species were collected from various breeding habitats
in the study site (Table I). Among important disease
vectors, An. stephensi and An. culicifacies, the urban
and rural malaria vectors, respectively, Cx.
tritaeniorhynchus, Cx. vishnui and Cx. pseudovishnui,
the potential vectors of JE in peninsular Indian region
and Ae. aegypti, vector of dengue were present. In rice
fields, no malaria or filaria vectors were observed
breeding. Similar results were obtained at Madurai
(John Victor and Reuben, 1999) and Pondicherry
(Chandrahas and Rajagopalan, 1979). Among vectors
of JE, Cx. tritaeniorhynchus was predominant being
encountered during all the 25 weeks of observation
carried out over a period of three rice growing seasons,
whereas Cx. vishnui and Cx. pseudovishnui were
observed breeding during seven and one occasions
only. Breeding pattern of Cx. tritaeniorhynchus
differed from observations of Reuben (1971) and
Rajendran and Reuben (1991) who reported intense
breeding at the latter stages of plant growth. In Tamil
Nadu, JE virus isolations have also been reported in
An. subpictus, Cx. infula, Cx. whitmorei and Cx.
fuscocephala, all of which breed in ricefields (Philip
Samuel et al., 2000). Except Cx. infula and Cx.
whitmorei, all other species were found breeding in
rice fields.
The density of immatures during the three cultivation
47
Mosquito breeding in riceland agro-ecosystem near Chennai
seasons is given in Fig. 1 and 2. Anopheles and Culex
density was high during first week after
transplantation. Thereafter, intensity in breeding
decreased. In Culex species, another peak in the
density of immatures was observed during fourth to
sixth week after transplantation. Standing water
maintained for a period of two weeks after
transplantation provided opportunity for intense
exploitation of the habitat for breeding. Reduction in
the density of immatures after this period, is due to
alterations in daily irrigation practices that are
controlled by various factors like soil texture, acreage
and the number of crops raised in a year, erratic power
supply and rainfall. Fields are usually irrigated
immediately after cessation of standing water to
manage adequate water supply. Such enforced
intermittent irrigation due to lack of sufficient
irrigation water has also been reported by Rajagopalan
et al. (1990) and Russell et al. (1942) in areas in the
adjacent erstwhile South Arcot district. In addition,
rice cultivation practices such as deweeding,
application of fertilizer and pesticides also reduce the
density of immatures. In the present study, no
pesticides were applied though application of urea as a
fertilizer was undertaken before and three weeks after
transplantation.
5000
4500
4000
3500
3000
2
Density/m 2500
2000
1500
1000
500
0
0
I
II
III
IV
VI
V
VII
VIII
IX
X
Weeks
Transplantation
Aug/Sep
Jan/Feb
Jun/Jul
Fig. 1. Anopheles immature density in different rice growing seasons.
5000
4500
4000
3500
3000
2
Density/m 2500
2000
1500
1000
500
0
0
I
II
III
IV
VI
V
VII
VIII
Weeks
Transplanta tion
Aug/Sep
Jan/Feb
Jun/Jul
Fig. 2. Culex immature density in different rice growing seasons.
IX
X
48
Ravindran and Williams
Table I. Mosquito species observed breeding in different habitats in the study area
Species
Rice
fields
Irrigation
wells
Draw wells
Puddles
Hoofprints
Bed
pools
Anophelines
An. subpictus
An. vagus
An. peditaeniatus
An. nigerrimus
An. barbirostris
An. stephensi
An. aconitus
An. splendidus
An. annularis
An. pallidus
An. culicifacies
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Culicines
Cx. tritaeniorhynchus
Cx. vishnui
Cx. pseudovishnui
Cx. bitaeniorhynchus
Cx. gelidus
Cx. fuscocephala
Cx. (Lutzia) fuscanus
Cx. (Lophoceraomyia) sp.
Cx. vegans
Cx. quinquefasciatus
Ae. vittatus
Ae. scathophagoides
Ae. aegypti
Ae. vexans
+
+
+
+
+
+
-
+
+
+
+
+
+
+
-
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
-
Irrigations wells supported breeding of 15 species of
mosquitoes. An. barbirostris, An. subpictus, An.
vagus, Cx. quinquefasciatus and Cx.
tritaeniorhynchus were potential breeders and were
encountered breeding for more than 20 weeks. An.
aconitus, An. splendidus, Cx. vishnui, Ae. vittatus,
were very poor breeders and were obtained in less than
five weeks during the course of observation. Among
malaria vectors, An. stephensi was predominant being
observed in 18 of the total 30 weeks of observation
when compared to An. culicifacies, which was
observed breeding only in two weeks and during postmonsoon season. Rapid urbanization in rice
cultivation area adjoining extended areas may be the
reason for prolific breeding of An. stephensi in these
irrigation wells. Seasonal breeding preference in
irrigation wells was noticed (Table II). An. aconitus
and An. splendidus were found breeding in pre-
monsoon season and An. culicifacies, An. nigerimus,
Cx. vishnui, Cx. (Lophoceraomyia) species and Ae.
vittatus were found breeding exclusively in postmonsoon season. All other mosquitoes were found
breeding in both seasons. An. stephensi, An.
subpictus, An.
splendidus, An. Aconitus, Cx.
quinquefasciatus and Cx. gelidus showed preference
to breed at a well-depth greater than 15 feet, whereas
An. barbirostris, An. vagus, An. nigerrimus, Cx.
tritaeniorhynchus and Cx. (Lophoceraomyia) sp. at
shallow levels with depth of less than five feet. Chi
square analysis performed on species obtained at all
well depths indicated preference of An. stephensi, An.
barbirostris, An. vagus, Cx. quinquefasciatus and Cx.
gelidus to breed at different well depths (Table III).
Irrigation wells may act as foci for mosquito breeding
during non-monsoon seasons and non-rice cultivation
periods.
49
Mosquito breeding in riceland agro-ecosystem near Chennai
Table II. Details of the density of immatures and larval emergence in irrigation wells
Particulars
Pre-monsoon
Immature density/dip-Anopheles
Immature density/dip-Culex
Well Depth (Min.-Max.)
Anophelines-No. emerged
An. stephensi
An. barbirostris
An. vagus
An. subpictus
An. aconitus
An. splendidus
An. culicifacies
An. nigerrimus
Culicines-No. emerged
Cx. quinquefasciatus
Cx. tritaeniorhynchus
Cx. gelidus
Cx. fuscanus
Cx. vishnui
Cx. (Lophoceraomyia) sp
Ae. vittatus
10.216.6
5.843.3
1417.3
918
210 (22.9)
431 (46.9)
34 (3.7)
238 (25.9)
2 (0.2)
3 (0.3)
0 (0)2
0 (0)
2544
1584 (62.3)
531 (20.9)
253 (9.9)
176 (6.9)
0 (0)
0 (0)
0 (0)
Monsoon /
Post-monsoon
12.722.2
5.323.9
2.623.2
1775
19 (1.1)
1094 (61.6)
322 (18.1)
285 (16.1)
0 (0)
0 (0)
(0.1)
53 (3)
1282
437 (34.1)
145 (50.3)
15 (1.2)
34 (2.7)
41 (3.2)
106(8.3)
4 (0.3)
(Figures in parenthesis denote percent)
Table III. Mosquito breeding at different well depths
Particulars
No. of wells with
mosquito breeding
Anophelines
An. stephensi
An. barbirostris
An. vagus
An. subpictus
An. aconitus
An. splendidus
An. culicifacies
An. nigerrimus
Culicines
Cx. quinquefasciatus
Cx. tritaeniorhynchus
Cx. gelidus
Cx. fuscanus
Cx. vishnui
Cx. (Lophoceraomyia) sp
Ae. vittatus
Well depth (ft)
X2
<5
515
> 15
80
62
14
9 (11.3)
60 (86.3)
23 (28.8)
19 (23.8)
0 (0)
0 (0)
1 (1.3)
12 (15)
18 (29)
41 (66.1)
7 (11.3)
9 (14.5)
0 (0)
0 (0)
1 (1.6)
1 (1.6)
5 (35.7)
9 (64.3)
1 (7.1)
4 (28.6)
1 (7.1)
1 (7.1)
0 (0)
0 (0)
8.95*
9.04*
8.25*
2.44
NP
NP
NP
NP
27 (33.8)
30 (37.5)
2 (2.5)
8 (10)
0 (0)
5 (6.3)
0 (0)
31 (50)
20 (32.3)
8 (12.9)
10 16.1)
1 (1.6)
0 (0)
4 (6.5)
9 (64.3)
4 (28.6)
2 (14.3)
1 (7.1)
0 (0)
0 (0)
0 (0)
6.62*
0.67
6.27*
1.59
NP
NP
NP
(Figures in parenthesis denote percent; NP - not performed, * p > 0.05)
-
50
Ravindran and Williams
Table IV. Details of species specific habitat positivity and survey particulars
Particulars
Domestic wells
Bed pools
Puddles
Hoof-prints
No. surveyed
No. positive for
Anopheles breeding
No. positive for
Culex breeding
Habitat positivity for breeding (%):
An. subpictus
An. vagus
An. nigerrimus
An. barbirostris
An. stephensi
An. aconitus
An. annularis
An. pallidus
An. culicifacies
Cx. tritaeniorhynchus
Cx. vishnui
Cx. bitaeniorhynchus
Cx. gelidus
Cx. fuscocephala
Cx. (Lutzia) fuscanus
Cx. (Lophoceraomyia) sp.
Cx. vagans
Cx. quinquefasciatus
Ae. scathophagoides
Ae. aegypti
Ae. vexans
225
175
275
217
87 (59)
93 (56)
136 (74)
62 (35)
49 (27)
51 (33)
79 (45)
88 (65)
1.7
8.5
3.4
11.9
88.1
1.7
1.7
0
5.1
77.8
0
0
3.7
0
3.7
18..5
0
77.8
0
3.7
0
82.1
32.1
3.6
16.1
1.8
0
17.9
1.8
17.9
15.1
0
3
0
0
0
0
0
15.1
0
0
0
55.4
32.4
10.8
21.6
1.4
40
65.7
5.7
3
5.7
0
0
2.9
8.6
13.9
7.7
0
0
0
4.6
0
3.1
13.9
0
0
0
4.1
5.4
13.5
26.7
6.7
0
4.4
2.2
6.7
0
0
26.7
4.4
0
4.4
(Figures in parenthesis denote the number of habitats where adult mosquitoes emerged)
Various mosquitoes sp. found breeding in puddles, bed
pools, hoof-prints and domestic wells are shown in
Table I. Puddles with rainwater were found to harbour
the maximum number of species (16 nos.). Except for
Ae. scathophagoides, all mosquitoes breeding in
irrigation wells were also observed breeding in these
habitats. An. stephensi was found to breed profusely in
domestic wells and An. culicifacies in bed pools and
puddles (Table IV). Breeding of An. stephensi in bed
pools, hoof-prints and puddles is rather unusual but
similar observations have been reported (Yadav et al.,
1989). Breeding of An. stephensi in
hoof-prints
with rainwater collections in Chennai outskirts has
been reported (Vasanthi, 1996). An. stephensi was
predominant breeder in domestic wells,
An.
subpictus in bed pools and puddles and An. vagus in
hoof-prints. Cx. tritaeniorhynchus, among JE vectors
was most commonly encountered in all breeding
habitats, whereas Cx. pseudovishnui was not
encountered in any of the breeding habitats. Cx.
vishnui was collected from puddles and hoof-prints
only. Reuben (1971) reported breeding of Cx.
tritaeniorhynchus and Cx. vishnui in wells, ponds,
ditches, irrigation channels, borrow pits and from rice
fields (fallow or planted) in North Arcot district of
Tamil Nadu.
Interspecific association (Table V) with regard to use
of habitats for breeding of vectors (An. culicifacies,
An. stephensi, Cx. tritaeniorhynchus and Cx.
quinquefasciatus) existed between An. stephensi and
An. culicifacies, and Cx. quinquefasciatus and Cx.
tritaeniorhynchus in bed pools, indicating bed pools to
be a very favourable source where all vector species
of various diseases co-existed. In canal irrigated area
51
Mosquito breeding in riceland agro-ecosystem near Chennai
Table V. Interspecific associations among important vector mosquitoes in study area
Habitat
Species
Interspecific
association (CAB)
Index of
association (I)
Domestic
well
An. stephensi An. culicifacies
An. stephensi - Cx. quinquefasciatus
An. stephensi - Cx. tritaeniorhynchus
An. culicifacies - Cx. quinquefasciatus
An. culicifacies - Cx. tritaeniorhynchus
Cx. tritaeniorhynchus - Cx. quinquefasciatus
- 0.008 ± 0.314
-0.060 ± 0.146
-0.093 ± 0.167
0.036 ± 0.377
0.75 ± 1.445
0.012 ± 0.071
-0.970
-0.860
Bed Pools
An. stephensi An. culicifacies
An. stephensi - Cx. quinquefasciatus
An. stephensi - Cx. tritaeniorhynchus
An. culicifacies - Cx. quinquefasciatus
An. culicifacies - Cx. tritaeniorhynchus
Cx. tritaeniorhynchus - Cx. quinquefasciatus
1.000 ± 0.393
-0.833 ± 3.690
-0.970 ± 1.259
0.035 ± 0.079
0.737 ± 0.372
0.461 ± 1.526
-0.954
-0.848
Puddles
An. stephensi An. culicifacies
An. stephensi - Cx. quinquefasciatus
An. stephensi - Cx. tritaeniorhynchus
An. culicifacies - Cx. quinquefasciatus
An. culicifacies - Cx. tritaeniorhynchus
Cx. tritaeniorhynchus - Cx. quinquefasciatus
-0.909 ± 2.795
-0.923 ± 2.519
-1.000 ± 0.786
-0.462 ± 0.64
-0.420 ± 0.370
0.180 ± 0.216
-1.000
-0.505
Hoof-Prints
An. stephensi An. culicifacies
An. stephensi - Cx. quinquefasciatus
An. stephensi - Cx. tritaeniorhynchus
An. culicifacies - Cx. quinquefasciatus
An. culicifacies - Cx. tritaeniorhynchus
Cx. tritaeniorhynchus - Cx. quinquefasciatus
0.600 ± 3.561
0.818 ± 1.98
0.111 ± 0.616
0.75 ± 1.591
0.938 ± 0.507
0.205 ± 0.277
-1.000
-0.762
in Gujarat, An. culicifacies was positively associated
with An. stephensi only in paddy fields (Bhatt et al.,
1990). Negative association was noticed in puddles,
which was also similar to present findings. Likewise,
in domestic wells a positive association existed
between An. culicifacies, Cx. quinquefasciatus and
Cx. tritaeniorhynchus. In puddles and hoof-prints, the
coexistence of vectors was relatively low.
Planned and systematic extension of Chennai city is
underway resulting in rapid environmental changes.
Both An. stephensi and An. culicifacies have been
observed in extended regions of the city. An.
stephensi, responsible for intense malaria
transmission in Chennai dominated indicating species
replacement on account of environmental change. Cx.
tritaeniorhynchus, among potential vectors of JE was
ubiquitous and was found to breed in all habitats
surveyed. Intensive exploitation of rice fields
throughout the rice growing season for breeding by
this species is a cause of concern. However, natural
enforced intermittent irrigation due to acute water
shortage and withdrawal of rice cultivation may
relatively contribute towards decreased risk in
outbreak of JE in this area. Investigations on mosquito
breeding in habitats in other extended areas may
provide information on other species that may be
prevalent in areas adjoining the city. Due to the
prevalence of disease factor and high labour
movement and activity, careful monitoring is
indispensable for prevention of disease outbreaks.
Bioenvironmental control, found to be successful in
many areas, can be planned and adopted in this
transition region before complete urbanization to
achieve effective species sanitation.
52
ACKNOWLEDGEMENTS
The authors wish to express their sincere thanks to Dr.
V. P. Sharma and Dr. Sarala K. Subbarao, former
Directors of National Institute of Malaria Research
(NMRI), Prof. Dr. A. P. Dash, Director, NMRI and Shri
Alex Eapen, Officer-in-charge, Integrated Disease
Vector Control Project (IDVC; NIMR), Field Station,
Chennai, for encouragement and facilities provided.
The authors also thank Dr. R. C. Dhiman, Deputy
Director, NMRI, for his critical review of the
manuscript. The technical assistance rendered by staff
of IDVC (NIMR), Field Station, Chennai, is also
gratefully acknowledged.
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Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 53–57
J PD
Random amplified polymorphic DNA of Trichomonas
vaginalis isolates from Tarbiz, Iran
1
1
2
2
2
R. Jamali, B. Zareikar , A. Kazemi , M. Asgharzadeh , S. Yousefee , R. Estakhri , S. Montazer and A.
1
Ghazanchaei
1
2
Tarbiz University of Medical Science, Faculty of Medicine, Parasitology Department.
Tarbiz University of Medical Sciences, Faculty of Medicine.
ABSTRACT. Trichomonas vaginalis, the causative agent of human trichomoniasis, is the most
common nonviral sexually transmitted disease. The infection may be asymptomatic or may cause
severe vaginitis and cervicitis in women. Despite its high prevalence, little is known about its genetic
variability and factors leading to asymptomatic infections. The random amplified polymorphic
DNA (RAPD) technique is a simple method to detect DNA polymorphism. RAPD was performed by
using four different random primers (OPD1, OPD2, OPD3 and OPD5) for the typing of 120 isolates
of T. vaginalis from Tarbiz. Phylogenetic analysis was performed using SPSS program, and
dendrogram with two distinct clusters was constructed. The asymptomatic isolates tended to form a
cluster, separate from symptomatic isolates. Further studies for better understanding the
relationship are suggested.
Keywords: polymorphism, random amplified polymorphic DNA, Trichomonas vaginalis
INTRODUCTION
Trichomonas vaginalis is a prevalent vaginal
pathogen, affecting 180 million persons worldwide
annually (Wang 2000). Evidence from published
studies exists that T. vaginalis is independently
associated with a variety of adverse health
consequences in both women and men, including
increased human immunodeficiency virus (HIV)
transmission, infertility, cervical intraepithelial
neoplasia (CIN) development in women, and
nongonoccocal urethritis and chronic prostatitis in
men (Jane and Edward, 2003; Soper, 2004).
Detection of T. vaginalis has traditionally relied on
wet-mount microscopy or culture. These methods are
highly specific but lack sensitivity. Nucleic acid
Corresponding author: Dr. Rasoul Jamali, Parasitology
Department, Faculty of Medicine, Tarbiz University of Medical
Sciences, Daneshgah St, Tarbiz, Iran. Telefax : 0411-3364665,
E-mail: [email protected]
amplification assays are highly desirable alternatives
to culturing, having both sensitivity and specificity for
detecting T. vaginalis DNA (Schee et al., 1999). The
random amplified polymorphic DNA (RAPD)
technique represents as an efficient tool for the study
of genetic polymorphism of DNA. It involves the
amplification of random segments of genomic DNA
by polymerase chain reaction (PCR) using short single
primers of arbitrary sequences (Fraga et al., 2002).
Different studies suggest that RAPD provides
powerful markers to analyze the genetic diversity in T.
vaginalis (Rojas et al., 2004; Kaul et al., 2004;
Vanacova et al., 1997). The aim of this study was to
genetically characterize by RAPD a collection of T.
vaginalis isolates from patients with clinical signs and
symptoms.
MATERIALS AND METHODS
A total of 2630 women visiting the health care centers
of Tarbiz with and without symptoms of T. vaginalis
54
(vaginal discharge, itching, dysuria and dyspareunia)
were selected and examined for the presence of T.
vaginalis. From each individual two samples were
collected from the posterior vaginal fornix by using
two sterile cotton swabs. First swab was used for wet
mount preparation and the second one was used to
inoculate the Kupferberg medium (Quelab
Laboratories, Canada). Culture tubes were incubated
at 37° C up to 7 days, and examined microscopically
on days 2, 3, 5 and 7 after inoculation.
DNA extraction: Log phase T. vaginalis cultures were
washed with phosphate buffered saline (pH 7.4) and
the cell pellet was suspended in 400 µl TE (10 mM
Tris, 1 mM EDTA) buffer (pH 8). To this suspension, 5
µl proteinase K (20 mg/ml) and 60 µl of 10% sodium
dodecyl sulphate solutions were added, and incubated
overnight at 55-65° C. Following incubation, 100 µl of
NaCl and 80 µl of pre-warmed (at 65° C) CTAB/NaCl
solution were added, vortexed well and incubated at
65° C for 10 min. DNA was cleaned by adding 700 µl
of chloroform-isoamyl alcohol (24:1) solution and
vortoxed for 20 s, and precipitated by 1ml of cold
ethanol (70%) and centrifugation at 12,000 x g for 5
min (×2) at 10° C. Finally, after air-drying, the DNA
pellet was dissolved in 50-100 µl TE buffer (pH 8).
RAPD PCR: Four different 10 base pair primers were
used for RAPD analysis (their sequence is shown in
Table I; Snipes et al., 2000). The DNA amplification
was performed at final volume of 25 µl containing: 2.5
µl of 10 x PCR reaction buffer (500 mM KCl and 200
mM Tris-HCl, pH 8.4), 1.25 µl MgCl2 (50 mM), 1 µl of
each primer (Cinnagen, Iran), 0.5 µl of mixed dNTP
(10 mM), 4 µl of template DNA, 15.35 µl of double
distilled water and 0.4 µl of Taq DNA polymerase (5
unit/µl; Cinnagen, Iran). Negative controls for each of
four primers used contained all components except
template DNA. The amplification protocol consisted
of an initial denaturation step at 94° C for 5 min
followed by 40 cycle's repetitions of 1 min at 94° C, 1
min at 36° C and 2 min at 72° C. The final cycle “the
extension step” was of 15 min at 72° C. The PCR
products were analyzed by electrophoresis in 1.2%
agarose gel in TBE buffer. The gels were then stained
with ethidium bromide (0.5 µg/ml) and visualized
under the UV transilluminator.
RESULT
Overall, 4.6% (120/2630) of specimens yielded a
positive T. vaginalis culture. Seventy four isolates
were obtained from symptomatic patients and 46 from
Jamali et al.
asymptomatic patients, axenically. DNA extracted
from these isolates was subjected to RAPD analysis
and amplified with 4 different random primers
(Fig. 1a-d) show the RAPD patterns obtained with the
primers used. All the primers provided distinct
patterns. For each primer, the banding pattern was
scored as presence (1) or absence (0) for each isolate
and matrix table was constructed by SPSS 11.0
software. Dendrogram was built based on RAPD-PCR
results for each primer, and for four primers, using
Wards method and SPSS 11.0 program (Fig. 2). The
isolates with similar banding pattern were assigned as
a single type. OPD1 had the least typing ability as it
gave 32 types for typing of 120 strains, whereas OPD5
had the highest typing ability that gave 58 types. OPD2
gave 56 and OPD3 gave 43 types. A total of 62
different types were obtained from 120 T. vaginalis
specimens analyzed. There was one cluster consisting
of four patients, seven with three patients, 41 with two
patients and 13 with one patient. According to the tree,
the isolates fell into two major groups (the
classification results based on discriminant analysis
are shown in Table II). The upper branch consisted of
65 isolates out of which 24 were from symptomatic
patients and 41 isolates belonged to asymptomatic
patients. The lower branch of tree consisted of 55
isolates, 50 of them from symptomatic patients and
only five from asymptomatic ones. The 11 isolates that
were from patients with the history of treatment
failure, showed a scattered format in the tree (isolates
99, 83, 84, 70, 107, 27, 14, 7, 74, 9 and 36).
Table I. The sequence of four primers used for RAPD
analysis
OPD
OPD2
OPD3
OPD5
Size (mer)
Squence (5' to 3')
10
10
10
10
ACCgCgAAgg
ggACCCAACC
gTCgCCgTCA
TgAgCggACA
Table II. Classification result based on discriminant
analysisa
Predicted group
membership
Ward method
Original count 1
2
%
1
2
1
2
Total
62
3
3
52
65
55
95.4
5.5
4.6
94.5
100.0
100.0
a. 95% of original grouped cases correctly classified.
55
Random amplified polymorphic DNA analysis
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Fig. 1-a: RAPD banding pattern of T. vaginalis isolates using OPD1 primer
Lane 1: Size marker 100 bp DNA ladder
Lane 2-23: Banding pattern of T. vaginalis isolates No.6 - 27
Lane 24: Negative control
Lane 25: Size marker lambda DNA/EcoRI+HindIII
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Fig. 1-b: RAPD banding pattern of T. vaginalis isolates using OPD2 primer
Lane 1: Size marker 100 bp DNA ladder
Lane 2-29: Banding pattern of T. vaginalis isolates No.75-102
Lane 30: Size marker lambda DNA/EcoRI+HindIII
1
2
3
4
5
6
7
8
9
10
11
12
Fig. 1-c: RAPD banding pattern of T. vaginalis isolates using OPD3 primer
Lane 1: Size marker lambda DNA/EcoRI+HindIII
Lane 2-13: Banding pattern of T. vaginalis isolates No.52-63
Lane 14: Size marker 100 bp DNA ladder
13
14
56
Jamali et al.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Fig. 1-d: RAPD banding pattern of T. vaginalis isolates using OPD5 primer
Lane 1: Size marker lambda DNA/EcoRI+HindIII
Lane 2-13: Banding pattern of T. vaginalis isolates No.52-63
Lane 14: Size marker 100 bp DNA ladder
DISCUSSION
Trichomoniasis is an important sexually transmitted
disease which may manifest with a wide range of
symptoms ranging from an asymptomatic
presentation to severe sequel. It is `not clear yet as to
why only a proportion of individuals infected with T.
vaginalis become symptomatic, whereas the rest
others remain asymptomatic. Strain variation and host
factors may play a role in leading to symptomatic or
asymptomatic infections (Kaul et al., 2004). Recent
studies have shown the ability of DNA fingerprinting
techniques in differentiating strains of various
organisms (Tibayrenc, 1998). Vanacova et al. (1997)
for the first time used RAPD technique for
phylogenetic analysis of T. vaginalis and found it a
useful method in epidemiological analysis. Their
results suggested a concordance between the genetic
markers with resistance to metronidazole and clinical
findings, but they found no concordance with the
presence of T. vaginalis virus (TVV) and the virulence
of strains.
Hample et al. (2001) assayed the relationship between
20 strains of T. vaginalis from eight countries using
RAPD analysis, and they found that the phylogenic
tree reflects the pattern of virulence, geographic origin
or infection by TVV. Rojas et al. (2004) used RAPD
technique in 40 isolates of T. vaginalis to find an
association between genetic polymorphism of
organism and its clinical characters. Their results
emphasize that the severity of infection depends on the
genetic type of T. vaginalis involved.
In the present study, we used RAPD method for the
genetic analysis of 120 clinical isolates of T. vaginalis,
and we investigated the association of T. vaginalis
genetic polymorphism and its clinical classification as
symptomatic or asymptomatic. In conclusion, our
results show that the isolates from asymptomatic
patients tend to form a distinct cluster separate from
symptomatic isolates, and that T. vaginalis isolates
from patients with or without symptoms are
genetically different. Further studies are necessary to
better understand the relationship between genetic
markers and the pathogenicity of the organism.
REFERENCES
Fraga J, Rojas L, Sariego I and Sarria CA. 2002. Optimization
of random amplified polymorphic DNA technique for its use
in genetic studies of Tricomonas vaginalis isolates. Infect
Genet E 2: 73-75.
Hample V, Vanacova S, Kulda J and Flegr J. 2001.
Concordance between genetic relatedness and phenotypic
similarities of Trichomonas vaginalis strains. BMC Evol
Biol 48:1-11.
Jane RS and Edward W. 2003. High rate of Trichomonas
vaginalis among men attending a sexually transmitted
Random amplified polymorphic DNA analysis
Rescaled Distance cluster Combine
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vaginalis: random amplified polymorphic DNA analysis of
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Rojas L, Fraga J and Seriego I. 2004. Genetic variability
between Trichomonas vaginalis isolates and correlation
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81
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84
111
113
55
119
106
45
70
103
Schee C, Belkum A, Zwijgers L, Brugge E, Oneill EL, Luijedjk
A et al.1999. Improved diagnosis of Trichomonas vaginalis
infection by PCR using vaginal swabs and urine specimen
compared to diagnosis by wet mount microscopy, culture
and fluorescent staining. J Cli Microbiol 37:4127-4130.
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104
105
109
107
107
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120
46
61
Snipes LJ, Gamard PM, Narcisi EM, Ben Beard C, Lehman T
and Secor EW. 2000. Molecular epidemiology of
metronidazole resistance in a population of Trichomonas
vaginalis clinical isolates. 38: 3004-3009.
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112
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29
24
Soper D. 2004. Trichomoniasis:under control or
undercontrolled. Am J Obstet Gynecol 190: 281-290.
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60
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1
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52
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14
Tibayrenc M. 1998. Beyond strain typing and molecular
epidemiology: integrated genetic epidemiology of
infectious disease. Parasitol Today 14: 323-329.
26
108
30
66
71
49
86
34
Vanacva S, Tachezy J and flegr J. 1997. Characterization of
trichomonad species and strains by PCR fingerprinting. J
Eukayot Microbiol 44: 545-552.
37
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11
13
3
4
6
7
5
10
16
59
17
2
18
15
9
67
77
62
69
64
58
72
73
74
54
56
76
63
75
48
51
50
23
57
38
40
41
42
47
25
43
65
78
79
9
36
85
33
53
31
35
19
20
22
92
Fig. 2. Dendrogram for 120 isolates of
Trichomonas vaginalis based on
RAPD-PCR data.
Wang J. 2000. Trichomoniasis. Prim Care Update Ob/Gyns
7:148-153.
Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 58–63
J PD
Two new species of Trypanosoma from freshwater fish
(Heteropneustes fossilis and Channa punctatus) from
Bareilly, India
1
D. K. Gupta, N. Gupta and R. Gangwar
Department of Zoology, Bareilly College, Bareilly.
Department of Animal Science, M. J. P. Rohilkhand University, Bareilly.
1
ABSTRACT. Haematological examination of fresh water fish Channa punctatus (115 specimens)
and Heteropneustes fossilis (163 specimens) revealed 10.6% and 14.6% infection, respectively, of
dimorphic species of Trypansoma. Both in the former and latter hosts, the parasite concentration
index was 112.4 and 110.5 Trypanosomes/100 RBCs, respectively. The flagellate from C. punctatus is
characterized by its dimorphic nature [small (18-33 µm) and large (30-52 µm) forms], and was
found to be distinct from the earlier reported species in total body length, flagellar length,
kinetoplast dimensions and granulation pattern. The trypanosome species from H. fossilis was also
observed to be dimorphic [small (23.5-30.5 µm) and large (33.1-43.8 µm) forms], and differences
existed in cell breadth, nuclear size, flagellar length and cytomorphological features from the
earlier recorded species. These two dimorphic species have been described herein as Trypanosoma
saulii n. sp and Trypanosoma heteropneusti n. sp from C. punctatus and H. fossilis, respectively.
Keywords: Channa, dimorphic, division, Heteropneustes, Trypanosoma
INTRODUCTION
The freshwater
fish, Channa punctatus and
Heteropneustes fossilis have served as favourable
hosts of Trypanosoma: T. ophiocephali Pearse, 1933;
T. striati Qadri, 1955; T. punctati Hasan and Qasim,
1962; T. elongatus Raychaudhuri and Misra, 1973; T.
bareilliana Gupta et al., 1987 and T. rohilkhandae
Gupta and Saraswat, 1991 have been reported from
various species of Channa. The dimorphic species
recorded are T. gachuii Misra et al., 1973 from C.
gachua and T. aligaricus Gupta and Jairajpuri, 1982a
from C. punctatus. On the other hand, T. saccobranchi
Castellani and Willey, 1905; T. danilewskyi Qadri,
1962; T. singhii Gupta and Jairajpuri, 1981; T.
Corresponding author: Prof. Neelima Gupta, Department of
Animal Science, M.J.P. Rohilkhand University, Bareilly 243
006, U.P., India. E-mail: [email protected]
kargenensis Gupta and Gupta, 1994 and T. karelensis
Gupta et al., 2001 have been recorded from H. fossilis;
however, T. mukundi Raychaudhuri and Misra, 1973 is
the only dimorphic species. Herein, two new species
of Trypanosoma have been recorded from C.
punctatus and H. fossilis.
MATERIALS AND METHODS
Live specimens of C. punctatus and H. fossilis,
collected from fresh waters of Bareilly were
transported to the laboratory and maintained in
separate aquaria under optimum conditions of food
and aeration. Blood was collected from their caudal
vein and examined immediately (hanging drop
preparation and micro-haematocrit) for the presence
of parasites. When positive, smears were made, air
dried, fixed in methanol and stained in Leishman +
phosphate buffer (pH 6.6) in the ratio of 1:7 for 40 min.
59
Two new species of Trypanosoma
After thorough washing, the films were air dried,
mounted in DPX, sealed and observed and
photographed at 1000 x magnification under LEICA
DMLB photoautomat. Camera lucida drawings were
made for statistical measurements of the parasites.
Nuclear index (NI), flagellar index (FI) and
kinetoplast index (KI) were calculated as follows:
NI = PN/NA; FI = CL/FL; KI = PN/KN
where PN = posterior end to nucleus ; NA = anterior
end to nucleus ; CL = body length ; FL = flagellar
length and KN = kinetoplast to nucleus.
RESULTS
Trypanosoma saulii n.sp. (Fig. 1 A-D; Table 1)
Host
- Channa punctatus Bloch
Locality
- Bans Mandi, Bareilly, India
Percentage of
infection/intensity - 10.6%/1-12.4
Trypanosomes/100 RBCs
(n = 115)
The live parasite in hanging drop preparation showed
an active wriggling movement amongst the blood
corpuscles. Out of the infected fish, 6.4% were
moderately infected (parasitaemia 5-12.4
Trypanosomes/100 RBCs), the remaining had scant
infection (parasitaemia 1-5 Trypanosomes/100
RBCs). In stained smears, two forms of the parasite
could be differentiated : minuta and magna.
Kinetoplast: Well developed structure and takes deep
stain upon fixation. Shape may be conical or spherical.
Division of the kinetoplast not observed.
Flagellum and undulating membrane: Flagellum
originates from kinetoplast, runs anteriorly bordering
the undulating membrane and finally extends beyond
body as a free flagellum. Flagellum well-developed in
larger forms than smaller forms. Undulating
membrane conspicuous and takes a light stain. It
extends throughout length of cell body. In smaller
forms, it has about 1-2 folds, whereas in larger forms it
has 1-3 folds. Width of undulating membrane greater
in larger forms.
Indices
FI
KI
NI
=
=
=
1.92
1.72
1.62
DISCUSSION
The genus Channa (=Ophiocephalus) has, from time
to time, been reported as a suitable host for protozoan
parasites (Haldar and Mukherjee, 1979). Various
trypanosome species have been put on record and a
comparison indicates that T. ophicephali, T. punctati
and T. elongatus are monomorphic forms and,
A
B
C
1. Morphology
Shape: Trypanomastigote elongated, anterior and
posterior ends tapering, the latter may assume a beaklike appearance. Configuration of parasite usually of
letter 'C', sometimes elongated 'S' and in rare cases,
almost a straight line. Posterior end extremity in both
(small and large) cases, more or less rounded.
Cytoplasm: Granular cytoplasm stains lightly. In the
smaller forms, the granules more concentrated in the
anterior region of the body commencing from just
anterior tip of the nucleus to approximately half the
distance from their commencement to the origin of the
flagellum. In large forms, granules occur along both
sides of nucleus.
Nucleus: Deep red stained nucleus oblong or beanshaped in middle of cell body, occupying almost entire
cell breadth.
D
E
F
G
H
J
I
K
10 µm
Fig. 1. A-D T. saulii n. sp. (A, B: minuta froms; C, D: magna
forms), E-K T. heteropneusti n. sp. (E, F: smaller forms; G, H:
larger forms; I-K: diving forms)
60
Gupta et al.
therefore, the present form is distinct from them. The
present species also can not be compared to T. striati,
T. channai and T. bareilliana which are polymorphic
species.
The dimorphic species recorded from C. punctatus are
T. gachuii and T. aligaricus (Table I) and the present
form warrants comparison with these species. A
critical examination indicates that the trypanosome
described herein is not comparable to both of these
species in the total body length (CF) as the present
form is a much smaller species, the larger form
approximates the smaller form of the above mentioned
species. Again, the length of the cell body of the
parasites is also not comparable as also the flagellar
length (FL). Moreover, the present form is also not
comparable with T. gachuii in breadth of cell body
(CB) although it may be comparable to T. aligaricus.
Interestingly, although the parasite is much smaller
than T. gachuii and T. aligaricus but the nuclear length
(NL) is comparable to T. aligaricus indicating a
relatively larger nuclear size in relation to its total
length. The kinetoplast dimensions of T. gachuii have
not been provided by the authors but the kinetoplast of
the present form is again comparatively larger than in
T. aligaricus. Apart from the above statistical
differences, the present parasite also differs from the
compared species in the granulation and vacuolation
pattern.
Although two other species have been described from
the same ecological niche, the present form cannot be
synchronized with them due to its dimorphic nature.
infected fish, 4.2% were moderately infected
(parasitaemia examined 5-10.5 trypanosomes/100
RBCs) and the remaining had scant infection
(parasitaemia 1-5 trypanosomes/100 RBCs). In
stained smears, the smaller and larger forms could be
clearly identified showing dimorphism.
MORPHOLOGY
Shape: 'C' or 'S' shaped body somewhat elongated but
usually takes twisted conformation upon fixation.
Cytoplasm: Lightly stained granular cytoplasm;
granules more prominent in posterior part of parasite.
Vacuoles were absent.
Nucleus: Deeply stained oval or bean-shaped nucleus
situated in the middle of body. Nucleolus not
observed.
Kinetoplast: Oval-shaped kinetoplast posteriorly
placed, takes a deep stain upon fixation.
Flagellum and undulating membrane: Flagellum
originates from kinetoplast. Free-flagellum
moderately well developed. Light pink stained
undulating membrane has 2-4 folds; 1-2 major folds
may also be present.
mi
Ia
sm
mg
The above discussion indicates that the present form is
different from any species reported either from the
same ecological niche or elsewhere. It is, therefore,
proposed that based on the geographical location,
morphometrics, cytological pecularities and host
status, the species discovered from the blood of C.
punctatus collected from Bareilly region, India,
should be given a new species status and the name T.
saulii n. sp is proposed.
Trypanosoma heteropneusti n.sp. (Fig. 1 E-H;
Table II)
Host
Locality
Percentage of
infection/intensity
- Heteropneustes fossilis
- Local fish market, Bareilly
- 14.6%/1-10.5
Trypanosomes/100 RBCs
(n = 163)
The live parasites showed an active wriggling
movement amongst the blood corpuscles. Out of the
E
Fig. 2. Photomicrographs of Trypanosoma. A. T. saulii n. sp. (miminuta form; mg - magna form), B-F. T. heteropneusti n.sp. B.
dimorphic forms (sm-small form, la - large form), C.
differentiating nucleus, D. longitudinal kinetosome division, E
and F. migrating nuclei.
61
Two new species of Trypanosoma
Indices
FI
KI
NI
=
=
=
1.82
1.72
1.16
DISCUSSION
H. fossilis has been considered to be an ideal host for
trypanosomes. A dimorphic form, T. mukundi was
discovered by Raychaudhri and Misra (1973) from the
fresh waters of Kolkata, India existing as “slender”
and “stumpy” forms. Table II shows a comparison of
dimorphic parasites recorded from the test fish. T.
mukundi Raychaudhuri and Misra (1973) is a
dimorphic species and the present form warrants a
critical examination of the said species. The contour of
the parasites differ, the present form being “slender”
and “elongated”, whereas T. mukundi has been
reported as “slender” and “stumpy” form. The present
forms have therefore contrastingly been described as
“small” (23-30.5 µm) and “large” (33.1-43.8 µm)
forms. Although CL of both parasites are comparable
but conspicuous difference in CB and nuclear size are
evident. Moreover, the flagellar lengths of both the
parasites also differ. The granulation pattern and the
vacuolation of both the parasites cannot be treated at
par. It is, therefore, proposed that the parasite
discovered from the blood of H. fossilis collected from
the fresh waters of Bareilly be designated as a new
species and, accordingly, the name T. heteropneusti
n.sp is proposed to accommodate the parasite with the
specific characters as given in this account.
DIVISION IN T. heteropneusti N.SP.
Some forms undergoing division showed peculiar
features in the blood of H. fossilis. However, only two
fish out of 163 specimens exhibited multiplying
forms. Division commenced in the nucleus showing
well differentiated chromatin prior to division (Fig.
1I). The two centrally-placed, round to ovoid nuclei
and the single, undivided kinetoplast was placed
Table I: Dimorphic trypomastigotes from Channa (= Ophiocephalus; all measurements in µm)
Host Parasite
Author(s)
Component
parts/forms*
O. gachua
T. gachuii
Misra et al., 1973
C. punctatus
T. aligaricus
Gupta and Jairajpuri, 1982
C. punctatus
T. saulii : n.sp (N=50)
Present study
Short
Slender
Small
Large
Minuta
Magna
CF
36.4
53.9
34.37
(34.25-35.5)
54.68
(53.25-55.3)
24.0
(18.0-33.0)
35.5
(30.2-52.0)
CL
24.9
42.1
23.50
(23.01-25.0)
46.18
(44.95-46.8)
18.5
(12.2-24.4)
28.2
(26.3-36.4)
CB
2.3
3.9
1.65
(1.5-1.8)
2.45
(2.1-2.6)
1.9
(1.0-2.5)
2.6
(2.0-3.6)
FL
11.1
10.9
10.87
(10.25-13.5)
8.50
(7.82-9.4)
6.6
(4.0-8.2)
8.8
(8.0-12.0)
NL
2.4
2.7
3.00
(2.5-3.5)
3.73
(3.6-3.8)
3.2
(1.0-6.0)
3.7
(1.0-6.0)
NB
2.2
2.7
1.12
(1.0-1.2)
2.02
(1.8-2.2)
1.6
(1.0-3.0)
2.0
(1.0-4.0)
KB
-
-
0.45
(0.4-0.5)
0.25
(0.21-0.3)
1.2
(0.5-2.0)
1.25
(0.5-2.0)
KL
-
-
10.5
(0.5-1.6)
0.51
(0.4-0.6)
1.6
(1.0-2.0)
1.9
(0.5-2.0)
* CB= Breadth of cell body; CF = Total length; CL = Length of cell body; FL = Flagellar length;
KB = Kinetoplast breadth; KL = Kinetoplast length, NB = Nuclear breadth; NL = Nuclear length
62
Gupta et al.
Table II: Dimorphic trypomastigotes from Heteropneustes fossilis (all measurements in µm)
Parasite
Author(s)
Locality
Component
Parts/forms
T. mukundi
Raychaudhuri and Misra
1973, India
T. heteropneusti n.sp
Present study
India (n = 50)
Slender
Stumpy
Small
Large
CF
32.5-43.5
22.0-28.0
27.9
(23.5-30.5)
37.83
(33.1-43.8)
CL
24.0-28.5
18.0-23.5
16.7
(14.4-19.0)
22.6
(20.1-24.2)
CB
2.0-2.5
1.9-2.5
1.68
(1.45-1.95)
1.40
1.3-1.5)
FL
9.0-15.5
4.0-6.5
8.48
(6.95-11.4)
16.05
(12.05-18.6)
NL
2.25-3.75
2.25-3.75
2.64
(1.41-3.35)
2.63
(1.51-3.30)
NB
1.75-2.25
1.75-2.25
1.14
(1.1-1.2)
1.23
(0.95-1.25)
KB
0.75-7.5
0.75
0.93
(0.90-0.95)
0.90
(0.8-0.95)
KL
-
-
1.18
(1.07-1.35)
1.16
(0.8-1.35)
* Abbreviations same as in Table I
posteriorly with a single, long free-flagellum and a
normal undulating membrane (Fig. 1J). The
kinetoplast usually divided subsequent to nuclear
division but in some instances, it was the first to
divide. Interestingly, both longitudinal as well as
transverse division occurred during the division of
kinetoplast although the former was more frequent
(Fig. 2D). The two nuclei now migrated, one in either
arm (Fig. 2E) and the parasite stretched between the
two kinetoplasts prior to cytokinesis (Fig. 2F). One
abnormal trypanosome with three kinetoplasts (Fig.
1K), dividing prior to nuclear division, could also be
observed which does not fit into the sequence of
divisional events of trypanosome multiplication and is
apparently an aberrant form.
Division in trypanosomes has been reported rarely.
Laveran and Mesnil (1907) reported absence of
division in T. remarki but occasionally observed forms
with two nuclei in the parva variety. T. danilewskyi
divided at 20° C satisfactorily in gold fish but the
process slowed down by increasing (30° C) or
decreasing (10° C) the temperature (Lom, 1973; Woo
et al., 1983; Islam and Woo 1992). Qadri (1955), Daly
and Degiusti (1971), Khan (1972), Grogl et al. (1980)
and Mukerjee and Haldar (1982) reported the absence
of divisional stages in the fish blood. Longitudinal
binary fission in culture form of T. striati (Qadri,
1955); binucleated trypomastigotes in T. batrachi
Qadri (1962), T. maguri and T. vittati (Tandon and
Joshi, 1973), T. mrigali (Joshi, 1976) and T. attii
(Gupta and Jairajpuri, 1982b) have also been
observed.
Two small trypanosomes possessing two nuclei and
two kinetoplasts (Baker, 1960), and two opposite
nuclei and a central kinetoplast (Raychaudhuri and
Misra, 1973) have also been put on record. Wenyon
(1926) and Lewis and Ball (1981) reported kinetoplast
division prior to nuclear, whereas Burreson (1982)
recorded a reverse condition in Trypanoplasma
bullocki. In T. heteropneusti n.sp, both instances have
Two new species of Trypanosoma
been recorded but prior nuclear division dominates
prior kinetoplast division. The nuclear division in our
specimens indicate only the transverse division. The
cytokinesis and flagellar division were not observed.
ACKNOWLEDGEMENT
Thanks are expressed to the University Grant
Commission, New Delhi, India, for providing
financial grant in the form of a research project.
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blood of freshwater fish in East Africa. Parasitology. 50 :
515 - 526.
Burreson EM. 1982. The life cycle of Trypanoplasma bullocki
(Zoomastigophorea: Kinetoplastida). Journal of
Protozoology. 29 : 72 - 77.
Castellani A and Willey A. 1905. Observations on haematozoa
in Ceylon. Quarternary Journal of Microscopic Science. 49 :
383 - 402.
Daly JJ and Degiusti DL. 1971. Trypanosoma catostomi n. sp.
from the white sucker Catostomus C. commersoni. Journal
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Grogl M, Marinerlle CJ, Suarez MF, Sanchez NH and Guhl F.
1980. Trypanosoma magdalenae n. sp. (Protozoa:
Kinetoplastida) from a fresh water teleost, Petenia kraussii
in Columbia. Journal of Parasitology. 66 : 1022 - 1026.
Gupta DK and Gupta N. 1994. A comparative study of culture
media for Trypanosoma kargenensis n. sp. from
Heteropneustes fossilis and its effect on the biochemical
composition of fish. In: Proc. Third Asian Fish. Forum.
Chou et al. (Eds) A. F. S. Philippines. pp320-323.
Gupta DK, Gupta N and Yadav P. 1987. New polymorphic
trypanosomes from two freshwater fishes. Himalayan
Journal of Environment and Zoology. 1 : 93 - 97.
Gupta N, Gupta DK and Saraswat H. 2001. Hypoglycemia in
Heteropneustes fossilis parasitized by two new species of
parasites Trypanosoma karelensis n.sp and Myxosoma
fossilii. n.sp. In: Proceedings of the National Symposium on
Fish Health, Management and Sustainable
Aquaculture.pp179-186.
Gupta N and Jairajpuri DS. 1981. Trypanosoma singhii n. sp.
from a fresh water teleost, Heteropneustes fossilis. Indian
Journal of Parasitology. 5 : 251 - 253.
Gupta N and Jairajpuri DS. 1982a. Trypanosoma aligaricus n.
sp. from the freshwater murrel, Ophiocephalus punctatus
Bloch. Archiv fur Protistenkunde. 125 : 109 - 114.
Gupta N and Jairajpuri DS. 1982b. A new polymorphic
trypanosome from an Indian freshwater fish Wallago attu
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Gupta N and Saraswat H. 1991. Trypanosoma rohilkhandae n.
sp. from fresh water teleost fish Channa (= Ophiocephalus)
punctatus. Himalayan Journal of Environment and Zoology.
5 : 29 -33.
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Haldar DP and Mukherjee M. 1979. An annolated list of
protozoan parasites from Ophicephalus punctatus Bloch, a
common food - fish. Archiv fur Protistenkunde. 121 : 392 400.
Hasan R and Qasim SZ. 1962. Trypanosoma punctati n. sp.
from the fish Ophiocephalus punctatus Bloch a common
fresh water murrel of India. Zeitschrift fur Parasitenkunde.
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Islam AKMN and Woo PTK. 1992. Effects of temperature on
the in vivo and in vitro multiplication of Trypanosoma
danilewskyi, Laveran and Mesnil. Folia Parasitologica. 39 :
1 - 12.
Joshi BD. 1976. Two new species of trypanosomes from fresh
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Khan RA. 1972. On a Trypanosoma from the Atlantic cod,
Gadus morhua. Canadian Journal of Zoology. 50 : 1051 1054.
Laveran A and
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1907. Trypanosomes and
Trypanosomiasis. Ist Ed. Bailliere, Tindall and Cox
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Lewis JW and Ball SJ. 1981. Observation on the division of
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Misra KK, Chandra AK and Choudhury A. 1973. Trypanosoma
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trypanosomes from India. Archiv fur Protistenkunde. 115 :
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of blood of freshwater fishes infected with two new forms of
trypanosomes. Zeitchrift fur Wissen schaftliche Zoologie.
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Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 64–67
J PD
Impact of anthelmintic therapy on live weight gain in
gastrointestinal nematode-infected goats*
A. K. Jayraw and Y. V. Raote
Department of Parasitology, College of Veterinary and Animal Sciences, Parbhani.
ABSTRACT. A trial was conducted to assess the effect of anthelmintic therapy on live weight gain
in gastrointestinal (GI) nematode-infected goats using three anthelmintics, viz. tetramisole
hydrochloride, morantel citrate and albendazole. Twenty four goats with heavy natural infection of
Strongyle, Strongyloides papillosus and Trichuris spp. were selected and divided into four groups of
six animals each. Group-I served as untreated control group, whereas Group-II, III and IV were
treated with tetramisole hydrochloride @ 15 mg kg-1 body weight (b.w.), morantel citrate @ 5.94 mg
kg-1 b.w. and albendazole @ 7.5 mg kg-1 b.w., respectively. Animals were weighed at weekly intervals
and their live weight was recorded for a period of 28 days post-treatment (PT). On 28 day PT,
overall live weight gain recorded was 9, 7.44 and 8.78% in animals of Group-II, III and IV,
respectively, whereas only 3.73% weight gain was recorded in goats of untreated control group. The
net profit obtained by the end of experimental study was Rs. 206, 138 and 174 in animals of GroupII, III and IV, respectively, as against a profit of Rs. 94 obtained in animals of untreated control
group.
Keywords: anthelmintic therapy, gastrointestinal nematode, goat, weight gain
INTRODUCTION
Owing to the growing demand for high quality animal
proteins for human consumption, small ruminants
occupy a special place as they are extremely efficient
in converting the indigestible cellulose and
hemicellulose to animal protein. India has a
population of 120 million goats, which contribute
greatly to rural economy (Abraham, 2001). Control of
gastrointestinal (GI) nematodosis is one of the most
serious challenges, as it poses a major constraint in the
growth of highly profitable animals due to the chronic
and insidious nature of parasitism (Sanyal, 1996). In
*Part of M.V. Sc. thesis by first author, Marthwada Agricultural
University, Prabhani.
Corresponding author: Dr. A. K. Jayraw, Department of
Parasitology, Nagpur Veterinary College, Nagpur-440 006,
Maharashtra, India.
the absence of a potent prophylactic agent,
chemotherapy continues to play a vital role against GI
nematodosis. Therefore, the present investigation was
undertaken to evaluate the live weight gain response in
goats following chemotherapy with commonly
available anthelmintics, viz. tetramisole
hydrochloride, morantel citrate and albendazole.
MATERIALS AND METHODS
The experimental study was conducted at the College
of Veterinary and Animal Sciences, Parbhani during
monsoon. A total of 49 female goats were maintained
under a semi-intensive system and each animal was
provided with 250 g concentrate daily. The helminthic
burden of each animal was ascertained after counting
eggs per gram (EPG) of faeces using Stoll's dilution
method (Soulsby, 1982). Out of 49 goats, 1-2 year old
naturally infected 24 animals were selected, showing
Effect of antihelmenthic therapy on live weight gain
the mean EPG counts of 3783 ± 1106.96; 404.16 ±
127.54 and 212.5 ± 55.01 for strongyle species, S.
papillosus and Trichuris spp., respectively. The
experimental animals were equally divided into four
groups (Gr.), where, Gr.-I served as infected, untreated
control group, Gr.-II animals were treated with
tetramisole hydrochloride (Nilverm soluble powder,
M/S ICI India Ltd., Kolkata) @ 15 mg kg-1 body
weight (b.w.) and Gr.-III animals with morantel citrate
(Banminth Tab., M/S Pfizer Ltd., Ahmedabad) @ 5.94
mg kg-1 b.w. The animals in Gr.-IV were drenched with
albendazole (Albomar suspension, M/S Glaxo India
Ltd., Mumbai) @ 7.5 mg kg-1 b.w. The experimental
goats were denied access to water and feed for
approximately 18 h, prior to recording their live
weight (weekly) using a weigh-bridge. The net profit
in terms of rupees (Rs.) was calculated taking into
account the final difference in body weight of the
experimental goats before and after chemotherapy,
cost of medication including cost of drug (tetramisole
hydrochloride - Rs. 10, morantel citrate - Rs. 29 and
albendazole - Rs. 24) and labour cost (Rs. 60 per head
for one day), except for the animals belonging to Gr.-I,
which served as infected untreated control and were
not treated with any anthelmintic. The average rate of
chevon was considered as Rs. 120 kg-1 at Parbhani
district of Maharashtra. The data obtained were
analysed following standard statistical procedures
(Snedecor and Cochran, 1994).
RESULTS
Animals were weighed at weekly intervals and their
live weight recorded for a period of 28 days. The mean
EPG counts recorded at 28 days post-treatment were
found to be 345.84 ± 290.47, 4.165 ± 4.165 and 154.16
± 37.50 for strongyle species, S. papillosus and
Trichuris spp., respectively. A maximum of 9% (mean
1.97 kg) live weight gain was recorded in tetramisole
hydrochloride treated animals (Gr.-II) followed by
8.78% enhancement (mean 1.82 kg) in experimental
goats belonging to albendazole treated group (Gr.-IV).
The morantel citrate treated group (Gr.-III) exhibited
7.44% weight gain (mean 1.56 kg). The lowest weight
gain of 3.73% (mean 0.79 kg) was evident in animals
of infected untreated group (Gr.-I). The mean daily
weight gain recorded is presented in the descending
order as 70 g day-1 in animals of Gr.-II, 65 g day-1 in Gr.IV, 55 g day-1 in Gr.-III and 28 g day-1 in animals of Gr.I. The net profit obtained at the end of experimental
study was higher (Rs. 206) in tetramisole
hydrochloride treated group, when compared with
65
albendazole (Rs.174) and morantel citrate (Rs. 138)
treated groups. On the other hand, the net profit
recorded in infected untreated control group was
Rs. 94 in comparison with the treated groups (Table I).
DISCUSSION
GI nematode infection is recognized as a major
constraint to small ruminant production worldwide. It
is well known to cause huge economic losses due to
mortality incidental to the severe parasitism and high
morbidity, reduction and/or spoilage of wool, meat
and milk production in chronic cases (Chowdhury,
1994). The continued presence of GI nematodes is
responsible for anorexia, reduced feed intake,
alterations in protein metabolism, low levels of
minerals, depressed activity of some intestinal
enzymes and diarrhoea (Soulsby, 1982). The
parasitism is also associated with loss of blood and
plasma proteins into the GI tract (Jayraw and Raote,
2004a) thereby lowering the weight gain in untreated
control group. Reduced skeletal growth brought about
by mineral deficiency affects growth rate, because
skeletal size ultimately determines the capacity of
growing animal to accumulate muscle (Sykes et al.,
1977). In addition, reduced levels of amino acid
incorporation in muscle protein results in reduced
weight gains (Soulsby,1982). Reduced weight gain in
GI nematode-infected goats was also reported by
Howlader et al. (1997) and Githigia et al. (2001). The
higher weight gain recorded in tetramisole treated
animals is attributable to its higher efficacy against
Trichuris spp. and strongyle species when compared
to efficacy of albendazole and morantel citrate against
these parasites (Jayraw and Raote, 2004b). The
present finding is in agreement with Sakhawat et al.
(1997), who also observed that levamisole treated
sheep gained more weight than albendazole and
morantel treated animals. The mean weight gain of
1.97 kg recorded in the present study corroborates well
with the findings of Sathianesan and Peter (1972), who
recorded 1.7 kg weight gain in tetramisole treated
goats over 30 days of observation. The 8.78% weight
gain observed in albendazole-treated animals is in
general agreement with Faizal et al. (1999) and
Githigia et al. (2001). Rajangam and Balchandran
(1989) earlier reported a mean weight gain of 38.3 g
day-1 in morantel citrate treated goats.
Correspondingly, a mean daily weight gain of 55 g
day-1 in morantel treated group was also evident in the
present study. Das et al. (2004) recorded the per head
loss of Rs. 351 and 377 in 3-6 and 6-12 months old GI
II
III
IV
2
3
4
21.05
±3.09
21.36
±2.96
21.73
±3.07
21.57
±2.4
22.79
±2.07
21.56
±3.45
II
week
22.09
±3.16
21.95
±2.4
23.26
±1.89
22.05
±3.44
III
week
22.54
±2.97
22.51
±2.18
23.84
±2.21
21.95
±3.21
IV
week
1.82
1.56
1.97
0.79
Mean
weight
gain (kg)
8.78
7.44
9.00
3.73
Percent
weight
gain
65
55
70
28
218
187
236
94
44
49
30
-
174
138
206
94
Gross profit
Cost of
Net profit
Mean
(Rs.) following medication
(Rs.)
daily
chemotherapy
following
weight
(except Gr.-I)
chemotherapy
gain
(except Gr.-I)
(g day-1)
Gr.-I: infected untreated control; Gr.-II: treated with tetramisole hydrochloride; Gr.-III: treated with morantel citrate and G r.-IV: treated with
albendazole
20.72
±3.08
20.95
±2.83
21.87
±1.85
22.25
±1.89
21.38
±3.7
I
1
21.18
±4.01
I
week
Sl. Group 0 day
No.
Table I. Effect of anthelmintic medication on live weight gain in natural GI nematode infection in goats
66
Jayraw and Raote
Effect of antihelmenthic therapy on live weight gain
nematode-infected Bengal goats, respectively, over a
period of nine months. Our findings are also consistent
with the findings of above workers, as a net gain of Rs.
206 was recoded in tetramisole treated group followed
by Rs. 174 and 138 in albendazole and morantel citrate
treated groups, respectively. However, the net profit
recorded in infected untreated control group was only
Rs.94, which may be attributed to the worm burden,
resulting in weight loss. The effective elimination of
GI nematodes in all treated groups has a direct bearing
on the improved feed utilization ratio and gain in body
weight by preventing blood, as well as, protein loss
into the GI tract. Tetramisole hydrochloride appears to
be superior to albendazole and morantel citrate in
effective elimination of GI nematodes and subsequent
improvement in weight gain.
67
goats in Kenya. Small Ruminant Research 42: 21-29.
Howlader M M R, Capitan S S, Eduardo S L, Roxas N P and
Sevilla C C.1997. Performance of growing goats
experimentally infected with stomach worm (Haemonchus
contortus). Asian Australasian Journal of Animal Sciences
10: 534-539.
Jayraw A K and Raote Y V. 2004a Effect of anthelmintic
treatment against gastrointestinal nematodes with a note on
haematology and plasma proteins in goats. Journal of
Veterinary Parasitology 18: 51-54.
Jayraw A K and Raote Y V. 2004b Comparative efficacy of
tetramisole hydrochloride, morantel citrate and albendazole
against gastrointestinal nematodes in goats. Journal of
Veterinary Parasitology 18: 131-134.
ACKNOWLEDGEMENTS
Rajangam R K and Balchandran S. 1989. Efficacy of morantel
citrate (Banminth- Pfizer) against gastrointestinal parasites
and its effect on body weight gain in stall-fed goats. Indian
Veterinary Journal 66: 919 -922.
The authors are thankful to the Associate Dean,
College of Veterinary and Animal sciences, Parbhani
and Farm Superintendent for providing necessary
facilities to do this work.
Sakhawat A, Anwar A H, Hayat B, Zafar I and Hayat C S. 1997.
Field evaluation of anthelmintic efficacy of levamisole,
albendazole, ivermectin and morantel tartrate against
gastrointestinal nematodes of sheep. Pakistan Veterinary
Journal 17: 114-116.
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Sykes A R, Coop R L and Angus K W. 1977. The influence of
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growing sheep. Journal of Comparative Pathology 87:521529.
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J PD
Ultrastructure, differential density and distribution
pattern of polymorphic microtriches in tegument of
Stilesia globipunctata infecting Ovis aries (sheep)
C. Venkatesh, K. Ramalingam and V. Vijayalakshmi
P.G. and Research Department of Zoology, Govt. Arts College, Chennai.
ABSTRACT. Scanning electron and transmission electron microscopic study of tegument of
Stilesia globipunctata revealed a complex pattern of microtrichial brush border. They show a wide
range of morphological variations. S. globipunctata shows species specific pattern of papilliform,
filiform, spiniform and blade-like microtriches. The scolex shows the presence of papilla-like,
spine-like and blade-like microtriches. The immature proglottid region is densely covered by
posteriorly directed filiform, spiniform and papilliform microtriches. In mature proglottid region,
papilla-like and filament-like microtriches are predominantly seen. The microtriches appear to be
non-uniform in density and size. In gravid segments, there is obviously an increasing degree of
disorder in the arrangement of microtriches. It increases the absorptive surface. It helps to resist
the peristaltic movement of intestine and maintains the parasite in position. Its absence in the
gravid region is due to the morphological changes in the tegument and the interaction of luminal
environment.
Keywords: density, distribution, microtriches, polymorphism, Stilesia globipunctata
INTRODUCTION
The external surface organization of the cestode
parasite not only confers protection to the parasite, but
also functions as a metabolically active tissue taking
up nutrients from the luminal environment and as an
outlet for elimination of its extracellular secretions
and the excretion phenomenon. It has been noticed
that in cestodes, the basic structure may be compared
with a gut turned inside out, with the external body
covering serving the absorptive functions normally
associated with the intestinal mucosa (Smyth and
McManus, 1989).
Corresponding author: Dr. V. Vijaylakshmi, P. G. and Research
Department of Zoology, Govt. Arts college, Men (Autonomous),
Nandanam, Chennai - 600 035, Tamil Nadu, India.
Ultrastructural studies have shown that the external
body covering of the adult tapeworm consists of a
naked cytoplasmic layer. There is no resistant cuticle.
This outer layer is a syncytium with no partitions of
cell walls. It is referred to as a tegument distinguishing
it from non-living cuticles of the type that occur in
nematode species. Before discussing the functional
morphology of the tegument, it is essential to have
some understanding of the structural properties of
absorptive surfaces in general. The structure,
physiology and biochemistry of the tegument are,
therefore, of fundamental importance in
understanding cestode physiology as a whole.
In cestodes, the tegument grows, not by cell division,
but by recruitment. Cells form the parenchyma come
up and attach to the tegument and vesicles pass up into
the tegument. However, the amplification of the
69
Ultrastructure of microtriches in Stilesia globipunctata
surface is achieved by the presence of delicate
cytoplasmic extensions called microtriches,
reminiscent of mucosal cell microvilli. The
microtriches on the tapeworm surface increase the
surface area of the parasite by about 20-times. The
most dominant feature of the cestode tegument is the
covering by microtriches, which are thought to be
responsible for nutrition and protection and possibly
also the mechanical functions of anchoring and
traction. They show a wide renge of morphlogical
variations.
Microtriches are unique in the cestodes and are
evident on the tegument and other epithelia that open
into the tegument. Microtriches have been widely
repeated among all major orders of Eucestoda. These
are present on the scolices and strobila of
representatives of most groups that have been
examined with SEM. Papilliform, spiniform and
filiform structures have been variously reported
among the Pseudophyllidea (Anderson, 1975,
Granath et al., 1983) Cyclophyllidea (Berger and
Mettrick, 1971; Ubelaker et al., 1973; Sampathkumar,
2001; Vijayalakshmi, 2001; Radha, 2003).
In the present study the characterization of
microtrichial structure, density, distribution and their
functional significance in S. globipunctata has been
attempted as the species of cestode has so far not been
exclusively studied due to its inconspicuousness in the
gut of sheep.
MATERIALS AND METHODS
Collection of tapeworms: The tapeworm S.
globipunctata (Rivolta, 1874) were collected from the
intestine of naturally infacted sheep autopsied in the
slaughterhouse at Perambur, Chennai. The sheep
intestines were transported to the laboratory within
half an hour of collection. In the laboratory, each
intestine was carefully dissected and the tapeworms
were collected. Then the worms were washed in
distilled water to render them free from intestinal
contents and rinsed quickly 3-4 times in normal saline.
The tapeworms were then observed through a
compound microscope to confirm their taxonomic
characters. The entire worm was spread out on a board
and the length was measured.The immature, mature
and gravid proglottid region of the worm was located
and separated as follows and dried on moist blotting
paper and used for scanning and transmission electron
microscopic studies.
a.
Immature proglottides containing scolex and
anterior region.
b.
Mature proglottides with functional
reproductive organs.
c.
Gravid proglottides containing eggs.
Scanning electron microscopic study : The scanning
electron microscopic (SEM) studies of the soclex,
immature, mature and gravid proglottides of S.
globipunctata were undertaken to understand its
ultrastructure. For this purpose, the specimens were
dissected in chilled glutaraldehyde (2.5%) and fixed
for 16 h at 4° C. The tissues were subsequently washed
thrice at an interval of 15 min each in phosphate buffer
at pH 7.0 and then dehydrated by passing through an
ascending series of alcohol from 30 to 100% an h in
each concentration. They were ultimately kept in
100% alcohol overnight. Following dehydration, the
tissues were air-dried in a desiccator for 7 to 10 days.
The dried samples were mounted on an aluminium
stub and gold sputtered in vacuum for 10 min, using an
Eiko IB-2 ion coater. The samples were observed
eventually on a Hitachi, S-415A scanning electron
microscope, scanned at 25 KV and micrographed at
different magnifications (Hayat, 1977).
Transmission electron microscopic study: The
scolex, mature and gravid proglottid regions of S.
globipunctata for transmission electron microscopy
(TEM) were immersed in 2.5% glutaraldehyde in
Millong's phosphate buffer (pH 7.3, 380 mOsm/L),
where they were diced into small pieces. After 3-4h
fixation at room temperature, the tissue was rinsed in
Millong's buffer. The tissue was then post-fixed in 1%
osmium tetroxide in Millong's buffer for 1.5 h, rinsed
quickly in distilled weter, dehydrated in an ethanol
series, infiltrated with propylene oxide, embedded in
Spurr's low-viscosity epoxy resin and polymerized at
60° C. Thin sections were cut at 70-90 nm with a
diamond knife, mounted on uncoated copper grids,
stained with uranyl acetate/ethanol and aqueous lead
citrate, and examined under a Philips 204 TEM at an
accelerating voltage of 40 or 60 kV (Conn, 1993).
RESULTS
SEM observations: Specimens of S. globipunctata
were found to have species-specific patterns of
papilliform, filiform, spiniform and blade-like
microtriches that are restricted to particular regions of
scolex and strobila. The central to peripheral regions
of the scolex are covered with papilliform and filiform
70
Venkatesh, Ramalingam and Vijaylakshmi
structures. The adherent surfaces of the sucker and
their cavities have a dense uniformly distributed
covering of spiniform and blade-like microtriches of
consistent structure (Fig. 1a). The tegument on the
margins and outer surface of the sucker is densely
covered with relatively long blade-like microtriches.
Similarly, different types of microtriches can also be
seen in neck and strobila. The microtriches seem to be
directed posteriorly. Microtriches in the center of
scolex measure about 2.0 µm and in the sucker region,
it measures 2.1µm.
The immature proglottid region is also densely
covered by posteriorly directed filiform, spiniform
and papilliform microtriches (Fig.1b). But the
microtriches are not distributed uniformly. This trend
continues as segments become older. The
polymorphic nature of the microtriches increases as
the segments become older. The mature proglottid
regions of S.globipunctata clearly reveal the decrease
in density and non-uniformity of microtriches
(Fig.1c). On gravid segments, however, there is
obviously an increasing degree of disorder in the
arrangement of microtriches (Fig.1d). The surface of
the posterior most part in the majority of worms
examined is in a stage of dissolution with on
microtriches being apparent. Thus the scanning
electron micrographs of the microtriches and their
distribution in the tegument of scolex, immature and
mature strobilar regions of the parasite revealed the
polymorphic nature of the microtriches.
Mt
Sb
Mt
(a)
(b)
Tf
Mt
Mt
(c)
(d)
Fig. 1. Scanning electron micrographs of scolex, immature, mature and gravid regions showing microtriches of S. globipunctata.
a. Adherent surfaces of sucker showing spiniform and blade-like microtriches (x80).
b. Tegumental surface of the immature region showing filiform, spiniform and papilliform microtriches (x15).
c. Mature region showing posteriorly directed microtriches (with less density; x25).
d. Microtriches of gravid region showing non-uniform and less density (x15).
Mt - Microtriches, Sb - segental boundary, Tf - tegumental folding.
71
Ultrastructure of microtriches in Stilesia globipunctata
TEM observations: In addition to the light and SEM
findings, ultrastructural observations were made by
TEM. The TEM picture of scolex (Fig. 2a-c) shows the
presence of papilla-like, spine-like and blade-like
microtriches. The microtriches may be divided into
three anatomical regions (Fig. 2b) viz. 1)
microfilament containing a base, 2) a dense cap and 3)
a complex junctional region between the base and cap.
Each base is found to contain an inner sleeve of dense
material, the core tunic. Distally, the core is found
connected individually to slightly curved tubule, the
junctional tubule.
As observed in SEM pictures, posteriorly directed
filament-like, spine-like and papilla-like microtriches
can be seen in the immature proglottid region (Fig.
2d). Whereas in the mature proglottid region, papillalike and filament-like microtriches are predominantly
seen (Fig. 3a). The microtriches appear to be nonuniform in density and size. A decreased microtrichial
density down the length of strobila and morphological
changes in the tegumental surface of the gravid
segments can be clearly observed (Fig. 3b). This
picture clearly reveals the stages of disintegration of
microtriches. Such changes involve surface
sculpturing accompanied by loss of all microtriches
and erosion of folds in the posterior region of the
parasite (Fig. 3c). The disintegrated microtriches can
also be seen in Fig. 3d.
Cp
Cr
Cp
Jr
Jr
B
Gx
Spl
B
(a)
Gx
Ct
Spl
(b)
Bm
Mt
Mt
Gx
Spl
(c)
(d)
Fig. 2. Transmission electron micrographs of tegument brush border of scolex and immature regions of S. globipunctata.
a. T.S. of the tegument showing different kinds of microtriches (x45,000).
b. Higher magnification of architecture of blade like mictothrix (x1,000,000).
c. L.S. of the margin of suckers showing microtriches (x7000).
d. Brush border of tegumental folds of immature region showing different types of microtriches (x20,000).
B - Base, Bm - basement membrance, Cp - cap, Ct - core tunic, Gx- glycocalyx, Jr - junctional region, Mt - microtriches, spl - sub
plasmalemmal layer.
72
Venkatesh, Ramalingam and Vijaylakshmi
Bm
Spl
Spl
(a)
(b)
Db
Mt
Spl
(c)
(d)
Fig. 3. Transmission electron micrographs of tegument brush border of mature and gravid regions of S. globipunctata.
a. Tegument under higher magnification of mature proglottid region showing different kinds of microtriches (x30,000).
b. Tegument (T.S.) of gravid region showing the dissolution of microtriches (x20,000).
c. L.S. of tegument of the gravid region showing smooth margin with complete lack of microtriches (x20,000).
d. Transmission electron micrographs showing disintegrated microtriches (x70,000).
Bm - basement membrane, Db - dense bodies, Mt - microtriches, Spl - subplasmalemmal layer.
DISCUSSION
The highly active outer surface of cestodes viz. the
tegument is a multifunctional entity serving for
absorption, digestion, protection, excretion
(Featherston, 1972; Thompson et al., 1980; Hayunga,
1991), anchoring (Rothman, 1963; Morseth, 1966;
Thompson et al., 1980) and traction for locomotion
(Rothman, 1963; 1966; Berger and Mattrick, 1971).
At the parasite host interface, it additionally serves for
chemical and tactile reception (Featherston, 1972;
Webb and Devey, 1974; Jones, 1988; Hass and
Guggenheim, 1977; Granth et al., 1983). Although
the tegument contains specific systems for the
transport of molecules and ions, especially the amino
acids, hexose sugars, vitamins, purnes, pyrimidines,
nucleotides and lipids, it also serves a number of other
vital functions as suggested by Podesta (1980): (a) it is
a major site of catalytic activity and contains enzymes
of parasite and possibly of host origin, (b) it may be a
site of volume regulation, (c) it serves a protective
function both against the host's digestive enzymes and
the host's immune reactions, (d) it may also function as
a site of matabolic transfer (Posdesta, 1982; Pappas,
1983; Threadgold, 1984).
Such diverse functions necessitate a high degree of
morphological specialization. Jones (1998) has
described three features for cestode epithelia, namely,
the occurrence of regional specialisation, microtriches
and secretory components. In addition he has also
suggested that some cestode epithelia are involved in
Ultrastructure of microtriches in Stilesia globipunctata
developmental processes such as the nurture of
embroyos and maintnance of surrounding tissues.
The microtriches of cestodes are more complex than
mammalian microvilli. An excellent model of their
structure is given by Holy and Oaks (1986). TEM
studies have, however, revealed that all cestode
species hitherto examined possess microtriches and
that they are probably of universal occurrence
(Morseth, 1966; Yamane, 1986). However, there are
differences in shape and density of microtriches
between larvae and adult worms (Yamane, 1968).
Novak and Dowsett (1983) have observed that during
asexual reproduction of T. crassiceps the metacestode
tegumental microtriches differentiate into at least
three morphologically distinct types. Berger and
Mettrick (1971) have described the size, shape and
number of microtriches in different parts of the worm
of three Hymenolepis species. The study of Anderson
(1975) shows that in Diphyllobothrium dendritucum
and D. ditremum there is a change in the shape and
length of microtriches during development from
plerocercoid to adult worms. In H. diminuta,
microtriches have been quoted as having maximum
diameter of 0.14-0.19 µm and maximum length of 0.91.08 µm (Threadgold, 1984).
The presence of microtriches in S. globipunctata has
been established in the present study by SEM and
TEM. These ordered structures have been linked by
many authors to mictovilli of mammalian brush
border (Read, 1955; Lumsden and Specian, 1980).
There is not only morphological but also functional
resemblance, as the parasite absorbs nutrients of low
molecular weight through its body surface. In addition
to the absorptive functions, the parasite epithlium
performs the function of body protection (Odland,
1966). This protective function of the tegument is also
attributed to a complicated structure as suggested by
Jha and Smyth (1969). The higher level of
architectural complexity may reflect a more complex
level of function for these surface specialisations of
cestodes.
The comparison of microtriches among different
species thus reveals that the ubuquitous nature of
microtriches in all cestodes, which have occupied the
luminal niche of the lost animals also have evolved
structural and functional features of homology in these
different species. The above homology of tegumental
structure in different cestode species thus reveals that
the distribution microtriches and their further
73
polymorphic modification confers not only
advantages to the parasitic species, but also represents
as an adaptation for multiple parasitisms. The host's
immunological reactions would have also acted as a
limiting mechanism to the multiple parasite loads with
differential organization.
Although the scolex is generally regarded largely as an
organ of attachment, in some cestodes such as
Echinococcus sp., may also have a 'placental' function
and absorb nutrients directly form the mucosal wall, a
condition which occurs in some trematodes (Smyth
and Halton, 1983). The root like projection with
rootlets increases the nutritional surface. Such a
nutritional function has been widely reported by
Lumsden (1975a) and Lumsden and Hildreth (1983).
The microtriches covering the scolex region revealed
a structure different from that of the strobila, a
situation presumable related to the topography of the
host mucosa.
The present study revealed marked difference in the
distribution pattern and density of microtrichial
structures between scolex, and immature and mature
segments. The socolex revealed more dense
distribution and uniform distribution of microtriches.
The immature proglottid region showed dense and
non-uniform distribution of microtriches, whereas in
the mature region, the microtriches appeared to be
non-unitorf in density and size. A decreased
microtrichial density down the length of strobila has
been noticed.
The scolex has also revealed differential nature of the
microtriches. The above microtrichial complexity in
the scolex region is of parasitic sognificance as it
represents the anchoring region and nodular region in
the host tissue.
In this context, the species S. globipunctata differs
from other cestode specieses, which show a simple
anchoring device over the intestinal mucosal region of
the host. The deep association of the Stilesia sp. in
groups, forming nodular regions in the host mucosal
tissue is of parasitic importance. Such group
association may not have only adverse consequences
to the host but it is also difficult to eliminate such
group associations than individual parasites
anchoring in the host lumen.
Berger and Mettrick (1971) have described
polymorphism of microtciehes all along the stobilar
length. Braten (1968a) has indicated that microtriches
74
form an almost continuous covering of the worm.
Earlier ultrastructural studies of T. hydatigena
(Featherston, 1972) have revealed three different
types of microthrix each associated with a particular
area of the strobila. Jha and Smyth (1971) have
examined the rostellum of E. graunlosus and reported,
"The microtriches and their branches are curved in
various directions to form a criss-cross pattern". The
surface of the scolex of Silurotaenia siluri is covered
with filiform microtriches and giant spine-like and
blase-like microtriches. They are also present on the
neck region and posterior margins and internal
cavities of the suckers (Scholz et al., 1999). Caira and
Ruhnke (1991) noted substantial changes in the
pattern and distribution fo microtriches during
ontogeny of the scolex in Calliobothrium
verticillatum. Vijayalakshmi and Ramalingam (2005)
observed filament-like, blade-like and intermediate
types of microtriches on the tegument of A. lahorea by
using SEM and TEM studies.
The present SEM and TEM study also clearly revealed
the existence of microtrichial polymorphism all along
the strobilar length of S. globipunctata. It revealed a
complex pattern of microtrochial brush border
showing wide range of morphological variations.
Species specific pattern of papilliform, filiform,
spiniform and blade- like microtriches were also
observed in S. globipunctata.
In the light of the observations, results and discussions
of previous studies, the results on TEM studies on the
tegument of S. globipunctata thus infer that the
adhesive and absorptive microtriches of the tegument
not only allow the diffusion and intake of various
nutrients, micro/trace elements and electrolytes
indispensable for the growth of the parasites but afford
firm positions inside the intestinal cavity wall against
the immune factors of the host which could reject the
parasite's holdfast. The above dense distribution of
microtriches in the scolex region of S. globipunctata
also strengthens the above suggestion. Its absence in
the gravid region is due to the morphological changes
in the tegument and the interaction of luminal
environment.
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Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 76–80
J PD
The protozoan fauna living in the digestive system of
Periplaneta americana in Kolkata, West Bengal, India
J. Ghosh and A. Gayen
Post Graduate Department of Zoology, Maulana Azad College, Kolkata.
ABSTRACT. The protozoan fauna living in the digestive system of Periplaneta americana in Kolkata,
West Bengal, India, was studied. Two ciliates, one amoeba and one mycetozoan were recognized. Of
these, Nyctotherus ovalis was the most common and prevalent species (frequency index 92.31,
concentration index 4.79). The other ciliate was Balantidium (frequency index 35.55, concentration
index 2.57) whose species designation was not very clear. This first study demonstrates the presence
of Endamoeba blattae from the hindgut (frequency index 60, concentration index 2.75) and of
Coelosporidium periplanetae from Malpighian tubules (frequency index 14.71, concentration index
2.8) that are new records for the protozoan fauna of Kolkata. Additionally, this study highlights
various morphological details as well as population distribution pattern of N. ovalis.
Keywords: distribution, gut, morphology, Periplaneta americana, protozoa
INTRODUCTION
Cockroach is a household insect that acts as a
mechanical carrier as well as vector to a large number
of pathogens. They harbour a variety of protozoans in
their digestive system wherein most of them live as
endocommensals. The major groups of protozoans
reported in the lumen of the gut of cockroaches are
ciliates, amoebas, flagellates and apicomplexans
(Kudo, 1926a; Kudo, 1926b; Kudo, 1926c; Kudo and
Meglitsch, 1938; Hoyte, 1961). Researchers have
examined various aspects of parasites including
morphological, cytological and ecological details.
Roth and Willis (1957) discussed the importance of
cockroaches as vectors of various vertebrate
pathogens. In the recent past, the contribution(s) of
anaerobic protozoans and methanogens to hindgut
metabolic activities of American cockroach has been
Corresponding author: Mrs. Jayati Ghosh, Post Graduate
Department of Zoology, Maulana Azad College,
Kolkata-700 013, W.B., India. E-mail: [email protected]
studied in detail (Gijzen and Barugahare, 1992).
Surprisingly, in India, especially in West Bengal,
Kolkata, only a few studies of this kind have been
reported. In 1922, two new ciliates Balantidium
ovatum and B. blattarum (Ghosh, 1922a; Ghosh,
1922b) were reported. In a description of Indian
ciliophorans, Nyctotherus ovalis was reported to be a
part of protozoan fauna of Periplaneta americana
(Bhatia and Gulati, 1927). But afterwards, apparently,
no detailed studies were undertaken on this aspect.
Therefore, presently, it is difficult to draw a list of the
fauna of cockroach gut for these areas without making
proper investigations. We, therefore, considered it
expedient to study the fauna of the digestive system of
P. americana in Kolkata. Such studies can be expected
to generate a new list of species, and the data related to
their prevalence (number of host infected) and
intensity (average number of parasites/host).
The goal of present investigation, therefore, was to
study the protozoan fauna of P. americana in Kolkata,
and to determine their morphology and distribution.
Our findings suggest that N. ovalis is the most
77
Protozoan parasites of Periplaneta americana
common and dominant specimen, which showed
considerable variation in its nuclear structure. Large
numbers of N. ovalis were observed in the colon of P.
americana with only a few specimens of Balantidium,
but its species status was not very clear because the
organisms had some morphological similarities and
dissimilarities with Balantidium blattarum. This is
the first study wherein we report the presence of
Endamoeba blattae and Coelosporidium periplanetae
in considerable numbers in the hindgut and
Malpighian tubules, respectively, of P. americana
collected from West Bengal, India. Nevertheless,
these two protozoan species have already been
reported earlier in P. americana from some other
countries (Kudo, 1926a, Sprague, 1940).
MATERIALS AND METHODS
The cockroaches used in this study were collected
from Beliaghata, East Kolkata households and
Belgachia market area during June 2004 to February
2006. The cockroaches were dissected within 24 h of
their collection. The mid-gut, ileum, colon and rectum
of P. americana were then taken out separately and
kept in different watch-glasses containing saline
(0.6% NaCl in distilled water) solution. The
population counts were made through
haemocytometer following Petroff-Hauser counting
method (Prescott et al., 1999). The gut smears were
first examined under a light microscope and then
permanent preparations were made. Fixation was
done by Schaudinns' fixative and the smears were
stained with iron-alum haematoxylin (Cable, 1963).
For the study of nuclear characters, Feulgen nuclear
technique (Feulgen and Rossenbeck, 1924) was
utilized. An ocular micrometer calibrated with an
objective micrometer was used for all the cell
measurements. All measurements given in the study
are in micrometers, and the figures were drawn by
using a camera lucida.
RESULTS AND DISCUSSION
Data in Table I show the population distribution
pattern of the parasites obtained from the gut of P.
americana. A comparative population distribution
pattern of the parasite species showed that mean
density range of N. ovalis was highest (4.79) followed
by C. periplanetae (2.8), E. blattae (2.75) and
Balantidium sp. (2.57). These data corroborate with
the previously published results by Gijzen and
Barugahare in 1992. But the maximum SD value was
attained by N. ovalis (2.83) and the lowest was
observed in E. blattae (1.55).
Table I: Comparative population distribution pattern of protozoan fauna in the hindgut of P. americana
Name of
specimen
Number of
host studied
Number of
host infected
Frequency
index
Density
range
Mean
Standard
deviation (SD)
Standard
error (SE)
N. ovalis
Balantidium sp.
E. blattae
C. periplanetae
26
90
40
34
24
32
24
5
92.31
35.55
60
14.71
(1-12) x 104
(1-7) x 104
(1-5) x 104
(1-6) x104
4.79
2.57
2.75
2.8
2.83
1.73
1.55
1.72
0.58
0.31
0.32
0.77
Table II: Data of various morphological characters of N. ovalis
Body length (L)
Body width (W)
L/W
Macronucleus length (ML)
Macronucleus width (MW)
ML/MW
No. of
Specimens
Range
(in µm)
Mean
(in µm)
Standard
deviation (SD)
Standard
drror (SE)
25
25
25
25
25
25
57-123.5
47.5-95
1.13-1.8
19-47.5
9.5-23.75
1-3
87.21
62.32
1.43
25.46
14.63
1.923
11.93
12.83
0.20
6.97
4.64
0.56
2.39
2.57
0.04
1.39
0.93
0.11
78
Ghosh and Gayen
Description of the specimens:
stains lightly. Also variable forms of macronucleus are
common in N. ovalis. Available forms here are
spherical, ovoid, cylindrical, club shaped, semi-ovoid
forms. Light micrograph of N. ovalis showing
variation in nuclear shape (Fig. 2).
N. ovalis Leidy, 1849
Habitat: Maximum number of specimens was found
in the colon of hind-gut.
Genus Balantidium Claparède and Lachmann,1858
Morphology: Various morphological characteristics
of N. ovalis have been studied including body length
(L) and width (W) and their ratios (L/W),
macronucleus length (ML) and width (MW) and their
ratios (ML/MW; Table II).
Habitat: Colon of the hind-gut.
Morphology: Body was pear shaped, anterior end
tapering, slightly bent to the side opposite to the
peristome. Small peristome observed about onefourth the body length. Endoplasm was coarsely
granular. Macronucleus was spherical (diameter 9.514.25 µm) and placed behind the peristome in the
center of the body. Body length varied from 57-76 µm
and width from 38-57 µm. Fig. 3 shows general
morphology of Balantidium sp. under a light
,
microscope, stained with Heidenhain s iron
hematoxylin. A large contractile vacuole was present
at the posterior end in B. blattarum (Ghosh, 1922b)
that was lacking in this specimen.
The body observed to be oval, comparatively wider in
posterior than anterior. Elongated macronucleus
situated at anterior one-third. Body length varied
from 57-123.5 µm and width from 47-95 µm.
Cytopyge terminal and oval or slit like in shape.
Peristome begins at the anterior end turns slightly to
the right and ends in cytostome located midway
between the ends. Nuclear length varied from 19-47.5
µm and width from 9.5-23.75 µm. In our study, body
dimensions of N. ovalis were relatively smaller than
those described by Leidy, 1849. When the L/W ratio
was considered, it can be said that the specimens were
slightly oval whereas ML/MW ratio indicated that
nuclear shape varied from spherical to ovoid and
elongated.
E. blattae Bütschli, 1878
Habitat: Colon and ileum of hind-gut.
Morphology: Amoeba with few, broad pseudopodia
and showed distinct ectoplasm and endoplasm.
Endoplasm was clear and homogeneous. Nucleus
with characteristic round or ovoid shape of this
parasite can be easily distinguished. Dark granules are
also found along its peripheral region with light
Fig. 1 shows the general morphology of N. ovalis
under a light microscope and stained with
Heidenhain,s iron hematoxylin (a) and using Feulgen
nuclear stain (b). Compact macronucleus gave strong
positive reaction in Feulgen test where micronucleus
An
V
Ma
Ma
CP
P
An
Cy
9.5 µm
9.5 µm
P
(a)
(b)
Fig. 1. General morphology of N. ovalis under a light microscope: (a) stained with Heidenhain,s iron hematoxylin, (b) using Feulgen
nuclear stain. An. anterior, P. posterior, V. vestibular opening, CP. cytopharynx, Ma. macronucleus, Cy. cytopyge.
79
Protozoan parasites of Periplaneta americana
An
An
19 µm
An
An
Ma
V
Ma
Ma
Cy
Ma
V
CP
19 µm
P
19 µm
19 µm
P
(a)
P
P
(c)
(b)
Cy
(d)
Fig. 2. Light micrograph of N. ovalis showing variation in nuclear shape: (a) ovoid, (b) club-shaped, (c) cylindrical , (d) semi-ovoid. An.
anterior, P. posterior, V. vestibular opening, CP. cytopharynx, Ma. macronucleus, Cy. cytopyge.
central part. Length of the amoeba varies from 66.5152 µm and width 47.5-104.5 µm. Nuclear diameter
was about 19-47.5 µm. Fig. 4 shows general
morphology of E. blattae under a light microscope
following
staining with Heidenhain , s iron
hematoxylin.
C. periplanetae Lutz and Splendore, 1903
19 µm
PS
N
Habitat: Malpighian tubule.
Morphology: Trophozoite stages are common with
developing spore inside. Shape of the trophozoite may
be spherical, ovoid or cylindrical. Diameter of
spherical trophozoites varies from 28-31.5 µm and in
En
Ec
An
Fig. 4. General morphology of E. blattae under a light
microscope, stained with Heidenhain,s iron hematoxylin. Ps.
pseudopodia, N. nucleus, En. endoplasm, Ec. ectoplasm.
V
Mi
Ma
19 µm
P
Fig. 3. General morphology of Balantidium sp. under a light
microscope, stained with Heidenhain,s iron hematoxylin. An.
anterior, P. osterior, V. vestibular opening, Mi. micronucleus, Ma
macronucleus.
case of ovoid ones observed length is 42 µm and width
35 µm. Fig. 5(a), (b) and (c) show light micrograph of
C. periplanetae stained with Heidenhain,s iron
hematoxylin showing trophozoites containing various
stages of developing spore.
The above findings indicate that the fauna of P.
americana collected from Kolkata is quite rich in
various species of protozoan parasites. For the last
fifteen years, ciliates in the cockroach gut have
received special emphasis due to the discovery of
hydrogenosome, a double-membrane sub-cellular
organelle, present in some anaerobic protist including
N. ovalis (Müller, 1993). Biochemical and molecular
genetic evidences argue that hydrogenosome share a
common ancestry with mitochondria (Embley et al.,
1997). However, all hydrogenosomes studied so far
80
Ghosh and Gayen
30 µm
30 µm
CB
CB
CB
T
DS
(a)
30 µm
(b)
(c)
,
Fig. 5. Light micrograph of C. periplanetae stained with Heidenhain s iron hematoxylin showing trophozoites containing various stages
of a developing spore: (a), (b) and (c), CB. chromatoid bodies, T. trophozoite, DS. developing spore.
lack a genome. Recently Akhmanova et al. (1998)
isolated hydrogenosomal DNA from N. ovalis.
Hydrogenosomes in N. ovalis are intimately
associated with endosymbiotic, methanogenic
bacteria that may have a major role in cockroach
metabolism. In vitro studies also suggest a major role
for hind-gut protozoa in cockroach metabolic
activities, especially during the insect growth period
(Gijzen and Barugahare, 1992).
ACKNOWLEDGEMENTS
The authors would like to acknowledge University
Grants Commission, New Delhi, for providing
financial support for this work [Minor project No.
PSW/032/04-05(ERO)]. The authors would also like
to thank Prof. Dipankar Sengupta, Head, Dept. of
Zoology, Maulana Azad College, Kolkata, for
providing departmental facilities and to other
colleagues for continuous cooperation. The authors
are especially thankful to Prof. Biswapati Dasgupta
and Dr. Tarak Khan for their valuable suggestions and
comments.
REFERENCES
Akhmanova A, Voncken F, van Alen T, van Hoek A, Boxma B,
Vogels G, Veenhuis M and Hackstein JHP. 1998. A
hydrogenosome with a genome. Nature 396:527-528.
Feulgen R and Rossenbeck H. 1924. Mikroskopischchemischer Nachweis einer Nucleinsarure von Typus der
Thymonucleinsaure und die darauf beruhende elective
Farbung von Zellkernen in mikroskopischen Praparaten.
Ztschr. Physiol. Chem. 135:203.
Ghosh E. 1922a. On a new ciliate, Balantidium ovatum sp. nov.,
an intestinal parasite in the common cockroach (Blatta
americana). Parasitology 14:371.
Ghosh E. 1922b. On a new ciliate, Balantidium blattarum sp.
nov., an intestinal parasite in the common cockroach
(Blatta americana). Parasitology14:15-16.
Gijzen HJ and Barugahare M. 1992. Contribution of anaerobic
protozoa and methanogens to hindgut metabolic activities
of the American cockroach, Periplaneta americana. Appl.
Environ. Microbiol. 58: 2565-2570.
Hoyte HMD. 1961. The protozoa occurring in the hind-gut of
cockroaches I. Parasitology 51:415-436.
Kudo R.1926a. Observations on Endamoeba blattae. Am. J.
Hyg. 6: 139-52.
Kudo R.1926b. Observations on Lophomonas blattarum, a
flagellate inhabiting the colon of the cockroach, Blatta
orientalis. Arch. Protistenk. 53: 191-214.
Kudo R. 1926c. A cytological study of Lophomonas striata
Bütschli. Arch. Protistenk. 55: 504-515.
Kudo RR and Meglitsch PA.1938. On Balantidium
praenucleatum n.sp. inhabiting the colon of Blatta
orientalis. Arch. Protistenk. 91:111-124.
Müller M. 1993. The hydrogenosome. J. Gen. Microbiol.
139:2879-2889.
Bhatia BL and Gulati AN. 1927. On some parasitic ciliates from
Indian frogs, toads, earthworms and cockroaches. Arch.
Protistenk. 57:85-120.
Prescott LM, Harley JP and Klein DA. 1999. Microbiology.4th
Edn. WCB/McGraw-Hill, USA. pp 117.
Cable RM. 1963. An illustrated laboratory manual of
parasitology, Allied Pacific Private Limited, Bombay, India,
pp133-134.
Roth LM and Willis ER. 1957. The medical and veterinary
importance of cockroaches. Smithson. misc. Collns.134:
no. 10
Embley TM, Horner DA and Hirt RP. 1997. Anaerobic
eukaryote evolution: hydrogenosomes as biochemically
modified mitochondria? Trends Ecol. Evol. 12: 437-441.
Sprague V. 1940.
Observations on Coelosporidium
periplanetae with special reference to the development of
the spore. Trans. Am. Microsc. Soc. 59: 460-474.
Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 81–84
J PD
Chelatrema neilgherriensis n. sp. (Trematoda:
Gorgoderidae) infecting the freshwater fishes from
Noolpuzha river in Wynad district, Kerala, India
K. T. Manjula and K. P. Janardanan
Department of Zoology, University of Calicut, Calicut.
ABSTRACT. A new species of digenetic trematode Chelatrema neilgherriensis (Gorgoderidae) was
recovered from the freshwater fishes Danio neilgherriensis and Labeo rohita, collected from
Noolpuzha river in Wynad district, Kerala. Herein, we have described this new species in detail,
discussed its systematic position and have compared it with the only other species in the genus C.
smythi Gupta and Kumari, 1973. We found C. neilgherriensis n. sp. to be different from C. smythi in
body measurements, size of oral and ventral suckers, sucker ratio, extent of uterine coils, nature of
testes and seminal vesicle, and shape and size of eggs. Apparently, this first study reports a second
species of the genus Chelatrema, a gorgoderid from Kerala.
Keywords: Chelatrema, Gorgoderidae, Kerala, trematoda
INTRODUCTION
The genus Chelatrema was erected by Gupta and
Kumari (1973) with C. smythi as the type species
which infect the freshwater fish, Chela bacala (Ham.)
from Satluj River at Ropar, Punjab, India. The authors
included the genus under the subfamily Arnolinae
Yamaguti, 1958 of the family Hemiuridae Luhe, 1901.
But Gibson and Bray (1979) stated that it is not a
hemiurid, and Gibson (2002) considered it as a
gorgoderid.
During an explorative study on the trematode fauna of
freshwater fishes in the hill streams of Wynad district
in Kerala, we came across a species of Chelatrema in
Danio neilgherriensis and Labeo rohita, collected
from Noolpuzha river. Detailed studies proved that
the trematode is significantly different from C. smythi,
Corresponding author: Dr. K. P. Janardanan, Department of
Zoology, University of Calicut, Calicut-673 635, Kerala, India.
E-mail: [email protected]
the only species in the genus Chelatrema, and is
reported here as a new species. This forms the second
species of the genus Chelatrema and the first report of
a gorgoderid from Kerala.
MATERIALS AND METHODS
The fresh water fishes Danio neilgherriensis (Day,
n=16) and Labeo rohita (Hamilton, n=6), collected
from Noolpuzha river in Wynad district of Kerala,
from April 2004 to May 2005, were examined for
trematode infections. The flukes recovered from the
intestine of D. neilgherriensis and the intestine and
body cavity of L. rohita were observed under phasecontrast microscope, with or without vital staining.
Those used for permanent preparations were fixed for
approximately 1 h in 10% formalin under cover glass
pressure at room temperature, and stained with alum
carmine as described by Cantwell (1981). The
descriptions are based on 20 whole mounts, and
measurements of 10 mature flukes fixed in 10%
formalin. Measurements are presented in
82
Manjula and Janardanan
micrometres, unless otherwise stated; the range is
followed by mean values in parentheses. The figures
were drawn with the aid of a camera lucida, and details
added free-hand from observations made on live
specimens.
RESULTS
Infections with adult flukes were observed in the
intestine of 10 out of 16 (62.5%) Danio
neilgherriensis, and intestine and body cavity of one
out of six (16.7%) Labeo rohita. The intensity of
infection varied from one to three.
Description (Fig. 1)
Body large, light red, elongate, ovoid, narrowing in
anterior body; measured 2.7146.5 x 1.0112.602 (4.413
x 1.747) mm; width 30-45 (40)% of body-length.
Tegument unarmed. Oral sucker round, sub-terminal;
390-625 (490) in diameter. Ventral sucker round,
muscular, large; 510-1,035 (801) in diameter. Sucker
length ratio 1: 1.69-1.82 (1.78). Anterior body 9002,192 (1,962) long; 30-37 (34)% of body length.
Prepharynx distinct, 20-72 (64) in length. Pharynx
elongate, ovoid; measured 125-232 x 94-215 (163 x
136). Oral sucker/pharynx width ratio 1: 0.30-0.38
(0.34). Oesophagus 18-42 (29) long. Many gland
cells found scattered around oesophagus and anterior
region of caeca. Intestinal bifurcation in anterior body;
800-1,205 (1,016) anterior to ventral sucker. Caeca
narrow, 3.0-4.985 (3.872) mm long; terminate blindly,
320-456 (389) from posterior extremity.
Testes two, symmetrical, spherical to ovoid, entire;
left testis 250-432, 209-382 (311 x 246); right testis
249-382 x 197-309 (305 x 243). Post-testicular region
820-1,198 (1,006) long; 18-27 (22)% of body-length.
Vas deferens traverses along left side of ventral sucker.
75 µm
800 µm
B
A
30 µm
300 µm
C
D
Fig. 1. A-D. C. neilgherriensis n. sp. A, Entire worm. B,
Terminal genitalia. C, Egg. D, Miracidium.
83
A new species of Chelatrema from fishes in Wynad
Cirrus-sac large, elongate, post-bifurcal, medially
placed; 279-415 x 107-193 (366 x 159) in size.
Seminal vesicle saccular, bipartite; 212-384 x 51-97
(348 x 77), reaches about ¾th of cirrus-sac, narrows
distally forming pars-prostatica.
Pars-prostatica
thick-walled, ensheathed in numerous gland cells.
Ejaculatory duct long, tubular, with narrow walls;
opens into base of genital atrium. Cirrus not visible.
Genital pore submedian, post-bifurcal; 162-283 (246)
from ventral sucker.
Ovary immediately posterior to ventral sucker,
equatorial, dextral, round to ovoid; 301-439 x 376-483
(345 x 406). Ventral sucker to ovary distance 84-129
(113); 2-3.1 (2.5)% of body-length. Oviduct leads
from lateral margin of ovary. Seminal receptacle
large, saccular, ovoid, placed lateral to ovary; 300-452
x 223-327 (361 x 255) in size. Vitellarium in the form
of a compact mass, situated behind seminal receptacle,
on the left side of median line; 180-362 x 150-286 (276
x 225) in size. Uterus fills the whole post-testicular
posterior body, extends into entire extra-caecal space,
up to the level of caecal bifurcation. Distal end of
uterus passes along left side of ventral sucker to form
distinct metraterm and opens at genital pore. Uterus
filled with numerous, small, oval, thin-shelled, nonoperculate, embryonate, and fragile eggs, measuring
78-86 x 70-81 (83 x 76).
232 (1,309 x 201), extends to midlevel of testes.
Taxonomic summary
Genus:
Chelatrema Gupta and Kumari,
1973
Species:
Chelatrema neilgherriensis n. sp.
Type host:
Danio neilgherriensis (Day),
Cyprinidae
Additional host:
L a b e o ro h i t a ( H a m i l t o n ) ,
Cyprinidae
Site:
Intestine and body cavity
Type locality:
India, Kerala, Wynad district,
Noolpuzha river
Holotype:
Deposited in the Department of
Zoology, University of Calicut,
Kerala, India. No: Z./Par./Dig2005-1a
Paratypes:
Z./Par./Dig-2005- lb-d
Date of collection: 13 April 2005
Etymology:
Named after the species name of
the type host.
Excretory bladder long, I-shaped; 1,190-1,570 x 104Table I. Comparison of C. neilgherriensis n. sp. with C. smythi Gupta and Kumari, 1973
Character
C. smythi Gupta and Kumari, 1973
C. neilgherriensis n. sp.
1.
Body size
6.9-7.0 x 2.5-2.75 mm
2.714-6.5 x 1.011-2.602 (4.413 x 1.747) mm
2.
Oral sucker
Round; 600 x 500-550
Round; 390-625 (490)
3.
Ventral sucker
Round; 1,000 x 900
Round; 510-1,035 (803)
4.
Sucker length ratio
1 : 1.6
1 : 1.78
5.
Testes
Entire or slightly lobed
Entire, round to ovoid
6.
Seminal vesicle
Ovoid; 66 x 88
Bipartite; 212-384 x 51-97 (348 x 77)
7.
Ovary
Entire; 500 x 400
Entire, round to ovoid; 301-439 x 376-483
(345 x 406)
8.
Uterine coils
Fill the space behind ventral sucker
Fill the space behind ventral sucker, and extend
extra-caecally up to the level of caecal
bifurcation.
9.
Eggs
Small, round; 45-60
Small, ovoid; 78-86 x 40-49 (83 x 46)
C. bacala (Hamilton)
D. neilgherriensis (Day) and L. rohita
(Hamilton)
10. Host (s)
Note: Measurements are in micrometres unless otherwise mentioned.
84
Miracidium
Freshly laid eggs hatched in 2-3 h. Miracidia
pyriform, with conical papilla at anterior end;
posterior end round; measured 132-176 x 46-73 (151 x
64). Cilia long, uniformly distributed. Eyespots two,
oval and conjoined; 19-23 x 8-13 (20 x 11.5) in size. A
large apical gland and a pair of lateral penetration
glands present. A pair of flame cells present behind
the eyespots. Miracidia swim actively in water,
changing direction intermittently. They exhibited
negative phototaxis.
DISCUSSION
In the present study, the fluke is characterised by the
presence of large ventral sucker, well-developed
pharynx, short oesophagus, symmetrical testes, pretesticular ovary, single compact vitellarium, strongly
convoluted uterus and embryonated eggs, and hence it
is included in the genus Chelatrema Gupta and
Kumari, 1973. These authors included the genus under
family Hemiuridae Luhe, 1901, but Gibson and Bray
(1979) treated it as a genus inquirendum and Gibson in
2002 considered it a gorgoderid. As the characters of
the genus Chelatrema agree fully with that of
Gorgoderidae, we agree with the arrangement made
by Gibson and place the genus under the family
Gorgoderidae.
The new species reported here needed to be compared
with the only species in the genus, C. smythi which
infects the freshwater fish Chela bacala. It resembled
C. smythi in the extent of caeca, position of gonads,
nature and position of vitellarium and position of
genital pore.
But it differed greatly in body
measurements, size of oral and ventral suckers, sucker
Manjula and Janardanan
ratio, extent of uterine coils which lead extra-caecally
up to caecal bifurcation, nature of testes and seminal
vesicle and the shape and size of eggs. A comparison
of the characters of C. smythi and of C.
neilgherriensis, presented in Table I, showed that the
two species were different from each other. Further,
they infect different hosts too. Therefore, the fluke
reported herein has been considered as a new species
and named Chelatrema neilgherriensis after the
specific name of the type host, Danio neilgherriensis.
ACKNOWLEDGEMENTS
The authors express their sincere thanks to Dr. D. I.
Gibson, Department of Zoology, The Natural History
Museum, London, for taxonomic identification of the
fluke to generic level, and to the Head of the
Department of Zoology, University of Calicut, for
providing facilities.
REFERENCES
Cantwell GE. 1981. Methods for invertebrates. In: staining
procedures. Clark G (Ed.) Williams and Wilkins, Baltimore.
pp 255-280.
Gibson DI. 2002. Family Derogenidae Nicoll, 1910. In: Keys to
the Trematoda. Volume 1. Gibson DI, Jones A & Bray RA
(Eds.) CAB International. pp 351-368.
Gibson DI and Bray RA. 1979. The Hemiuroidea: terminology,
systematics and evolution. Bull. Br. Nat. Hist. (Zool.). 36:
35-146.
Gupta N K and Kumari A. 1973. Chelatrema smythi n. gen. n.
sp. (Trematoda, Hemiuidae: Arnolinae) from a fresh-water
fish, Chela bacala, from Ropar. Res. Bull. (N.S.) of the
Panjab Univ. 24:109-112.
Short communication
Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 85–88
J PD
Haemato-biochemical studies on fowl coccidiosis in
layer birds*
N. D. Hirani, J. J. Hasnani, R. S. Joshiand K. S. Prajapati
Department of Veterinary Parasitology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand.
ABSTRACT. This study on haemato-biochemical profile of 48 coccidia-infected and 24 healthy
birds from commercial layer farms revealed that the infected-birds had significantly (p < 0.05)
lower mean haemoglobin concentration (7.62 ± 0.14 vs 10.59 ± 0.19 g%) and total erythrocyte count
(1.63 ± 0.04 vs 3.10 ± 0.05 million cells/cmm), apparently lower packed cell volume (23.00 ± 0.71 vs
31.46 ± 0.70 unit %) and significantly (p < 0.05) higher mean total leucocyte count (16.41 ± 0.37 vs
10.40 ± 0.16 thousand cells/cmm) than the healthy ones. The mean values of blood glucose (221.01 ±
2.66 vs 176.03 ± 0.75 mg%) and serum total cholesterol (271.71 ± 4.71 vs 112.68 ± 0.60 mg%) were
significantly (p < 0.05) higher and serum total protein was lower (2.98 ± 0.08 vs 4.42 ± 0.10 g%) in
coccidia-infected birds as compared to healthy birds, whereas the activities of serum aspartate
aminotransferase (77.84 ± 1.10 vs 65.62 ± 0.63 U/L), alanine aminotransferase (9.20 ± 0.28 vs 7.07 ±
0.25 U/L) and alkaline phosphatase (902.23 ± 9.30 vs 816.58 ± 25.95 KAU%) were non-significantly
higher in infected-birds than the healthy ones. Almost a similar trend was observed for all the
parameters in layer birds managed under both deep litter as well as cage system. These findings
reflected anaemic condition with depressed metabolism due to tissue damage and increased
immune response in infected-birds.
Keywords: coccidiosis, deep litter vs cage system, haematology, layer birds, serum
Coccidiosis is a parasitic disorder, which can have an
acute or chronic course of infection in birds. It
produces a deviation in the various haematological,
biochemical and enzymatic components of the body
(Padmavathi and Muralidharan, 1986a,b).
Therefore, measurement of biochemical and enzyme
activities is useful in determining the pathologic
conditions in the tissues. Serological profiles of
certain enzymes are altered greatly in cell membrane
degeneration, and inflammatory and diffuse tissue
* A part of M. V. Sc. thesis of the first author; approved by
A A V, Anand.
Corresponding author: Dr. N. D. Hirani, Department of
Veterinary Parasitology, College of Veterinary Science and
Animal Husbandry, Anand Agricultural University, Anand-388
001, Gujarat, India.
degeneration and loss (Deger et al., 2002). Scanty
literature is available on haemato-biochemical
alterations due to coccidiosis in layer birds from round
the globe.
This study was, therefore, conducted on 99 layer farms
in the middle of Gujarat, involving fecal sampling of
594 birds managed under deep litter and/or cage
system. All the samples were examined by the
standard methods. From all coccidia positive farms,
two infected and one healthy birds were selected for
haemato-biochemical studies. In all, 48 blood samples
from coccidia-infected birds and 24 samples from
normal healthy birds were studied. Data thus
generated were statistically analyzed using unpaired 't'
test (Snedecor and Cochran, 1980).
In this study, significantly (p < 0.05) lower mean
86
haemoglobin concentration and total erythrocyte
count, apparently lower packed cell volume, and
significantly (p < 0.05) higher mean total leucocyte
count were found in coccidia-infected birds than the
healthy ones (Table I). These findings were in
conformity with the earlier reports (Joshi et al., 1974;
Padmavathi and Muralidharan, 1986a; Jaipurkar et al.,
2004). The reduction in the value of haemoglobin and
total erythrocyte count observed in the infected-birds
might be attributed to haemorrahges in the intestine
followed by development of intestinal lesions. There
may be injury to tissue and liberation of large
quantities of histamine, which increase the local blood
flow and also increase the permeability of capillaries
and venules allowing large quantities of fluid to come
out (Padmavathi and Muralidharan, 1986a). The
reduction observed in packed cell volume during the
acute-phase of infection might be due to severe blood
loss resulting in anaemic condition (Joshi et al., 1974).
The increased total leucocyte count in coccidiainfected birds might be due to immune suppression of
infection. This was suggestive of increased
leucopoiesis as a first step of defense mechanism to
infection (Padmavathi and Muralidharan, 1986a).
In the present study, significantly (p < 0.05) higher
blood glucose and serum total cholesterol and lower
serum total proteins were found in coccidia-infected
birds as compared to healthy ones. Joshi et al. (1974)
reported similar findings on blood glucose. However,
Padmavathi and Muralidharan (1986b) recorded
serum hypoglycaemia in birds with experimental
Emeria tenella infection, whereas Basith et al. (1998)
failed to see any change in plasma glucose. This high
level of blood glucose observed in coccidia-infected
birds may be either due to stress conditions leading to
the liberation of adrenal corticoids which induce
hyperglycaemia or disturbed carbohydrate
metabolism due to interference with phosphorylative
carbohydrate dissimilation by unidentified material
present in the intestine of infected-fowls. The apparent
hyperglycaemia was undoubtedly due to loss of
erythrocytes. The observations on protein were in
conformity with those of Turk (1972), who reported
marked reduction in protein during the acute-phase of
E. necatrix infection. The significant (p < 0.05)
reduction in serum total protein observed in infectedbirds might be due to reduced feed intake and/or
haemorrahges through the gut and formation of
inflammatory exudates rich in blood proteins (Basith
et al., 1998). The decrease might also be due to leakage
of protein resulting from the increased permeability of
Hirani et al.
the intestinal mucosa, which was necessary for the
passage of plasma protein at the height of the disease.
These lower protein levels might be due to reduced
absorption of amino acids derived from the protein
constituents of feed due to reduced feed consumption
(Padmavathi and Muralidharan, 1986b). The results
on cholesterol are in agreement with the observations
of Singh et al. (1976), Padmavathi and Muralidharan
(1986b) and Basith et al. (1998), whereas
Constantinescu (1976) could not see significant
changes in serum cholesterol values in both infected
and healthy birds. The hypercholesteremia observed
in the present study among the infected-birds might be
due to disturbed fat metabolism and loss of fluid
resulting in apparent increase (Padmavathi and
Muralitharan, 1986b) or due to impaired liver function
consequent to injury to intestinal epithelium in
coccidiosis (Basith et al., 1998).
Mean activities of serum aspartate aminotransferase,
alanine aminotransferase and alkaline phosphatase
observed in infected-birds were non-significantly
higher as compared to the healthy ones. Singh et al.
(1976) reported comparable findings, while Kumar
and Rawat (1975) found no significant difference, but
values were slightly less in infected group as
compared to normal group. The increase in SGOT and
SGPT activities observed in coccidia-infected birds
under study might be due to extensive damage to
intestine and liver by the parasites and thereby loss of
appetite. Constantinescu (1976) reported significant
increase in serum AKP activity among infected-birds.
On the contrary, Kumar and Rawat (1975) reported
significant decrease in serum AKP of E. necatrix- and
E. acervulina-infected 3-4 months old cockerels.
It can be concluded that haemoconcentration, reduced
haemoglobin and total erythrocyte count and an
increase in total leucocyte count in coccidia-infected
birds suggest haemorrhage and increased leucopoiesis
as a first step of defense mechanism to infection. The
high level of blood glucose observed in coccidiainfected birds may be either due to stress leading to the
liberation of adrenal corticoids which induce
hyperglycaemia concomitant with loss of
erythrocytes. Reduced serum total protein and
increased serum enzyme activity in coccidia-infected
birds indicate damage to intestine and vital organs,
increased cell membrane permeability and loss of
intracellular contents.
1.70 ± 0.08
15.33 ± 0.94
230.53 ± 6.15
3.22 ± 0.20
278.55 ± 7.11
76.13 ± 2.61
9.49 ± 0.53
Erythrocytes count
(million cells/cmm)
Leukocytes count
(thousand cells/cmm)
Blood glucose (mg %)
Serum total protein
(gm %)
Serum cholesterol
(mg %)
SGOT (U/L)
SGPT (U/L)
N = number of birds , * Significant at p < 0.05,
884.58 ± 15.79
24.68 ± 1.69
Packed cell volume (%)
Alkaline phosphatase
(KAU %)
7.87 ± 0.39
12
Infected birds
Haemoglobin (gm %)
N
Parameter
834.91 ± 29.41
7.18 ± 0.32
65.63 ± 0.69
112.43 ± 0.59
4.48 ± 0.13
175.21 ± 0.74
10.47 ± 0.20
3.03 ± 0.04
31.06 ± 0.77
10.48 ± 0.24
18
ns = non-significant.
908.11 ± 11.18
9.10 ± 0.33
78.41 ± 1.19
269.43 ± 5.82
2.90 ± 0.08
217.84 ± 2.74
16.77 ± 0.37
1.61 ± 0.05
22.44 ± 0.76
7.54 ± 0.13
36
Healthy birds
Cage system
Infected birds
** Significant at p < 0.01,
761.57 ± 52.57
6.77 ± 0.19
65.58 ± 1.52
113.40 ± 1.70
4.26 ± 0.14
178.50 ± 1.70
10.20 ± 0.17
3.30 ± 0.11
32.67 ± 1.61
10.93 ± 0.16
6
Healthy birds
Deep litter
902.23 ± 9.30
9.20 ± 0.28
77.84 ± 1.10
271.71 ± 4.71
2.98 ± 0.08
221.01 ± 2.66
16.41 ± 0.37
1.63 ± 0.04
23.00 ± 0.71
7.62 ± 0.14
48
Infected birds
816.58 ± 25.95
7.07 ± 0.25
65.602 ± 0.63
112.68 ± 0.60
4.42 ± 0.10
176.03 ± 0.75
10.40 ± 0.16
3.10 ± 0.05
31.46 ± 0.70
10.59 ± 0.19
24
Healthy birds
Overall total
Table I. Haemato-biochemical profile of coccidia-infected and healthy layer birds managed under deep litter and cage system in Gujarat
0.60ns
0.93ns
1.49ns
4.85**
1.93*
2.40*
2.24*
3.79**
1.41ns
2.22*
't' value
Haemato-biochemical studies on fowl coccidiosis
87
88
ACKNOWLEDGEMENTS
We thank the Principal and Dean of the College, and
the poultry farmers of the region for the facilities,
support and co-operation extended for this work on
their birds. The facility of autoanalyzer provided by
ADIO, Navsari, Gujarat State is also gratefully
acknowledged.
Hirani et al.
Joshi HC, Singh BP, Prasad B and Prasad RK. 1974. Variations
in certain blood constituents during caecal coccidiosis in
poultry. Indian J Parasitol. 9:22-24.
Kumar A and Rawat JS. 1975. A note on the effect of
coccidiosis on serum enzymes, blood glucose and
cholesterol in chicken. Indian J Anim Sci 45:154-156.
REFERENCES
Padmavathi P and Muralidharan SRG. 1986a. Alteration in
haematological parameters in chicken during Eimeria
tenella infection. Indian Vet J 63:716-722.
Basith Abdul S, Rajavelu G and Murali Manohar B. 1998.
Biochemical studies in experimental Eimeria necatrix
infection in chickens. Indian Vet J 75:876-878.
Padmavathi P and Muralidharan SRG. 1986b. Studies on the
alteration in the serum metabolites during the Eimeria
tenella infection in chicks. Indian Vet J 63:530-536.
Constantinescu V. 1976. Biochemical and histoenzymic
changes in coccidiosis in chickens. Buletinul Institutului
Agronomic Cluj Napoca 30:115-117.
Singh CV, Joshi HC and Shah HI. 1976. Biochemical studies in
intestinal coccidiosis of poultry. Pantnagar J Res 1:63-66.
Deger Y, Dedo S and Deger S. 2002. Enzyme activity changes in
the sera of chickens treated with coccidiostatic agents.
Indian Vet J 79:912-916.
Jaipurkar SG, Deshpande PD, Narladkar BW, Rajurkar SR and
Kulkarni GB. 2004. Caecal coccidiosis in broiler chicks:
haematological, pathological changes during treatment
with herbal antidiarrhoels. J Vet Parasitol 18:135-138.
Snedecor GW and Cochran WG. 1980. Statistical Methods. 8th
Edn. Iowa State Univ. Press, Ames, Iowa, USA.
Turk DE. 1972. Protozoan parasitic infections of the chick
intestine and protein digestion and absorption. J Nutr
102:1217-1222.
Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 89–91
Short communication
J PD
Re-redescription of Dissurus farrukhabadi Verma, 1936
(Digenea - Echinostomatidae) with a discussion of the
genus Dissurus Verma, 1936
P. C. Gupta and R. B. Singh
Department of Zoology, D. A. V. College, Kanpur.
ABSTRACT. Dissurus farrukhabadi Verma, 1936 from the large intestine of a White-necked
stork, Dissurus episcopus (Boddaert) from Unnao, India is re-redescribed. The validity of the
species of Dissurus, Verma, 1936 is discussed. D. xenorhynchi Wahid, 1962 is considered a
synonym of Stephonoprora gigantica Gupta, 1962 and Psilocollaris guptai Gupta and Saxena,
1986 is considered a synonym of D. farrukhabadi.
During the survey of digenetic termatode parasites of
birds, nine specimens of Dissurus farrukhabadi
Verma, 1936 were collected and described.
The trematodes were fixed in 70% alcohol under slight
cover glass pressure for 24 h, stained with acetic alum
carmine, dehydrated in graded series of alcohols,
cleared in clove oil and mounted in Canada balsam.
The drawings were made with the aid of a camera
lucida, and unless otherwise stated, all measurements
are in mm.
DESCRIPTION
Dissurus farrukhabadi Verma, 1936
Body long, slender, delicate, spinose, 5.34-5.71 x
0.57-0.69, with rounded extremities. Head collar
reniform, 0.30-0.34 x 0.41-0.45; with 24 spines,
interrupted dorsally, arranged in a single row with no
end group. Oral sucker terminal, avoid 0.10-0.12 x
0.08-0.09. Prepharynx 0.06-0.09 long. Pharynx
subglobular 0.11-0.13 x 0.09-0.16. Oesophagus
tubular, with thick irregularly sinuous walls, 0.48Corresponding author: Dr. P.C. Gupta, 8/200-A, Arya Nagar,
Kanpur-208 002, U. P., India.
0.58 long. Intestinal caeca simple, extending to hind
end of body. Ventral sucker sub-spherical, much larger
than oral sucker, 0.40-0.46 x 0.43-0.50 at 0.87-1.14
from anterior extremity.
Excretory bladder Y-shapped; arms bifurcation just
behind posterior testis and their extension beyond
anterior testis not traceable due to vitallariaum
follicles, excretory pore subterminal.
Testes oval, entire or notched, subequal, tandem,
apart, in last fifth of body. Anterior testis 0.30-0.55 x
0.23-0.28 at 3.61-4.30 from anterior extremity.
Posterior testis, 0.47-0.60 x 0.31-0.32 at 0.48-0.52
from posterior extremity. Vasa efferentia arising from
testis, join together to form vas deferens, running
anteriorly to form vesicula seminalis. Cirrus sac small,
heart shaped, 0.21-0.27 x 0.14-0.17, extending
posteriorly either upto anterior margin of ventral
sucker or overlapping anterior third of it. Vesicula
seminalis bipartiate, 0.12-0.15 x 0.09-0.16. Parsprostatica small, 0.03-0.06 long surrounded by large
number of prostate gland cells. Cirrus muscular, 0.010.02 long. Genital pore median, between intestinal
bifurcation and ventral sucker at 0.72-0.99 from
anterior extremity.
90
Gupta and Singh
Ovary rounded, entire, submedian, postequatorial,
pretesticular, intercaecal, 0.14-0.18 x 0.16-0.18 at
2.73-3.22 from anterior extremity. Receptaculam
seminalis oval, 0.044 x 0.035. Laurer's canal present.
Vitellaria follicular, dense extending from a level
anterior to ovary up to hind end of body. Transverse
vitelline ducts from either side meet and form a yolk
reservoir to open at ootype, surrounded by a large
number of Mehlis' gland cells. Uterus long,
intercaecal lying between ootype and venteral sucker,
Egg large, oval, few, 0.09-0.10 x 0.04-0.05.
Host
:
White-necked stork
Dissurus episcopus (Bodd.)
Location
:
Large intestine
Locality
:
Unnao, U.P., India
Material
:
Nine specimens (incidence
2/3)
Verma (1936) erected the genus Dissurus with D.
farrukhabadi as its type, recovered form the intestine
of a White-necked stork. D. espicopus from
Farrukhabad, U.P. Wahid (1962) added D.
xenorhynchi from the large intestine of Black-necked
stork. Xenorhynchus asiaticus at London zoo, and
Dwivedi (1967) described D. setheae from the type
host at Chindwara (M. P.). Srivastava (1974) reexamined Verma's specimens and gave illustrations
together with an emended generic diagnosis.
Singh (1954) established another genus Psilocollaris
with P. indicus as its type for his specimens from the
intestine of D. episcopus from Lucknow, U.P. The
genus Psilocollaris was characterized by an aspinous
collar.
Gupta (1980) critically studied P. indicus Singh, 1954
in detail and raised question on its validity, doubting
its conspecificity with D. farrukhabadi. Srivastava
2
LC
0.25mm
MG
RC
3
0.05mm
4
0.2mm
5
6
0.05mm
1
Fig. 1-6. Dissurus farrukhabadi Verma, 1936.
1. entire ventral view; 2. head collar, (enlarged); 3. extension of cirrus sac up to anterior third of ventral sucker; 4. cirrus sac,
enlarged; 5. female genital complex, enlarged; 6. showing entire testes.
OV - ovary, U - uterus, OO-ootype, RS - receptaculum seminalis, LC - Leurar's canal, YR - yolk reservoir.
Re-redescription of Dissurus Farrukhabadi Verma
(1982) re-examined the type specimen of P. indicus
and found the presence of collar spines and accepted
the views of Gupta (1980). Further, he considered P.
singhi Pandey, 1975 as species inquirenda and also
transferred D. setheae to the genus Stephanoprora
Odhner, 1902, due to presence of 22 collar spines,
tandam testes lying in mid-third of body, vitellaria
extending form posterior testis to hind end and short
uterus. Srivastava (1982) further observed that D.
setheae is probably a synonym of S. nigerica Gupta,
1983. The authors are in agreement with the views of
Gupta (1980) and Srivastava (1982).
In the opinion of present authors D. xenorhynchi
Wahid, 1962 better fits under the genus Stephanoprora
Odhner, 1902 and shares most of its characters viz. the
extension of the vitellaria, 22 collar spines, the
position of the testes and thickening in the wall of the
oesophagus with S. gigantica Gupta (1962). The
former, therefore, is considered a synonym of later.
Gupta and Saxena (1986), overlooking the synonymy
of Psilocollaris, described P. guptai form the intestine
of an Indian stork, Leptoptilos dubius form Lucknow
and differentiated it from the type species on such
characters as the muscular pharynx, position of the
testes and the ovary. These characters are merely
intraspecific variations and, therefore, P. guptai is a
synonym of D. farrukhabadi.
The present specimens of D. farrukhabadi differed
from the previous descriptions in having a smaller
body, in the extensions of vitellaria from a little
anterior to overy instead of from the level of it, and in
having a smaller post-testicular space. These
differences are considered as intraspecific variations.
REFERENCES
Dwivedi MP. 1967. A new species of the genus Dissurus Verma,
1936 (Trematoda Echinostomatidae). Natural and Applied
Science Bulletin, 20: 267-275.
91
Gupta PD. 1980. Further observations on Psilocolaris indicus
Singh, 1954 with a note on its systematic position
(Trematoda: Psilostomatidae). Bulletin of the Zoological
Survey of India. 2: 217-218.
Gupta R. 1962. Studies of trematode parasites of Indian birds.
II. On Stephanoprora gigantics sp. nov. from the Blacknecked storck, Xenorhynchus asiaticus (Latham).
Proceeding of the National Academy of science, India.
32:381-386.
Gupta R. 1963. On Stephanoprora nigerica sp. nov. with a brief
review of the genus Stephanoprora
Odhner, 1905
(Trematoda : Echinostomatidae). Zoological Anazica.
170:117-130.
Gupta V and Saxena AM. 1986. On Psilocollaris guptai sp. nov.
(Psilostomatidae : Trematoda) from the intestine of an
Indian Stork, Leptonhilos dubius (Gmelin) from Lucknow.
Indian Journal of Helmonthlogy. 37: 1-3.
Pandey KC. 1975. Studies on some known and unknown
trematode parasites. Indian Journal of Zootomy. 14: 197219.
Singh KS. 1954. Psilocollaris indicus n.g., n. sp.
(Psilostomatidae) from an Indian stork, Dissurus episcopus
episcopus. Journal of the Washington Academy society. 44:
24-26.
Srivastava CB. 1974. A critical study of Verma's "Notes on
trematode parasites of Indian birds" based on his trematode
collection on Part 2. Family Echinostomatidae Dietz, 1909.
Journal of the Zoological society of India. 24:160-191.
Srivastava CB. 1982. The fauna of Indian and the adjacent
countries. Platyhelminthes Vol. I (Supplement) Trematoda Digenea. Addition to Prof. H. R. Mehra's volume on
trematoda - Digenea. Publ. by the Director, Zoolgical
Survey of India, Calcultta, 163pp.
Verma SC. 1936. Notes on trematode parasites of Indian birds.
Part I Allababad University Studies. 12: 148-188.
Wahid S. 1962. On a new trematode from Black-necked stork,
Xenorhynchus asiaticus. Journal of Helminthology. 36:
211-214.
Short communication
Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 92–93
J PD
A case of vaginal bleeding due to leech bite
R. P. Ganguly, M. S. Mukhopadhyay and K. K. Patra
Department of Obstetrics and Gynaecology, R. G. Kar Medical College and Hospital, Kolkata.
Medically important leeches belong to Phylum
Annelida and Class Hirudinea. It is a worm like
creature that attaches to their hosts by means of
chitinous cutting jaws and draws blood through
muscular suckers. Haemadipsa sylvastris, H. zelanica
and H. montana, H. ornata etc. are land leeches
available in India. The most prevalent species is H.
sylvestris. Hirudo birmanica and Limnatis nilotica are
water leeches. L. nilotica is notorious for internal
hirudiniasis. Internal hirudiniasis may involve
pharynx, larynx, vocal cord, trachea, oesophagus and
genitourinary tract. Attachment is usually painless.
Continued bleeding from the site takes place after the
leech has detached. Death due to exhaustion has been
reported. Leech gets dislodged from its site of
attachment if saline water, strong vinegar or match
flame is applied.
CASE REPORT
A six year old girl was admitted as an emergency case
in the Department of Gynaecology and Obstetrics at
R. G. Kar Medical College and Hospital, Kolkata, with
a complaint of bleeding per vagina for 4 h. Patient's
mother gave history that her daughter had gone to a
local pond in the evening to wash herself after
defecation. She came back home and after sometime
noticed that her undergarments were wet with blood.
Patient denied any history of local trauma. She was
then brought to the hospital for medical help.
On examination, the patient was conscious but very
pale. On local examination, fresh blood was seen
coming out through vagina. The bleeding point could
not be seen as it was inside vagina. There was no
Corresponding author: Dr. R. P. Ganguly, BC 45/7, Bichitra
Abason, Sector 1, Saltlake, Kolkata-700 064, W.B., India.
evidence of trauma anywhere. Per rectal examination
revealed a boggy mass of about 5 cm x 4 cm which was
appreciated anteriorly. On milking the swelling
towards introitus blood clot came out along with a
leech and the swelling disappeared. Under intra
venous sedation vaginal douching was done with
normal saline. The hysteroscope was introduced
inside vagina which revealed a bleeding point on the
lateral wall of vagina about 1.5 cm from introitus. No
other trauma in the vagina could be seen.
A paediatric Foley's Catheter (No.10) was introduced
inside vagina and the bulb of Foley's Catheter was
inflated to 20 cc with distilled water. Three hundred ml
(300 ml) of blood was transfused to the patient.
Catheter was kept in vagina for 4 h. During the period
there was no bleeding from vagina. Routine blood
examination and coagulation profile showed normal
picture. Patient was kept admitted for 1 week before
she was discharged as leech bite patient often gets
recurrent episodes of bleeding. This patient too had
little trickle on the third day but did not require any
intervention .
We present this unusual case of vaginal bleeding in a
six-year old girl because this is a rare cause of vaginal
bleeding and its management by Foley's bulb
tamponade avoids unnecessary and complicated
intervention under general anaesthesia in a child. It
also maintains anatomical integrity of local parts.
The leech is a parasite which has a habit of entering
anatomical orifice of human being and animals. It
releases an anticoagulant (heparin like substance,
hirudinin) during the process of sucking blood which
causes bleeding from bite site. Deka and Rajeev
(2001) have reported leech bite as an unusual cause of
haematuria. Severe rectal bleeding from leech bite
case of vaginal bleeding
was reported by Raj et al. (2001), and vaginal bleeding
resulting from leech bite was reported by Hernandez
and Ramirez (1998).
REFERENCES
Deka P M and Rajeev T P. 2001. Unusual cause of haematuria.
Urol Int 66:41-42.
93
Hernandez M and Ramirez RE. 1998. Vaginal bleeding
resulting from leech bite. Ginecol Obstet Mex 66:246-248.
Raj S M, Radzi M and Tee M H. 2000. Severe rectal bleeding
due to leech bite. Gastroenterol 95:1607.
Short communication
Journal of Parasitic Diseases: June 2006, Vol. 30, No. 1, 94–97
J PD
Field evaluation of a rapid immunochromatographic
test kit for the diagnosis of Plasmodium falciparum and
non-falciparum malaria parasites from Sonitpur
district, Assam
C. Rajendran and S. N. Dube
Biotechnology Division, Defence Research Laboratory, Tezpur.
ABSTRACT. The sensitivity and specificity of ICT Parascreen test kit device (rapid test for
malaria pan/Pf) was compared with conventional microscopic method as the gold standard. A total
of 126 patients were tested with ICT and microscopic blood-smear examination. Sixty one
(48.41%) patients were found to be positive for malaria infection in microscopic examination. With
the ICT kit, the sensitivities and specificities for Plasmodium falciparum and non-falciparum (P.
vivax) parasites were 96.30 and 88.88%, and 98.48 and 98.48 %, respectively. The ICT kit gave a
little lower sensitivity as compared to microscopic examination.
Keywords: HRP II, ICT Parascreen test kit, malaria, pLDH
Globally, malaria still remains a major parasitic
disease with high morbidity and mortality. In the
Northeast region of India, the endemicity of malaria
infection and the predominance of Plasmodium
falciparum over P. vivax is now well documented (Dev
and Phookan, 1996; Kamal and Das, 2001). Accurate
diagnosis and proper treatment, which are important
to the control of malaria, are the main aims of the
global malaria control strategy (WHO, 2000a). Based
on clinical symptoms alone, malaria and non-malaria
febrile cases and the species of Plasmodium can not be
distinguished (Chandramohan et al., 2001). Further,
accurate diagnosis of malaria is essential to make a
rational choice of drug for the treatment. The
development of Rapid Diagnostic Tests (RDT) for
malaria based on immunochromatographic test (ICT)
strips provides a valid alternative to conventional
Corresponding author: Dr. Rajendran, Biotechnology Division,
Defence Research Laboratory, Post Bag No. 2, Tezpur-784 001,
Assam, India. E-mail: [email protected]
microscopic method (WHO, 1996). The ICT is a RDT
for P. falciparum specific HRP II antigen (Parra et al.,
1991) and a pan malaria antigen (Garcia et al., 1996).
RDT is very much useful where microscopy is lacking
and where the malaria is severe (WHO, 2000b). The
conventional microscopy is the gold standard test to
diagnose malaria infections worldwide. Even though
it is sensitive and economical, several disadvantages
make it inconvenient for use in the field viz.,
requirement of electricity, trained microscopy
personals and difficulties in accurate species
identification as evidenced by sensitive molecular
methods. Currently, rapid ICT cards are available to
detect malaria infection upto species level
(Wangsrichanalai, 2001). The first generation of
detection kits was designed to diagnose only P.
falciparum, but now the newer devices have been
designed to diagnose both P. falciparum specific
antigens as well as Plasmodium genus specific
antigens. Early detection and differentiation of
malaria is of paramount importance due to the
Immunochromatographic test kit evaluation for malaria diagnosis
incidences of cerebral malaria and drug resistance
associated with falciparum malaria, and due to the
morbidity associated with the other malaria forms. So,
ICT kits are considered for malaria diagnosis as
microscopic method has limitations. So far, field
evaluation of ICT kits has not been done for its use in
immediate clinical management of malaria in Sonitpur
district, Assam. Therefore, the present study was
undertaken to study the performance of the ICT kit in
the field by comparing it with the gold standard
microscopy method.
The study was conducted at seven different places of
Sonitpur district, Assam, between August 2004 and
May 2005. Each patient, who was suffering from
fever, was finger pricked by using a sterile lancet.
Thick and thin blood-smears were prepared and
stained with Giemsa according to standard
procedures. The thick smear was used to detect the
infection, whereas the thin smear was used for species
identification. A blood sample was considered
negative, when no parasite could be detected in 100
fields of an oil immersion (x1000 magnification)
objective lens of a microscope (Fernando et al., 2004).
At the same time, approximately 5 µl of whole blood
from finger prick of the patient was transferred
directly to a sample pad. For testing the sample, ICT
Parascreen test kit (Zephyr Biomedicals, India) was
used as rapid diagnostic device (Lot No: 101003, Mfg
Dt: 08-2004 and Exp. Dt: 07-2006). Parascreen
utilizes the detection of P. falciparum specific
histidine rich protein II, which is water soluble protein
that is released from parasitized-erythrocytes of
infected individuals, whereas for the detection of pan
malaria, Parascreen detects the presence of pan
malaria specific pLDH released from the parasitised
erythrocytes. Then, a drop of buffer was added and
allowed to react for 2 min. This buffer was added to
induce cell lysis and allow PfHRP II and pan-malarial
antigens to bind to colloidal gold-labeled monoclonal
antibodies. All tests were considered as valid if a
control line was observed. The sensitivity and
specificity was calculated as per the formula given by
Mason et al. (2002). The sensitivity was calculated as
the number of true positives by the test divided by total
positives by Giemsa [TP/(TP+FN)], and the
specificity was determined as true negatives divided
by the false positives (TN/TN+FP).
A total of 126 blood samples were collected. Among
these, 61 (48.41 %) samples were found positive for
malaria infection by standard microscopical blood-
95
smear examination. Out of these 61 positive samples,
52 were found positive for P. falciparum (85.24 %) and
9 were positive for non-falciparum parasite (P. vivax;
14.75 %), whereas the Parascreen ICT kit showed 54
samples positive for P. falciparum and eight for P.
vivax. A summary of the findings by these two tests has
been given in Table I. The sensitivity and specificity
values of the ICT were calculated using the results of
microscopic examination as the gold standard. As per
the Parascreen ICT kit, the sensitivity and specificity
for P. falciparum and non-falciparum (P. vivax)
parasites were 96.30 and 88.88 %, and 98.48 and 98.48
%, respectively.
The present field study revealed that the ICT kit could
detect P. falciparum from 54 cases, whereas light
microscopy showed 52 positive cases. Among the 54
cases, two blood-smears were found negative for P.
falciparum but the ICT was positive for them. The
possible reason could be due to the persistence of
PfHRP II following the clearance of P. falciparum
(Wongsrichanalai et al., 1999). But for the case of nonfalciparum (P. vivax) parasite, the ICT kit could detect
only eight cases out of nine found by microscopical
method. Generally, irrespective of the manufacturers,
the sensitivity and specificity for non-falciparum,
with the available ICT kit, is varying from 50-70% and
37.580%, respectively, as compared to microscopy.
But the present study revealed 88.88% sensitivity and
98.48% specificity. This study was conducted based
on qualitative assessment but not quantitative one as
the accurate counting of parasites was not carried out
to assess the correlation of the sensitivity. But
increasing sensitivity of the test with increasing
parasite densities is one of the main factors in
detecting parasites (Mason et al., 2002). Various
authors have reported different degrees of sensitivity
and specificity of P.f/P.v test kit manufactured from
different countries and from different localities.
Palmer et al. (1998) evaluated OptiMAL test for the
diagnosis of P. falciparum and P. vivax malaria, and it
uses a monoclonal antibody to the intracellular antigen
parasite lactate dehydrogenase (pLDH). It
differentiates species by the use of a P. falciparum
specific and a genus specific antibody. It has shown 88
and 94% sensitivities on symptomatic Honduran
patients and specificities of 100 and 99% for the
diagnosis of falciparum and vivax malarias. Beatriz E
ferro et al. (2002) evaluated OptiMAL and gave a
higher efficiency of 98.1% for P. vivax than 94.9% for
P. falciparum, in a malaria referral center in Colombia.
But Mason et al. (2002) evaluated two test kits viz,
96
OptiMAL and ICT of which both gave lower
sensitivities than the earlier report. ICT gave 86.2 and
2.9% sensitivity for P. falciparum and P. vivax and
specificity of 76.9 and 100%, but OptiMAL gave 42.6
and 47.1% sensitivity and, 97 and 96.9% specificity
for P. falciparum and non-falciparum parasites,
respectively. Kolaczinski et al. (2004) evaluated
OptiMAL for the diagnosis of P. vivax and P.
falciparum with 34% as compared to 36% by gold
standard microscopy. For OptiMAL 48 test, cross
checking of the corresponding smears at the reference
laboratory gave a sensitivity of 79.3% and a specificity
of 99.7% for P. falciparum, and the corresponding
values of 86.1% and 98.7% for P. vivax infections. The
performance of the field microscopy was better, with a
sensitivity and specificity of 85.2 and 99.7% for P.
falciparum, and 90.4 and 98.7% for P. vivax,
respectively. According to Tjitra et al. (1999), the ICT
malaria P.f /P.v test was 96% sensitive and 90%
specific for P. falciparum, and 75% sensitive and 95%
specific for P. vivax even though the blood samples
were collected in an anticoagulant, EDTA, from the
veins but not from the finger prick. Forney et al.
(2000) conducted a study in Thailand and Peru with
P.f/P.v test kit and recorded 95% sensitivity and 85%
specificity for P. falciparum, and 68% sensitivity and
87% specificity for P. vivax and concluded that the
sensitivities were closely correlated with parasite
densities. Richter et al. (2004) evaluated ICT (Malaria
ICT Now, Binax, Portland, USA) for its performance
on 2547 patients, and ICT was positive in all of 204
patients with symptomatic P. falciparum infections,
whereas microscopy revealed parasites in 202 of 204
of these patients. But, from these two cases,
trophozoites were detected by microscopy only in
samples taken after 6 and 12 h. So ICT showed a
specificity of 99.74%, whereas for non-falciparum
malaria, the sensitivity and specificity of ICT was only
50 and 37.5%, respectively, as compared to
microscopy. In our present study, one out of nine cases
was found negative for non-falciparum (P. vivax) by
ICT. Moreover, this particular blood smear had a
comparatively very low density of parasites (2
parasites/100 microscopic fields). Even though the
parasite could be seen on blood smear, the ICT missed
to detect the infection. Moody et al. (2000) conducted
a study and concluded that though parasite may look
healthy in the blood smear but it may be non-viable
and not producing pLDH. It is known that pLDH
activity gets declined with therapy. However, the ICT
kit may be a useful device where microscopy is not
available, and immediate clinical diagnosis of malaria
Rajendran and Dube
is required especially for P. falciparum cases which
may develop cerebral complications. But for nonfalciparum malaria cases, the present study has given a
moderate percentage of sensitivity and specificity but
it is difficult to suggest on this aspect, as the number of
cases studied was small. With the ICT P.f/P.v the
sensitivity for the detection of P. vivax was 96% at
parasitaemias greater than 500 parasites/µl blood but,
at parasitaemias lower than this the sensitivity was
reduced to only 29% (Tjitra et al., 1999; Hunt-Cooke
et al., 1999). The ICT can not replace the microscopy
method for the determination of parasitaemia. Several
factors may be responsible for a little lower
performance of the test kit viz, human error though the
manufacturer's instructions were strictly followed.
The information regarding the history of selfmedication by the patients and the storage condition of
the kits also need consideration. Moreover, increasing
sensitivity of the test with increasing parasite densities
is one of the main factors in detecting parasites at low
densities rather than the human error (Mason et al.,
2002). So, therefore, to assess the sensitivity and
specificity of ICT for non-falciparum parasites,
further studies should be done with larger number of
samples for the evaluation of its detection limit.
Table I. Showing the number of cases studied for the
evaluation of ICT with standard microscopic method
Parasite
Giemsa
method
ICT (Parascreen)
kit
P. falciparum
52
54
Non-falciparum
(P. vivax)
9
8
Negative
65
-
Total
126
-
ACKNOWLEDGEMENTS
The authors are grateful to The Director, Defence
Research Laboratory, Tezpur, Assam, for providing
necessary facilities and constant encouragement. The
study was not supported by the manufacturer of the
ICT kit.
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THE INDIAN SOCIETY FOR PARASITOLOGY
Executive Committee
President
Secretary
Professor Veena Tandon
Department of Zoology
North-Eastern Hill University
Shillong
Dr. J. K. Saxena
Division of Biochemistry
Central Drug Research Institute
Lucknow
Vice-President
Joint Secretary
Dr. J. Mahanta
Regional Medical Research Centre
Dibrugarh
Dr. Vas Dev
Malaria Research Center
Sonpur
Treasurer
Dr. L. M. Tripathi
Division of Parasitology
Central Drug Research Institute
Lucknow
Members
Dr. Wasim Ahmed, Aligarh
Dr. P. Prakash Babu, Hyderabad
Dr. S. C. Dutta, Kolkata
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Prof. Neelima Gupta, Bareilley
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Dr. A. M. Khan, Dibrugarh
Dr. Ashwani Kumar, Goa
Prof. G. G. Mani, Visakhapatnam
Dr. S. K. Puri, Lucknow
Prof. R. Kaleysa Raj, Trivandrum
Dr. K. K. Saxena, Bareilley
Prof. Prati Pal Singh, S. A. S. Nagar
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