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Title
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Antigenic Diversity among Oocysts of Clinical
Isolates of Cryptosporidium parvum
Author(s)
Griffin, K., Matthai, E., Hommel, M., Weitz, J.
C., Baxby, D., Hart, C. A.
Citation
The Journal of Protozoology Research, 2(3): 97101
Issue Date
URL
1992
http://ir.obihiro.ac.jp/dspace/handle/10322/169
Rights
帯広畜産大学学術情報リポジトリOAK:Obihiro university Archives of Knowledge
J. Protozool. Res., 2. 97-101 (1992)
Copyright © 1992 , Research Center for Protozoan Molecular Immunology
Antigenic Diversity among Oocysts of Clinical Isolates of
Cryptosporidium parvum
K. GRIFFIN1,2, E. MATTHAI1, M. HOMMEL1, J.C WEITZ3,
D. BAXBY2 and C. A. HART2
Department of Tropical Medicine and Infectious Diseases and Department of Medical
Microbiology, University of Liverpool, Liverpool, N. K.
and 3Insitute de Salud Publica, Santiago, CHILE
Received 11 November 1991/ Accepted 2 February 1992
Key words: Cryptosporidium parvum, monoclonal antibody, antigenic diversity
ABSTRACT
A panel of monoclonal antibodies has been produced against the protozoan parasite Cryptosporidium
parvum which is a major cause of diarrhoeal disease in man and other animals. C. parvum oocysts from
both a human and a bovine case of cryptosporidiosis were used as immunogens. A total of 11 different
monoclonal antibodies were obtained which could bind to formalin-fixed oocysts. One was IgA but the
remainder were all of IgM isotype. The reactivity of these monoclonal antibodies against a series of C.
parvum oocysts obtained from 25 patients in Chile was examined using indirect immuno-fluorescence.
Although a mixture of all of the 11 antibodies would have detected oocysts in each of the samples, no one
monoclonal antibody recognised all oocysts. Each antibody showed a different recognition pattern. Thus
by using these monoclonal antibodies we have demonstrated that there is tremendous antigenic variability
among C. parvum oocysts. These antibodies should prove most useful in examining the epidemiology of
C. parvum infections.
INTRODUCTION
Cryptosporidium is a coccidian parasite that infects the intestinal cells of many animal species including
man (Hart and Baxby 1987; Crawford and Vermund 1988), domestic animals and birds (Angus 1983;
Tzipori 1983). Only recognised as a pathogen of man in 1976 (Nime et al 1976; Meisel et al 1976), it has
since been found to be a significant cause of diarrhoeal disease in both immunocompetent and
immunodeficient individuals (Soave and Armstrong 1986).
Originally it was thought that Cryptosporidium was species specific and at least 20 species were named
according to the animals from which they were first isolated. This is currently under review but it has
been suggested that five species be recognised C. parvum and C. muris that infect mammals, C.
meleagridis and C. baileyi that infect birds and C. nasorum that infects reptiles (Levine 1984; Current
1986). C. parvum is the cause of most, if not all human infections.
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MONOCLONAL ANTIBODIES AGAINST C. PARVUM
Monoclonal antibodies (McAbs) have been produced that recognise an epitope present on all C. parvum
oocysts and these have been used in immuno-diagnostic tests (Sterling and Arrowood 1986; McLaughlin
et al 1987). These epitopes are also apparently present on C. muris and C. baileyi oocysts. In contrast,
panels of McAbs have been used to study antigenic diversity in several protozoan parasites (Knowles et al
1984), including Cryptosporidium (McDonald et al 1991). In order to further investigate antigenic
diversity among C. parvum oocysts, we have produced panels of McAbs against a human and a bovine
isolate and tested their reactivity against a panel of 27 human isolates obtained from diarrhoeic patients in
Chile.
MATERIALS AND METHODS
Oocysts
Human clinical isolates were prepared from faeces by sucrose phenol flotation and preserved in 10%
formol saline. Monoclonal antibodies were produced against a human isolate from the South of England,
R(c), provided by Dr. V. McDonald (London School of Hygiene and Tropical Medicine, UK) and a
bovine isolate from Scotland provided by Dr. D. Blewett (Moredun Research Institute, Edinburgh, UK).
Production of Hybridomas
Oocysts of the human R(c) and bovine isolates were excysted by incubation with 1% trypsin (Flow
Laboratories, UK) for 60 min at 37OC followed by incubation with equal volumes of 1% sodium
deoxycholate (BDH Chemicals, UK) and 2.2% sodium hydrogen carbonate (BDH, UK) in distilled water
at 37OC for 30-40 min or until 50-70% excystation had been achieved. Female BALB/c mice were
injected intramuscularly with 1 x 107 excysted R(c) oocysts which had been suspended in phosphate
buffered saline and emulsified with an equal volume of Freund's complete adjuvant. This was followed by
1 x 107 excysted oocysts on days 108 (intraperitoneally), 118 (subcutaneously with incomplete Freund's
adjuvant) and 123 (intravenously). On day 132 the spleen was removed and a lymphocyte rich suspension
obtained. Half the lymphocytes were fused with P3-NSl-Ag4-l myeloma cells using polyethylene glycol
as the fusogen. The remaining splenic lymphocytes from the immunised mouse and some from an
unimmunised mouse were given an in vitro challenge with the excysted oocysts (1 x 107) and fused on
day 136. Hybridomas against the bovine isolate were raised using a similar methodology except that 1 x
108 oocysts were employed. Hybridoma culture supernatants were stored at –20OC until used.
Indirect immunofluorescent antibody tests (IFAT)
The Cryptosporidium oocysts preparations (R(c) and bovine) were air-dried onto slides and stored at
–20OC. Prior to use slides were thawed, air dried and fixed with 3% HCl in methanol. IFAT was carried
out as described by Danforth (1982) and the slides examined using an epifluorescent microscope
(Fluorophot, Nikon) with x 40 and x 100 objectives. Experiments were repeated on a minimum of three
occasions. To determine the reactivity of the monoclonal antibodies against the formol saline fixed
materials, IFATs were carried out using fixed immunizing antigen and non-reactive hybridomas
eliminated from the study.
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MONOCLONAL ANTIBODIES AGAINST C. PARVUM
RESULTS AND DISCUSSION
Out of a total of 15 hybridomas that produced reactive antibodies, 11 were chosen for further study since
they were able to bind to formol fixed oocyst preparations with minimal background fluorescence. Each
of the monoclonal antibodies was of IgM isotype except for 4H5 which was IgA. The reactivity of these
11 monoclonal antibodies against the Chilean isolates are shown in Table 1. All were C. parvum on the
basis of oocyst size.
The recognition patterns of the individual monoclonal antibodies were extremely variable. For example,
1F4 recognised none of the 27 isolates, 3G11 one isolate and 1C7 three isolates, whereas 2G1 recognised
IS isolates, 4H5 16 isolates and IIIE8 22 isolates. Each of the 11 monoclonal antibodies had unique
recognition patterns and no one antibody recognised each of the isolates. One antibody (1F4) did not
recognise any of the isolates although it did bind (in IFAT) to the bovine and human R(c) isolates used as
immunogens. However, the immunofluorescence produced by this antibody was punctate over the oocyst
surface which might be less readily detected on oocysts in faecal samples. Others (Sterling and Arrowood
1986; McLaughlin et al 1987) have raised monoclonal antibodies that apparently react with all C. parvum
oocysts (as well as C. muris and C. baileyi) which indicates that there is at least one common epitope on
C. parvum oocysts. Our results show that there is also tremendous antigenic variation among C. parvum
oocysts. Virtually all of the isolates had a unique recognition pattern. Only isolates 3 and 9, 5 and 13, and
11 and 12 respectively had identical reactivities within the whole panel of monoclonal antibodies.
In any one faecal sample a binding monoclonal antibody recognised most of the oocysts present. However,
some apparently bound to a minority of oocysts present in the faecal sample. This could result from
variability of antigenic expression on oocysts, from lability of epitopes or even to dual infection with
different strains of C. parvum gastroenteritis in immunocompetent individuals do occur (Hart and Baxby
1988). This panel of monoclonal antibodies should provide useful tools for determining epidemiological
similarities and differences between C. parvum isolates infecting animals in man.
ACKNOWLEDGEMENTS
We are grateful to the Wolfson Foundation and the University of Liverpool for financial support.
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MONOCLONAL ANTIBODIES AGAINST C. PARVUM
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MONOCLONAL ANTIBODIES AGAINST C. PARVUM
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