1. business and industrial
2. agriculture and forestry
3. livestock

Rev. sci. tech. Off. int. Epiz., 1993,12 (3), 909-922
Successful therapeutic regimens for treating
Brucella melitensis and Brucella abortus
infections in cows
A.I. RADWAN *, S.I. BEKAIRI *, A.M. AL-BOKMY *, P.V.S. PRASAD *,
O.M. MOHAMED ** and S.T. HUSSAIN **
Summary: Three therapeutic regimens were evaluated in 121 cows naturally
infected with Brucella melitensis or Brucella abortus, using a combination of
long-acting Oxytetracycline (LA-OTC),
streptomycin
(ST) and OTCintramammary infusion (IMI). Cessation of shedding of b r u c e l l a in udder
secretions and absence of Brucella in selected tissues were considered criteria
for successful treatment.
Regimen A (tested on 35 cows) consisted of LA-OTC 25 mg/kg
administered intramuscularly (i.m.) every 3 days for 42 days, ST 25 mg/kg i.m.
daily for 8 days, and OTC-IMI 20 ml/teat daily for 4 days. Regimen B (tested on
53 cows) was similar to regimen A, except that ST was administered every
2 days for 16 days and OTC-IMI every 2 days for 8 days. Both regimens were
equally effective in eliminating Brucella organisms from all cows involved in
the tests and no relapses were recorded. However, regimen C, which was similar
to regimen A, except that ST was administered every 3 days for 24 days and
OTC-IMI every 3 days for 12 days, resulted in the elimination of Brucella
organisms from only 30 (91%) of 33 cows.
Before commencement of the therapeutic regimens, B. melitensis biovar 1 or
2 had been repeatedly isolated from udder secretions of 103 cows and
B. abortus biovar 1 from mammary secretions of 18 cows.
KEYWORDS: Antibiotics - Brucella abortus - Brucella melitensis - Cattle Oxytetracycline - Streptomycin - Therapy.
INTRODUCTION
Several c h e m o t h e r a p e u t i c agents have b e e n employed in recent decades for the
treatment of Brucella abortus infection in cows; however, n o n e of these has been
entirely successful. N u m e r o u s chemical agents, general antimicrobials (phenols or
dyes), trace elements, minerals and mixtures of vitamins (A and E ) have b e e n tried
unsuccessfully. F u r t h e r m o r e , a t t e m p t s using antibiotics such as penicillin or
sulfonamides failed to cause cessation of the shedding of B. abortus from the mammary
secretions of infected cows, or caused only temporary cessation (7,11,37).
* Animal Production and Health Section, National Agriculture and Water Research Centre,
Ministry of Agriculture and Water, P.O. Box 17285, Riyadh 11484, Saudi Arabia.
** National Agriculture Development Co., P.O. Box 2557, Riyadh 11461, Saudi Arabia.
910
The use of broad-spectrum antibiotics such as aureomycin, terramycin, tetracyclines
and streptomycin (ST), singly or in combination, has resulted in the reduction of
abortions in infected herds or individual cows (20,21). However, the cost of therapy, the
presence of antibiotic residues in milk and the failure to cure udder infections in many
cases have led to the general conclusion that such treatment is unsuitable for the control
of bovine brucellosis.
With the development of Oxytetracycline (OTC) and long-acting (LA)-OTC, the use
of these agents alone or in combination with ST has succeeded in eliminating the
symptoms of this disease and reducing the shedding of brucellae by infected cows.
Consequently, such therapeutic regimens have been applied in infected herds to prevent
abortions and reduce the spread of brucellosis. However, these regimens have failed to
result in complete cure. Furthermore, serious local reactions were reported in cows, due
to repeated intraperitoneal (i.p.) inoculations with O T C (12,13,24,25,29). In addition,
the regimens previously tried involved small doses and few injections of the antibiotics
used. In one of these studies, injections of L A - O T C (20 mg/kg intramuscularly [i.m.]
every 3 days for 2 weeks) combined with ST (20 mg/kg i.m. daily for 1 week) resulted in
cessation of shedding of B. abortus in 10 (71.4%) of 14 cows (24). In a n o t h e r study,
inoculation of L A - O T C (20 mg/kg i.m. every 3 days for 2 weeks) alone was successful in
curing 3 (21%) of 14 cows. However, when L A - O T C was combined with ST (20 mg/kg
intravenously [i.V.] or i.m. daily for 1 week) 14 (67%) of 21 cows w e r e successfully
treated (25).
With regard to brucellosis in sheep, 9 sheep experimentally infected with B. abortus
were injected daily for 6 days with 1 g Chlortetracycline, then 20 days later with 1.4 g
t o g e t h e r with i m m u n e serum for 3 days. Only one sheep was found to be
bacteriologically positive at slaughter, 46 days after infection (30). Furthermore,
Radwan and colleagues (34) conducted a long-term treatment trial using a variety of
doses of O T C , alone or in combination with ST, on 118 sheep naturally infected with
B. melitensis. In this trial, groups of infected Najdi sheep were injected with 250,500 or
1,000 mg O T C daily for 6 weeks, by the i.p. route. In the respective groups, 52%, 69%
and 100% of the sheep were found to be Brucella-îre-c at the end of the trial. In addition,
treatment with OTC (250 mg i.p. daily for 6 weeks) combined with ST (1 g i.m. daily for
3 weeks) showed a synergistic effect, increasing the percentage of Brucella-tree sheep to
82%. Moreover, when 8 infected Najdi sheep were inoculated with 1,000 mg LA-OTC
i.p. every 3 days for 6 weeks, 6 sheep were Brucella-iiee. at necropsy (34). In this trial,
the majority of the treated sheep developed subcutaneous sterile abscesses in the flank
at the site of repeated i.p. inoculation with OTC. Recently, Radwan and colleagues (35)
evaluated six different long-term treatment regimens on 480 sheep and goats naturally
infected with B. melitensis, using high doses of L A - O T C combined with ST. One of
these regimens ( L A - O T C 25 mg/kg i.m. every 2 days for 4 weeks combined with
ST 20 mg/kg i.m. every 2 days for 2 weeks) proved to be the most practical, safe,
effective and least expensive means of achieving complete elimination of B. melitensis in
the 80 sheep and goats treated (35).
In view of the encouraging results in cows (24,25) and the successful results in sheep
and goats (35), the present study was undertaken to evaluate the efficacy of three
long-term therapeutic regimens using a combination of LA-OTC, ST and OTC
intramammary infusion (IMI) in eliminating B. melitensis or B. abortus from naturallyinfected cows. The aim of the study was to obtain a therapeutic regimen which is
effective, practical, without side-effects and relatively inexpensive, in order to make
treatment of Briicella-infected cows with superior genes a viable alternative to slaughter.
911
MATERIALS AND METHODS
Animals
A total of 121 cows were selected, which gave positive results for brucellosis after
serological and bacteriological tests. These animals were selected to provide a m o r e
conclusive evaluation than that obtained in previous studies. The cows originated from
three large commercial dairy herds raised in the central and eastern provinces of Saudi
Arabia. T h e animals belonged to two breeds (108 Holstein x Friesian and 13 Jersey).
The cows were aged 3-10 years and weighed between 450 kg and 750 kg. The three herds
were composed of highly valuable breeding cows originally imported from the United
States of America (USA) and Europe. Herd records indicated that none of the cows had
been vaccinated during calfhood.
In two herds, B. melitensis was isolated from udder secretions of 103 cows (biovar 1
from 97 cows and biovar 2 from 6 cows). These two herds originally tested seronegative
on arrival in Saudi A r a b i a , approximately four years prior to this study. T h e cows
contracted brucellosis from sheep and goats infected with B. melitensis (biovars 1 and 2)
which had been introduced to the two farms 8-12 m o n t h s before bovine brucellosis
occurred in the two cattle herds (unpublished findings).
In the third herd, B. abortus biovar 1 was isolated from the mammary secretions of
18 cows. This herd contained few seropositive cows on arrival in Saudi A r a b i a , eight
years prior to the present study.
Shortly before initiation of the present treatment regimens, 89 cows were found not
to be pregnant (having already aborted or calved 4-8 months before t r e a t m e n t ) and
32 cows were in various stages of pregnancy. Milking was ceased in lactating cows. The
121 cows were divided into three treatment groups (A, B and C), without regard to age,
number of previous pregnancies, date of recent pregnancy and n u m b e r of previous
abortions due to Brucella. The reason for the division into three treatment schedules
was to choose the most practical and effective regimen, particularly because L A - O T C is
a long-acting antibiotic, in contrast to ST and OTC-IMI which are not. Since the number
of available cows infected with B. abortus was small, these were placed in g r o u p B
together with 35 cows with B. melitensis. All cows in the study were isolated from other
animals. Non-treated cows were not included as controls, as several previous studies
have indicated that Brucella infections persist in cattle and s p o n t a n e o u s recovery is
uncommon (10,22,28).
One m o n t h after t r e a t m e n t , all n o n - p r e g n a n t cows were artificially inseminated.
Three months later, a total of 12 pregnant cows (4 from each t r e a t m e n t group) were
sacrificed, and selected tissues were collected for bacteriological examination. In
addition, immediately after birth, a total of 3 calves (1 from a cow in each treatment
group) were sacrificed, and selected tissue samples were cultured.
Therapeutic agents
The pharmaceutical products used in this study were as follows:
- L A - O T C injectable solution (from F i a n c e ) containing 200 mg O T C base p e r
millilitre
- ST sulphate (from Giza, E g y p t ) supplied in vials, each containing l g or 4 g,
dissolved (just before use) in 3 ml or 12 ml sterile distilled water, respectively
912
- OTC-IMI (from Athens, Greece) in 10 ml syringes, each containing 200 mg OTC
hydrochloride, 100 mg neomycin sulphate, 100 mg oleandomycin p h o s p h a t e , 5 mg
Prednisolone and 10 ml special excipient.
Therapeutic regimens
The treatment regimens of the three groups of infected cows are shown in Table I.
Both ST and OTC-IMI were administered with L A - O T C at the c o m m e n c e m e n t of
therapy. The L A - O T C and ST were injected i.m. in the cervical, thigh and shoulder
muscles, at a dosage of 20-30 ml per site. The date of previous abortions in relation to
the initiation of treatment was not recorded on the farms involved.
Serological testing
All animals were subjected to three successive serological examinations at monthly
intervals. A t each examination, animals were initially screened for the presence of
Brucella agglutinins by the rose bengal test (3). T h e rose bengal test antigen was
obtained from the Central Veterinary Laboratory, New Haw, Weybridge, United
Kingdom. Animals testing positive to the rose bengal test were retested by the standard
US plate agglutination procedure for the determination of Brucella antibody titres (3).
The standard Brucella plate antigen (made of B. abortus strain 1119-3) was obtained
from the United States Department of Agriculture in Ames, Iowa, USA. Agglutination
at 1:100 or greater was considered positive.
Following the three serological examinations, milk samples from all seropositive cows
were subjected to bacteriological examination for identification of Brucella shedders.
Furthermore, sera from the selected 121 serologically- and bacteriologically-positive
cows were serologically tested again immediately before initiation of the present
treatment regimens and at monthly intervals after completion of treatment (for 4 months
in 12 sacrificed cows and for 16 months in the remaining 109 cows). In addition, sera from
all calves born from treated cows were examined immediately after birth.
Bacteriological examination
T h e m e d i u m for Brucella culture was p r e p a r e d as described previously (35).
Immediately before initiation of the therapeutic regimens, cultures of udder secretions
were m a d e from the selected 121 positive cows to confirm shedding of Brucella
organisms. Separate 30 ml samples of udder secretions from each quarter were also
cultured every week during treatment, every month after completion of treatment until
calving and again every 2 weeks after calving for 6 m o n t h s . In addition, from the
12 pregnant sacrificed cows (4 cows from each treatment group which were slaughtered
at 4 months post-treatment) the following tissue specimens were aseptically collected
for bacteriological examination: supramammary, prescapular, iliac, precrural,
mediastinal, mesenteric, popliteal and head lymph nodes, udder secretion, and sections
of udder, uterus, ovary, brain, liver and spleen. In addition, samples of stomach contents,
liver, lung and spleen of foetuses from the sacrificed animals were cultured. Similar
selected tissue specimens were cultured from 3 sacrificed newborn calves (1 from a cow
in each treatment group).
Aliquots from each udder secretion sample were spread with sterile cotton swabs on
2-4 freshly prepared plates of culture medium. Each tissue specimen was individually
homogenised in a tissue grinder and aliquots were spread with sterile cotton swabs on
several (4-8) freshly prepared plates of culture medium. The plates were incubated at
37°C for 7 days in the presence and absence of 10% C 0 a t m o s p h e r e . T h e isolated
2
i.m.:
IMI:
a)
b)
c)
d)
e)
f)
g)
C
i.m.
25mg/kg
i.m.
25mg/kg
IMI 20 ml/teat
3
3
3
3
™°
25mg/kg
2
2
E
Y
8
1
4
3
4/4
4
4
^¿J.
FA
v
3
14
14
8
b
3/4
8
27/29(91%)
14
49/49(100%)
4/4 31/31 (100%)
No of
Success/treated
Tissuesa)Udder secretions < >
intramuscular (injection)
intramammary infusion
number of sacrificed cows from which selected tissues were cultured for Brucella
number of cows from which mammary secretions were repeatedly cultured for Brucella
cows naturally infected with B. melitensis
cows naturally infected with B. abortus
long-acting Oxytetracycline injectable solution (from France) 200 mg/ml
streptomycin sulphate (from Egypt)
Oxytetracycline intramammary infusion (from Greece)
LA-OTC( )
STW
&OTC-IMl(s)
e
33W
B
e
LA-OTC( )
i.m.
i.m.
25mg/kg
&OTC-IMlig)
IMI 20 ml/teat
35(c)
ST(f)
18( )
d
Dosage
25mg/kg
i.m. 25 mg/kg
IMI 20 ml/teat
1
i.m.
35(c)
f
Route
A
e
No.of Periodicity
treated
Therapeutic agents
cows
LA-OTC( )
ST( )
& OTC-IMI(g)
Regimen
Efficacy of three treatment regimens in eliminating Brucella melitensis or B. abortus
from naturally-infected cows
TABLE I
913
914
Brucella cultures were identified morphologically, microscopically, biochemically and
serologically (3). The biotyping of the identified isolates was performed at the Central
Veterinary Laboratory, New Haw, Weybridge, United Kingdom.
When Brucella organisms could no longer be recovered from udder secretions nor
from any of the selected tissue specimens collected at necropsy, the respective cow was
considered to be cured (successful t r e a t m e n t ) . C o n t i n u e d or r e s u m e d shedding of
Brucella organisms in udder secretions or the isolation of Brucella organisms from any
of the tissue specimens obtained at necropsy were considered as conclusive evidence of
treatment failure.
In this study, aborted foetuses from the three cattle herds involved were cultured for
Brucella isolation, in addition to routine bacteriological examination for listeriosis,
salmonellosis, mycoplasmosis and mycotic abortion.
RESULTS
Breeding performance
A comparison was made between breeding performance in the three herds prior to
identification of the infected cows and that in the treated animals in the breeding season
following treatment. Before t r e a t m e n t , the cows had b e e n living in an infected
environment for periods of between 1 and 8 years. The abortion rate in the three herds
ranged between 5% and 1 5 % and the infertility r a t e was approximately 1 5 % . In
addition, there were unusually high rates of metritis, retained placenta and mastitis,
with milk production reduced by 10-15%. However, in the breeding season following
treatment, the cows were living in an uninfected environment. Of the cows treated with
regimens A, B and C, 94% b e c a m e p r e g n a n t and experienced n o r m a l calving. No
abortions were observed a m o n g the treated cows. On the whole, the treated cows
expelled placenta from the womb without requiring any special manual manipulation,
or the use of c h e m o t h e r a p e u t i c measures. F u r t h e r m o r e , the 32 cows which were in
various stages of pregnancy at the time of initiation of the therapeutic regimens A, B
and C experienced n o r m a l calving within 4 m o n t h s following completion of the
treatment. Moreover, there were no cases of metritis or mastitis in the treated cows, and
milk production was within the normal range for uninfected animals of the same breed.
Side-effects of the therapeutic agents
In 8 cows, there was very light swelling and signs of painful sensations at the site of
r e p e a t e d L A - O T C injections, particularly in the thigh muscles. T h e s e slight local
reactions disappeared within 36 hours. Meat inspection of the twelve cows which were
slaughtered (4 months post-treatment) revealed complete absence of any detectable
abnormalities in the muscles at the site of intramuscular inoculations of L A - O T C and
ST. Furthermore, no systemic reactions were observed in any of the treated cows during
the treatment period.
Cost of therapeutic agents and animals
The weight of the treated cows ranged between 450 kg and 750 kg, and the average
cost of the therapeutic agents alone used (regimen A, B or C) in treating a cow weighing
600 kg was US$186 (SR700).
915
The treated cows were imported a few years previously as pregnant heifers, at a cost
of US$3,600 each, including the cost of air freight to Saudi Arabia.
Serological findings
Among the treated animals, there was a decrease of only one dilution in the US plate
agglutination test (from 400 to 200) from the beginning of t r e a t m e n t until 4 months
post-treatment in 4 of 12 slaughtered cows. T h e remaining 8 slaughtered cows did not
show any decrease in titre over the same period.
The Brucella agglutinin titres w e r e also c o m p a r e d with results o b t a i n e d p r e treatment and 16 months post-treatment in the remaining 109 treated cows. There was a
decrease of two, t h r e e and four dilutions in 4 2 % , 4 3 % and 10% of the treated cows
respectively. In addition, only 5% of the 109 cows became seronegative 16 months posttreatment, while all calves born from treated cows were seronegative for brucellosis.
Bacteriological findings
In the present study, no Listeria, Salmonella, Mycoplasma or pathogenic fungi were
isolated from the aborted foetuses examined in the three herds; only B. melitensis or
B. abortus w e r e isolated. T h e efficacy of the t h r e e t r e a t m e n t regimens applied in
eliminating B. melitensis or B. abortus from 121 naturally-infected cows is shown in
Table I.
In regimen A, the selected tissue specimens from 4 sacrificed cows (at 4 months posttreatment) and all repeated udder secretion samples from the remaining 31 cows (for
16 months post-treatment) were all found to be free from Brucella organisms.
When 53 cows (of which 35 were naturally infected with B. melitensis and 18 infected with
B. abortus) were treated with regimen B, all selected tissue samples from 4 slaughtered
cows and the repeated udder secretion samples were found to be Brucella-free.
However, when 33 cows (naturally infected with B. melitensis) were treated with
regimen C, B. melitensis biovar 2 was isolated (1-5 colonies per plate) from mammary
secretions, udder tissue and supramammary lymph nodes of only 1 of 4 sacrificed cows
(at 4 months post-treatment). In addition, B. melitensis biovar 1 was isolated from the
repeated udder secretion samples from only 2 of 29 cows. The average number of
Brucella colonies recovered per plate from the udder secretion samples of these 2 cows
prior to treatment was approximately 30 times higher than the number recovered
following treatment, which ranged between 2 and 8 colonies per plate. However, when
these 2 Brucella-shedding cows were re-treated with regimen B, they ceased shedding
Brucella.
All selected tissue specimens from the 3 newborn calves (1 calf from a cow in each
treatment group) were found to be free from Brucella organisms.
In all successfully treated cows in the present study (118 cows treated with regimen
A, B or C), shedding of Brucella organisms in mammary secretions had ceased when
these secretions were cultured (1 week after initiation of the antibiotherapy) and did
not recommence. However, in the 3 cows of regimen C where t r e a t m e n t failed, the
amount of shedding declined after the initiation of treatment and remained low until
4 months post-treatment in 1 slaughtered cow, or after calving in the other 2 cows (these
animals were then re-treated with regimen B).
916
DISCUSSION
In order to increase milk production, commercial dairy production was introduced to
Saudi Arabia through the importation of valuable and highly productive exotic cattle at
a very high cost. These cattle were originally imported as p r e g n a n t heifers from the
USA and some E u r o p e a n countries, namely: the Netherlands, Germany and France.
On arrival in Saudi Arabia, the cattle were raised in large n u m b e r s u n d e r intensive
management systems and sometimes together with small ruminants.
Brucellosis has been reported and confirmed in both livestock and humans in Saudi
Arabia. B. melitensis biovars 1, 2 and 3 were responsible for all infections in sheep,
goats, camels and dairy cattle, except cows in one dairy herd which had B. abortus.
Consequently, B. melitensis accounted for 92% of human cases and B. abortus for 8%,
while B. suis was not found in animals or humans (1,2,4,5,6,16,18,19,23,31,32,33,34,
35,39).
In the present study, two of the cattle herds contracted B. melitensis infection from
sheep and goats raised on the same farms. Transmission of this infection to cattle
p r o d u c e d an infection similar to that p r o d u c e d by B. abortus. Such infection was
directly responsible for the high rate of abortions, infertility, metritis, retained placenta
and mastitis with reduced milk production, leading to serious consequences for cattle
performance and cattle handlers. However, in B. abortus infection, it is recognised that
the economic consequences of infection are e n c o u n t e r e d principally during initial
infection and become less prominent in further pregnancies.
In a campaign against brucellosis, it is impossible to apply only one method which
would suit every country. The "test and slaughter" m e t h o d combined with calfhood
vaccination no doubt contributes to considerable reduction in the number of infected
herds; however, for economic reasons this method cannot be applied in all cases. An
eradication campaign through slaughter and compensation is not within the economic
scope of developing countries, especially where it is difficult to change agricultural
customs or social habits. On the other hand, mass vaccination of infected herds protects
only uninfected animals without altering the course of infection. T h e infected
vaccinated animals continue to present a serious public health risk. In addition, one
worker assessed the average cost of an infected cow at approximately US$3,200, not just
for abortion or stillbirth, but from the subsequent problems or irregular breeding, loss
of milk production, and the reduced h u m a n productivity resulting from a spread of
infection (38). Moreover, with the development and widespread use of techniques such
as artificial insemination and embryo transfer, cattle with superior genetic potential
have become increasingly valuable. Slaughter of infected animals with superior genetic
potential has serious economic and genetic consequences (24). Livestock producers in
Saudi Arabia and in many other developing countries cannot afford the traditional "test
and slaughter" approach used in developed countries. A n effective, practical and safe
antibiotherapy would be of e n o r m o u s benefit to producers in these countries as an
alternative to slaughter of infected animals of high value. Although this has been the
goal of several workers, none has been fully successful.
T h e following explanations were given for the previous t r e a t m e n t failures and
difficulties:
a) Brucella organisms are known to survive within phagocytic cells of the
reticuloendothelial system, particularly in lymph nodes, liver, spleen, bone marrow, the
917
mammary glands, reproductive organs and tissues with a weak blood supply, and are
therefore p r o t e c t e d from antibodies, c o m p l e m e n t and antibiotics. T h e ability of
Brucella organisms to survive intracellularly can result in long-term chronic infection.
In addition, relapses after antimicrobial t r e a t m e n t w e r e also explained by the
intracellular location of brucellae (9,24,25,26).
b) Incorrect choice of antibiotics (7,11,37).
c) Insufficient doses of effective antibiotics (12,13,24,25,26,29).
d) Insufficient duration of treatment (8,12,13,24,25,26,29).
e) Use of the intraperitoneal route for administration of antibiotics created serious
reactions in some cases (17,34).
f) Use of only one antibiotic (20,21,24).
g) High cost of antibiotherapy (24,25,34).
h) Failure to cure udder infections in many cases (24,25,26).
i) Presence of antibiotic residues in milk and m e a t of t r e a t e d animals, and the
possible harmful effect on consumers (27).
The present study aimed to overcome the above problems and involved work under
particularly difficult conditions with regard to the level of infection among the cows
used, which were all serologically and (particularly) bacteriologically positive.
Consequently, the following points were taken into account when attempting to obtain
more successful results. Since successful therapy in human beings appears to depend on
the permeability of cell walls to drugs (24), the O T C base was used because it is capable
of penetrating intracellularly and inhibits bacterial protein synthesis at the level of the
ribosomes. The L A - O T C variant was selected to save time and effort and to provide
long-lasting O T C concentration in blood plasma, as one injection gave an effective
concentration of 0.6 ug/ml for three days (26). ST was also used, because it is known to
inhibit p r o t e i n synthesis of G r a m - n e g a t i v e bacteria. F u r t h e r m o r e , ST acts
synergistically with O T C to inhibit growth of B. abortus within bovine cells cultured
in vitro. O T C at 0.5-1.0 µg/ml, p e r m i t t e d growth or was bacteriostatic for brucellae
within tissue cells. However, O T C proved to be effectively bactericidal when combined
with 10 µg/ml of ST (15, 25, 34, 36). In addition, a combination of L A - O T C and ST
proved very effective in curing sheep and goats naturally infected with B. melitensis
(35). However, early studies (20,24,25) on the treatment of bovine brucellosis indicated
poor success rates when L A - O T C or ST was used alone. For these reasons, L A - O T C
combined with ST was used for the t r e a t m e n t of cows naturally infected with
B. melitensis or B. abortus.
Furthermore, in infected cows, despite the use of a combination of L A - O T C and ST,
several authors failed to cure udder infections in many cases (24,25,26). For this reason,
before initiation of the present treatment regimens, milking was stopped in lactating
cows and udders were completely evacuated before each injection, in order to decrease
the n u m b e r of Brucella organisms in u d d e r tissues and secretions. Consequently,
OTC-IMI was used locally in combination with systemic (i.m.) inoculation of L A - O T C
and ST. This c o m b i n a t i o n p r o v e d to be highly effective in eliminating Brucella
organisms from all infected cows in regimens A and B. However, O T C - I M I and ST,
when administered every 3 days in regimen C, were less effective in eliminating Brucella
organisms in infected cows. This may be due to the'fact that both ST and OTC-IMI are
918
not long-acting antibiotics. Also, it seems unlikely that O T C - I M I alone would be
effective for the treatement of lactating cows due to the continuous flushing of OTC by
milking, and the protection of Brucella organisms in the presence of milk.
Previous studies in cows and sheep (17, 34) concluded that i.p. inoculation of OTC
was not safe, due to the development of several serious local reactions at the site of
injection. In the present study, i.m. inoculation of L A - O T C did not result in harmful
systemic effects or any obvious lasting local reactions at the site of inoculation.
The doses and duration of application of selected antibiotics in the present study
indicate that the long-term therapy and the drug combinations used yielded better
results than several other regimens (8,12,13,24,25,26,29).
In humans, although O T C has been considered the most effective antibiotic for
treatment of brucellosis, a small number of relapses do occur. Relapses have also been
reported in cows (14,24,25). In the present study, relapses occurred in only 3 of 33 cows
treated with regimen C. However, relapses did not occur in any of the 88 cows treated
with regimen A or B (provided that the treatment protocol is strictly observed). In the
present trial, the majority (approximately 90%) of the cows treated were monitored
bacteriologically for 16 months post-treatment in order to detect possible relapses.
Even in relapsed cows (3 animals in regimen C), only a small n u m b e r of Brucella
colonies were recovered from large tissue samples.
In the present study, all t r e a t e d cows were dried before initiation of treatment;
consequently, there was no risk of harmful effects on h u m a n beings from antibiotic
residues in the milk from these cows during and after treatment. However, the residues
of antibiotics in milk and meat of treated animals should be investigated to determine
accurately when milk and/or meat can be safely consumed following completion of the
treatment regimens.
The present results of serological examinations were of some use in evaluating the
effectiveness of the treatment regimens. However, the results were not conclusive, as
animals were only monitored for 16 months post-treatment, and t h e r e was no nontreated control group for comparison. T h e results obtained were in agreement with
findings on successful treatment of brucellosis in sheep and goats when monitored for
8 months post-treatment (35). Other investigators, however, reported that serological
findings were not useful in evaluating the effectiveness of treatment regimens (24,34).
In Saudi Arabia, the treated cows were originally imported as pregnant heifers at an
average cost of US$3,600, including the cost of air freight. The cost of antibiotics used
was calculated according to sale prices in the Saudi market at the time of the experiment
(US$1 = SR3.75). T h e average cost of the antibiotics used in regimen A, B or C for
treating a cow of an average weight of 600 kg was US$186 (SR700). This did not include
the cost of e q u i p m e n t or interventions, because all serological and bacteriological
examinations were performed free of charge at Government institutions. However, in
other situations, the cost of supplies and intervention should be considered. The cost of
successful treatment of a cow represents approximately 5% of the original price of the
animal, which is considered economically feasible in Saudi Arabia.
In conclusion, when treatment protocols were strictly observed, regimens A and B
proved to be effective, practical, without side-effects and relatively inexpensive in
causing cessation of the symptoms of brucellosis and eliminating Brucella organisms
from infected cows. Consequently, at the present time, this treatment would probably
be limited to valuable breeding animals. However, further research may yield a regimen
919
which could b e used for the t r e a t m e n t of all infected animals in a h e r d and for
prophylaxis of those animals recently exposed to Brucella infection. Furthermore, it is
concluded that the failure of a small number of cows to respond to regimen C cannot be
accounted for by t h e d e v e l o p m e n t of resistance by Brucella. T h e t r e a t m e n t of
seropositive animals should be performed together with vaccination of all seronegative
animals in the same herd to achieve effective control and minimise public health risks.
ACKNOWLEDGEMENTS
The a u t h o r s wish to express their sincere t h a n k s to M.S. Bin Salamah, D i r e c t o r
General of the Saudi National Agriculture and Water Research Centre and National
Director of Project 016/UTFN/SAU/FAO, for his encouragement and co-operation. The
authors also acknowledge the technical assistance of A . Mukayel and A . Al-Taher.
Thanks are also extended to the owners and veterinary staff of the three dairy farms
involved, for their s u p p o r t and c o - o p e r a t i o n in t h e i m p l e m e n t a t i o n of the p r e s e n t
treatment regimens.
*
* *
DES PROTOCOLES THÉRAPEUTIQUES EFFICACES POUR LE TRAITEMENT
DE L'INFECTION PAR BRUCELLA
MELITENSIS ET BRUCELLA
ABORTUS
CHEZ LES VACHES. - A.I. Radwan, S.I. Bekairi, A.M. Al-Bokmy, P.V.S. Prasad,
O.M. Mohamed et S.T. Hussain.
Résumé : Trois protocoles thérapeutiques ont été évalués chez 121 vaches
atteintes d'une infection naturelle par Brucella melitensis ou Brucella abortus,
combinant à la fois l'Oxytétracycline à action prolongée
(long-acting
Oxytetracycline : LA-OTC), la streptomycine (ST) et l'infusion intramammaire
d'OTC (OTC-intramammary infusion : OTC-IMI). Deux critères ont été jugés
déterminants pour conclure au succès du traitement : l'arrêt de l'excrétion de
brucelles dans les sécrétions mammaires et l'absence de brucelles dans des tissus
spécifiques.
Le protocole A (appliqué à 35 vaches) consistait en une administration
intramusculaire (i.m.) de 25 mg/kg de LA-OTC tous les trois jours pendant
42 jours, de 25 mg/kg de ST (i.m.) quotidiennement pendant huit jours et en une
OTC-IMI quotidienne de 20 ml par trayon pendant quatre jours. Le protocole
B (appliqué à 53 vaches) était similaire au précédent, sauf pour la ST qui était
administrée un jour sur deux pendant 16 jours et pour l'OTC-IMI, pratiquée un
jour sur deux pendant huit jours. Les deux modes de traitement se sont révélés
aussi efficaces l'un que l'autre pour l'élimination totale des brucelles des vaches
objet de l'expérimentation et aucune récidive n'a été constatée. Quant au
protocole C, similaire au protocole A, hormis pour la ST qui était administrée
tous les trois jours pendant 24 jours et l'OTC-IMI, pratiquée également tous les
trois jours pendant 12 jours, il n'a permis l'élimination des brucelles que sur 30
(91 %) des 33 vaches traitées.
Avant l'application de ces trois protocoles thérapeutiques, les bio types 1 ou 2
de B. melitensis ont été régulièrement isolés dans les sécrétions mammaires de
103 vaches et le biotype 1 de B. abortus dans celles de 18 vaches.
920
MOTS-CLÉS : Antibiotiques - Bovins - Brucella abortus - Brucella
melitensis - Oxytétracycline - Streptomycine - Thérapie.
*
PROTOCOLOS TERAPÉUTICOS EFICACES PARA EL TRATAMIENTO DE LA
INFECCIÓN POR BRUCELLA MELITENSIS Y BRUCELLA ABORTUS EN VACAS.
- A.I. Radwan, S.I. Bekairi, A.M. Al-Bokmy, P.V.S. Prasad, O.M. Mohamed y S.T. Hussain.
Resumen: Se evaluaron tres protocolos terapéuticos en 121 vacas infectadas por
vía natural por Brucella melitensis o Brucella abortus, a partir de una
combinación
de oxitetraciclina
de acción prolongada
(long-acting
Oxytetracycline: LA-OTC), estreptomicina (ST) e infusión intramamaria de
OTC (OTC-intramammary infusion: OTC-IMI).
Para considerar el
tratamiento exitoso se tuvieron en cuenta dos criterios: el cese de la excreción de
brucelas en las secreciones mamarias y la ausencia de brucelas en tejidos
específicos.
El protocolo A, que fue experimentado en 35 vacas, consistía en la
administración intramuscular (i.m.) de 25 mg/kg de LA-OTC cada tres días
durante 42 días, de 25 mg/kg de ST (i.m.) diarios durante ocho días y en una
OTC-IMI diaria de 20 ml por pezón durante 4 días. El protocolo B, que se
experimentó en 53 vacas, era similar al anterior excepto en cuanto a la ST, que
se administró cada dos días durante 16 días, y ala OTC-IMI, que se administró
cada dos días durante ocho días. Uno y otro tratamiento se mostraron
equivalentemente eficaces para la eliminación total de las brucelas en las vacas
tratadas y no se constató ninguna recidiva. Por último, el protocolo C, similar al
protocolo A salvo en que la ST se administraba cada tres días durante 24 días y
la OTC-IMI también cada tres días durante 12 días, sólo permitió eliminación
de brucelas en 30 de las 33 vacas tratadas, es decir, un 91%.
Antes de aplicar estos tres protocolos terapéuticos, los biotipos 1 o 2 de
B. melitensis fueron regularmente aislados en las secreciones mamarias de
103 vacas y el biotipo 1 de B. abortus en las secreciones mamarias de 18 vacas.
PALABRAS CLAVE: Antibióticos - Bovinos - Brucella abortus - Brucella
melitensis - Oxitetraciclina - Estreptomicina - Terapéutica.
REFERENCES
1. AL-MARSHEDY A.R. & HARLAND P.S.E.G. (1986). - Brucellosis in children. Ann. Saudi
Med., 6,25-27.
2. A L - M E Z A I N I S., SINOUSSI Y , CHANG S.H., HAWARI A., CHEN M . C . , WANG C F . & CHOU
N.Y. (1984). - A study on brucellosis among animals of some farms and rural areas of
Saudi Arabia. Proc. Saudi biol. Soc, 7,351-357.
3. ALTON G.G., JONES L . M . , A N G U S R.D. & V E R G E R J . M . (1988). - Techniques for the
brucellosis laboratory. Institut National de la Recherche Agronomique, Paris, 190 pp.
921
4. AL-ZEFTAWI N . M . , AL-ISSA M . A . , BEKAIRI S.I., RADWAN A.I. & H A F E Z S.M. ( 1 9 8 5 ) . -
Epidemiology of brucellosis among abattoir workers in Saudi Arabia. Ann. Saudi Med.,
6,29-31.
5. ARRIGHI H . M . ( 1 9 8 6 ) . - Brucellosis surveillance in Saudi Arabia's eastern province. Ann.
Saudi Med., 6,5-10.
6. BASHIR R., A L - K A W I M . Z . , H A R D E R E . J . & JINKINS J . ( 1 9 8 5 ) . - Nervous system
brucellosis: diagnosis and treatment. Neurology, 3 5 , 1 5 7 6 - 1 5 8 1 .
7. BERMAN D . T . , IRWIN M . R . & B E A C H B.A. ( 1 9 4 6 ) . - T h e effect of penicillin on cows
infected with Brucella abortus. Cornell Vet., 3 6 , 3 1 1 - 3 1 3 .
8. COLE J . R . , HENDRICKS J . B . & H E R R O N D . B . ( 1 9 8 0 ) . - Efficacy
of
injectable
Oxytetracycline in the prevention of bovine brucellosis. Proc. Ann. Meet. US Anim. Hlth
Assoc., 8 4 , 1 1 2 - 1 1 7 .
9. COLLINS F . M . & CAMPBELL S.G. ( 1 9 8 2 ) . - Immunity to intracellular bacteria. Vet. Immun.
Immunopath., 3, 5-66.
10. COTTON W.E. ( 1 9 1 3 ) . - The persistence of the bacillus of infectious abortion in the tissues
of animals. Am. vet. Rev., 44,307-318.
11. DUMARESQ J.A. ( 1 9 4 0 ) . - Effect of sulfanilamide on Brucella abortus infection in guinea
pigs and cows. Aust. vet. J., 1 6 , 1 0 2 - 1 0 7 .
12. FENSTERBANK R. ( 1 9 7 6 ) . - Traitement de vaches atteintes de brucellose ancienne par
l'oxytétracycline. Ann. Rech. vét, 7 , 2 3 1 - 2 4 0 .
13. FENSTERBANK R., PLOMMET M . & PARDON P. ( 1 9 7 5 ) . - Traitement de la brucellose bovine
par l'oxytétracycline. Ann. Rech. vét., 6 , 4 3 - 6 6 .
14. GUERRA M . A . & NICOLETTI P. ( 1 9 8 6 ) . - Comparison of susceptibility of Brucella abortus
isolates obtained before and after cows were treated with Oxytetracycline and
streptomycin. Am. J. vet. Res., 4 7 , 2 6 1 2 - 2 6 1 3 .
15. HALL W.H. & MANION R.E. ( 1 9 7 0 ) . - In vitro susceptibility of Brucella to various
antibiotics. Appl. Microbiol., 2 0 , 6 0 0 - 6 0 4 .
16. HASHIM N.H., GALIL G.A., HULAIBI M . A . & AL-SALEEM E . M . ( 1 9 8 7 ) . - T h e incidence of
brucellosis and species of Brucella organisms isolated from animals in Al-Hassa. Wld
Anim. Rev., 6 1 , 3 2 - 3 5 .
17. HELFNE M . M . ( 1 9 7 4 ) . - Preliminary results of a field trial of Oxytetracycline for the
treatment of brucellosis. Bull. Soc. Sci. vét. Méd. comp. Lyon, 7 6 , 3 9 3 - 3 9 9 .
18. KAMBAL A . M . , M A H G O U B E.S., JAMJOOM G . A . & CHOWDHURY M . N . H . ( 1 9 8 3 ) . -
Brucellosis in Riyadh, Saudi Arabia. A microbiological and clinical study. Trans. R. Soc.
trop. Med. Hyg., 11,820-824.
19. KIEL F.W. & KHAN M . Y . ( 1 9 8 7 ) . - Analysis of 5 0 6 consecutive positive serological tests
for brucellosis in Saudi Arabia. J. clin. Microbiol, 2 5 , 1 3 8 4 - 1 3 8 7 .
20. KING N.B., V E N Z K E W . G . & E D G I N G T O N B.H. ( 1 9 5 2 ) . - Studies on the experimental
treatment of bovine brucellosis. Am. J. vet. Res., 1 3 , 1 5 2 - 1 5 7 .
21. LARSEN P.H. & GILMAN H . L . ( 1 9 5 0 ) . - Aureomycin as a treatment of acute brucellosis of
cattle. Cornell Vet., 4 0 , 2 5 9 - 2 7 2 .
22. MCCULLOUGH N.B., EISELE C.W. & BYRNE A . F . ( 1 9 5 1 ) . - Incidence and distribution of
Brucella abortus in slaughtered Bang's reactor cattle. Publ. Hlth Rep., 6 6 , 3 4 1 - 3 4 5 .
23. MADKOUR M . M . , MOHAMED A.E., SAMAD TALUKDER M . A . & KUDWAH A.J.N. ( 1 9 8 5 ) . -
Brucellosis in Saudi Arabia. Saudi med. J., 6 , 3 2 4 - 3 3 2 .
922
24. MiLWARD F.W., NicoLETTi P. & HOFFMAN E. (1984). - Effectiveness of various
t h e r a p e u t i c r e g i m e n s for bovine brucellosis. Am. J. vet. Res., 45,1825-1828.
25. NICOLETTI P., MILWARD F.W. & HOFFMAN E. (1985). - Efficacy of a long-acting
O x y t e t r a c y c l i n e a l o n e or combined w i t h s t r e p t o m y c i n in the t r e a t m e n t of bovine
brucellosis.J. Am. vet. Med. Assoc., 187,493-495.
26. NICOLETTI P., LENK R.P., POPESCU M . C . & SWENSON C.E. (1989). - Efficacy of various
t r e a t m e n t r e g i m e n s , u s i n g l i p o s o m a l s t r e p t o m y c i n in cows w i t h brucellosis.
Am. J. vet. Res., 50,1004-1007.
27. PETZER I.M., STADEN J . J . VAN & GIESECKE W.H. (1984). - Tissue reaction and residues in
s l a u g h t e r cattle after a d m i n i s t r a t i o n of long-acting Oxytetracycline f o r m u l a t i o n s . Jl S.
Afr. vet. Assoc., 55,107-111.
28. PHILLIPPON A., RENOUX G . & PLOMMET M . (1970). - Brucellose b o v i n e expérimentale :
II. Répartition d e Brucella abortus dans l'organisme six semaines après le part et trois
mois à cinq mois et demi après l'épreuve infectante. Ann. Rech, vét., 1,203-213.
29. PHILLIPPON A., PLOMMET M . & RENOUX G . (1971). - Brucellose b o v i n e expérimentale :
VII. Influence sur l'évolution d e l'infection d'une faible concentration d'oxytétracycline
dans l'organisme au moment d e l'inoculation. Ann. Rech. vét., 2,147-157.
30. PIVNYAK I . G . (1958). - Treatment with biomycin (Chlortetracycline) and immune serum
of sheep experimentally infected with brucellosis. Vet. Bull., 28, Abstract No. 364.
31. RADWAN A.I., ASMAR J.A., FRERICHS W.M., EL-BEKAIRI S.I. & AL-MUKAYEL A. (1983).
- Incidence of brucellosis in domestic livestock in Saudi Arabia. Trop. Anim. Hlth Prod.,
15,139-143.
32. RADWAN A.I., BEKAIRI S.I. & AL-MUKAYEL A. (1983). - Occurrence of brucellosis in
sheep and sheep handlers in Haradh, Saudi Arabia. Proc. Saudi biol. Soc, 6,583-593.
33. RADWAN A.I., BEKAIRI S.I., AL-MUKAYEL A. & A B U - A G L A O.S. (1984). - Control of
ovine brucellosis in Najdi sheep in Saudi Arabia. Trop. Anim. Hlth Prod., 16,213-218.
34. RADWAN A.I., H A F E Z S.M., AL-ASKA A.K., AL-YAMANI M . J . , BEKAIRI S.I., AL-JULAIFI
M . & AL-MUKAYEL A. (1989). - Experimental t r e a t m e n t of Brucella melitensis infection
in sheep with Oxytetracycline a l o n e or c o m b i n e d with s t r e p t o m y c i n . Trop. Anim. Hlth
Prod., 21,211-216.
35. RADWAN A.I., BEKAIRI S.I. & MUKAYEL A.A. (1992). - Treatment of Brucella melitensis
infection in sheep and goats with Oxytetracycline c o m b i n e d with s t r e p t o m y c i n . Rev. sci.
tech. Off. int. Epiz., 11 (3), 845-857.
36. RICHARDSON M . & HOLT J.N. (1962). - Synergistic a c t i o n of s t r e p t o m y c i n with other
antibiotics on intracellular Brucella abortus in vitro. J. Bact., 84,638-646.
37. SCHUHARDT V.T., R I C H G . C . & B E A L G . A . (1945). - Sulfadiazine t h e r a p y in bovine
brucellosis. J. Bact., 50,127-131.
38. SHEPHERD A.A., SIMPSON B.H. & DAVIDSON R . M . (1979). - An economic evaluation of
t h e New Zealand b o v i n e b r u c e l l o s i s e r a d i c a t i o n s c h e m e . In Proc. 2nd International
Symposium on Veterinary Epidemiology and Economics, 443-447.
39. TALUKDER M.A., ABOMELHA M.S. & H I G H A M R . H . (1984). - Brucellosis in farming
community in Saudi Arabia. Dev. biol. Stand., 56,593-595.