J. Comp.
Path.
1992
Vol.
107,
239-242
SHORT
Isolation F. M.
PAPER
of Brucella melitensis from Ruminants in Jordan M. Aldomy,
K. L. Jahans
and
Aborting
Y. H. Altarazi
Animal Health Institute, PO Box 2395, Ministry of Agriculture, Amman,Jordan
Summary Thirty-four isolates (16.5 per cent) of Brucellamelitensis were cultured from 206 samplesfrom aborted, still-born or weak full-term animals and vaginal swabs from aborted animals. Twenty-six of 31 isolatesfrom sheepwere B. melitensis biovar 3. Two isolateswere biovar 1 and one of thesewas the vaccine strain Rev 1. The three remaining isolateswere from vaginal swabsand were not biotyped sinceB. melitensis biovar 3 had been isolated from the aborted fetuses. One of two isolatesfrom cattle and one isolate from a goat were biovar 1. The remaining cattle isolate was biovar 3. Introduction
Jordan has a small, but important, ruminant livestock industry. Amman, the capital, is one of the main livestock rearing areas for meat and dairy products. Animal resources represent an important part of the agricultural sector and account for over 42 per cent of national agricultural income. Generally, sheep and goats are raised on every farm and supply farmers’ households with milk, cheese, ghee, meat and wool. The surplus of these products is sold to earn farmers a regular income throughout the year. The Department of Animal Production and Health, Ministry of Agriculture (1989) has estimated the ruminant population in Jordan as 1 523 000 sheep, 474 950 goats and 28 953 cattle and has reported the prevalence of brucellosis to be 12 per cent in sheep and goats and 2 per cent in cattle. This report arose from an investigation into the causes of perinatal mortality of small ruminants in Jordan. Materials
and
Methods
BacteriologicalSamples Post-mortem examinations were carried out on all aborted, premature stillborn and weak full-term lambs, kids and calves from the Amman region. One hundred and sixty-five samplescomprising abomasal content, liver, lung and heart were examined. In addition 41 vaginal swabswere cultured from dams that had aborted to give a total of 206 samples. Address Central
for correspondence: Veterinary Laboratory,
K. L. Jahans, Weybridge,
FAOjWHO Surrey
KT15
Collaborating 3NB, U.K.
Centre
for Reference
on Brucellosis,
240
F. M.
M.
Aldomy
et al.
Culture, Identification and Typing Post-mortem samples and vaginal swabs were cultured in duplicate by plating on blood agar and on Brucella agar containing cycloheximide (100 mg per litre), Bacitracin (25 000 I.U. per litre) and Polymixin B (6 000 I.U. per litre) (Life Technologies, Paisley, U.K.). Inoculated plates were incubated at 37 C in air plus 5 to 10 per cent CO, for 2 to 7 days. Brucella colonies were initially identified by morphology, Gram stain and agglutination with A and M monospecific antisera (Murex Diagnostics, Dartford, Kent, U.K.). Brucella cultures were identified and typed at the Central Veterinary Laboratory by the methods of Corbel and Hendry (1983). Blood Samples and Serology Blood samples were taken from 12 ewes on six farms in the Amman region and sera examined by the rose bengal plate test (RBPT) and the complement fixation test (CFT) (Alton and Jones, 1967).
Results Brucella Isolation and Typing Twenty-nine strains (17.6 per cent) of Brucella melitensis were isolated from 165 post-mortem samples from aborted, stillborn or weak full-term animals. A further five strains (12 per cent) were isolated from 41 vaginal swabs from aborted dams. Twenty-seven isolates were biovar 3 and four biovar 1. Three vaginal swab isolates were not biotyped since B. melitensis biovar 3 had been isolated from the aborted fetuses (Table 1). Of the twenty isolates that were typed from sheep in Amman, 18 were biovar 3 and one biovar 1. The remaining isolate was identified as the vaccine strain Rev 1 and was isolated from a flock of 350 ewes inoculated with 0.5 ml B. meZiten.sis Rev 1 vaccine (Pendik, Turkey) containing 5 X lo* organisms (half the recommended dose). One month after vaccination, eight ewes aborted within a period of 20 days and three gave birth to weak, full-term lambs which died within 24 h (Table 1). In the Mafraq and Zerka regions, eight B. melitensis strains all of biovar 3 were isolated from sheep. One strain each of B. melitensis biovar 1 and 3 were isolated from a bovine calf and fetus, respectively in Amman. B. melitensis biovar 1 was also isolated from an aborted goat fetus in Amman (Table 1). The Brucella melitensis biovars had the following characteristics: CO, independent; agglutination by type M monospecific antisera (biovar I), agglutination by types A and M monospecific antisera (biovar 3); did not produce H,S; grew in the presence of basic fuchsin (20 pg per ml), thionin (20 pg per ml) and safranin 0 ( 100 pg per ml); sometimes lysed by bacteriophages Bk2, Iz and Wb but not by Tb. Rev 1 was differentiated from Brucella melitensis biovar 1 by the following characteristics: it did not grow in the presence of basic fuchsin (20 pg per ml), thionin (20 pg per ml) and safranin 0 (100 pg per ml) in air, or in the presence of 5 units of penicillin per ml, but grew in the presence of 2*4pg per ml of streptomycin.
Brucella
melitensis
241
in Jordan
Serology Eleven ranging fetus of (Table
of 12 sera from aborted ewes from six farms in Amman gave CFT titres from 1 in 40 to 1 in 320. B. melitensis biovar 3 was isolated from the one sheep (ewe number 11)) which was negative to the RBPT and CFT 2).
Brucella
No. of isolates
melitensis
biovars
Table isolated
1 from
ruminants
in Jordan
No. offarms
Location
Source of isolations
Biovars isolated
17 3 2 1
17 2 1 1
Amman Amman Amman Amman
ovine ovine ovine ovine
3 (3) 3 and 1 (Rev 1
5 3
2 1
Mafraq Zerka
ovine lambs* ovine (2 vaginal
1
1
Amman
caprine
1 1
1 1
Amman Amman
bovine bovine
* Died within minutes of birth. (3) The isolates from these animals aborted fetuses.
Serology
Farm
and
clinical
history
were not biotyped
of 14 aborted melitensis
Ewe no.
RBPT
Table ewes biovar
fetuses/lambs* vaginal swabs fetuses fetus
swabs, 1 lamb*)
3 3
fetus
1
calf* fetus
since biovar
2 from six f-s 3 was isolated
CFT
1 3
3 had been isolated
in Amman
from
from
their
which
Clinical his&y
1 2 3 4 5
+ + + + +
l/320 l/l60 l/40 l/320 l/80
2
6
NK
l/40
15 of 300 ewes aborted
3
7 8 9
+ +
l/l60 l/320 l/40
14 of 220 ewes aborted
4
10
NK
l/40
Farmer’s wife contracted brucellosis
5 6
11 12
+
1
f positive;
- negative;
NK=
+
not known.
l/80
8 of46 ewes aborted
B.
1)
242
F. M. M. Aldomy
et al.
Discussion The prevalence of human brucellosis in Jordan has risen dramatically during the past 5 years. The number of casesreported during the years 1985 to 1990 was 17, 521, 503, 463, 628 and 729, respectively (Directorate of Primary Health Care, Ministry of Health, 1991). Eight randomly selected B. m&ten&s isolates from 106 casesof human brucellosis studied at the Jordan University hospital were identified as either biovar 1 or 3 (Shehabi, Shakir, El-Khateeb, Qubain, Fararjeh and Shamat, 1990). The present study is the first record of these biovars in ruminants in Jordan and shows that the majority of cases of Brucetta abortion in sheep in the three districts studied are due to B. melitensis biovar 3. The isolation of Rev 1 from aborted fetuses following vaccination with Rev 1 vaccine (Pendik, Turkey) containing 5 X lo8 organisms (half the manufacturer’s recommended dose) illustrates the known danger of vaccinating pregnant sheep. This dose vastly exceeds the recommendation that “a lower dose of Rev 1 vaccine (5 to 10 x lo4 cells) given to adult sheep and goats might play a useful role at the beginning of a vaccination campaign where some of the animals in a herd may be pregnant” Woint FAO/WHO Expert Committee on Brucellosis, 1986). Eleven of 12 sera examined by RBPT and CFT showed diagnostically significant titres. One (ewe 1l), from which B. melitensis was isolated, was negative to both tests providing further illustration of the limitations of serology. On a larger scale, MacMillan (1990) states that of 300 sera from bacteriologically confirmed brucella abortion, 91-4 per cent were positive to the RBPT and 92-7 per cent positive to the CFT. Acknowledgments
The authors wish to thank the Ministry of Agriculture, Government of the Hashimite Kingdom of Jordan, the British Council and the GTZ VEEP Project in Jordan for supporting and financing this study. We also thank Dr Safi Hussien for assistance with the serology and Dr E. S. Broughton for comments on the manuscript. References
Alton, G. G. and Jones, L. M. (1967). Laboratov Organisation, Geneva.
Techniques in Brucellosis. World Health
Corbel, M. J. and Hendry, D. McL. F. D. (1983). Methodsfor the IdentiJication of Brucella. Ministry of Agriculture, Fisheries and Food, Alnwick. Department of Animal Production and Health, Ministry of Agriculture, Jordan ( 1989). Annual Report. Directorate of Primary Health Care,’ Ministry of Health, Jordan ( 1991). Report on Brucellosis in Jordan. Joint FAO/WHO Expert Committee on Brucellosis (1986). Technical Report Series 740. World Health Organisation, Geneva. Macmillan, A. P. (1990). Conventional serological tests. In Animal Brucellosis. Nielsen and Duncan, Eds, CRC Press, Boca Raton, Florida. Shehabi, A., Shakir, K., El-Khateeb, M., Q u b ain, H., Fararjeh, N. and Shamat, A. R. A. (1990). Diagnosis and treatment of 106 cases of human brucellosis. Journal of Infection, 20, 5-l 0. Received, February 5th, 1992 Accepted, May 23rd, 1992
1
Path.
1992
Vol.
107,
239-242
SHORT
Isolation F. M.
PAPER
of Brucella melitensis from Ruminants in Jordan M. Aldomy,
K. L. Jahans
and
Aborting
Y. H. Altarazi
Animal Health Institute, PO Box 2395, Ministry of Agriculture, Amman,Jordan
Summary Thirty-four isolates (16.5 per cent) of Brucellamelitensis were cultured from 206 samplesfrom aborted, still-born or weak full-term animals and vaginal swabs from aborted animals. Twenty-six of 31 isolatesfrom sheepwere B. melitensis biovar 3. Two isolateswere biovar 1 and one of thesewas the vaccine strain Rev 1. The three remaining isolateswere from vaginal swabsand were not biotyped sinceB. melitensis biovar 3 had been isolated from the aborted fetuses. One of two isolatesfrom cattle and one isolate from a goat were biovar 1. The remaining cattle isolate was biovar 3. Introduction
Jordan has a small, but important, ruminant livestock industry. Amman, the capital, is one of the main livestock rearing areas for meat and dairy products. Animal resources represent an important part of the agricultural sector and account for over 42 per cent of national agricultural income. Generally, sheep and goats are raised on every farm and supply farmers’ households with milk, cheese, ghee, meat and wool. The surplus of these products is sold to earn farmers a regular income throughout the year. The Department of Animal Production and Health, Ministry of Agriculture (1989) has estimated the ruminant population in Jordan as 1 523 000 sheep, 474 950 goats and 28 953 cattle and has reported the prevalence of brucellosis to be 12 per cent in sheep and goats and 2 per cent in cattle. This report arose from an investigation into the causes of perinatal mortality of small ruminants in Jordan. Materials
and
Methods
BacteriologicalSamples Post-mortem examinations were carried out on all aborted, premature stillborn and weak full-term lambs, kids and calves from the Amman region. One hundred and sixty-five samplescomprising abomasal content, liver, lung and heart were examined. In addition 41 vaginal swabswere cultured from dams that had aborted to give a total of 206 samples. Address Central
for correspondence: Veterinary Laboratory,
K. L. Jahans, Weybridge,
FAOjWHO Surrey
KT15
Collaborating 3NB, U.K.
Centre
for Reference
on Brucellosis,
240
F. M.
M.
Aldomy
et al.
Culture, Identification and Typing Post-mortem samples and vaginal swabs were cultured in duplicate by plating on blood agar and on Brucella agar containing cycloheximide (100 mg per litre), Bacitracin (25 000 I.U. per litre) and Polymixin B (6 000 I.U. per litre) (Life Technologies, Paisley, U.K.). Inoculated plates were incubated at 37 C in air plus 5 to 10 per cent CO, for 2 to 7 days. Brucella colonies were initially identified by morphology, Gram stain and agglutination with A and M monospecific antisera (Murex Diagnostics, Dartford, Kent, U.K.). Brucella cultures were identified and typed at the Central Veterinary Laboratory by the methods of Corbel and Hendry (1983). Blood Samples and Serology Blood samples were taken from 12 ewes on six farms in the Amman region and sera examined by the rose bengal plate test (RBPT) and the complement fixation test (CFT) (Alton and Jones, 1967).
Results Brucella Isolation and Typing Twenty-nine strains (17.6 per cent) of Brucella melitensis were isolated from 165 post-mortem samples from aborted, stillborn or weak full-term animals. A further five strains (12 per cent) were isolated from 41 vaginal swabs from aborted dams. Twenty-seven isolates were biovar 3 and four biovar 1. Three vaginal swab isolates were not biotyped since B. melitensis biovar 3 had been isolated from the aborted fetuses (Table 1). Of the twenty isolates that were typed from sheep in Amman, 18 were biovar 3 and one biovar 1. The remaining isolate was identified as the vaccine strain Rev 1 and was isolated from a flock of 350 ewes inoculated with 0.5 ml B. meZiten.sis Rev 1 vaccine (Pendik, Turkey) containing 5 X lo* organisms (half the recommended dose). One month after vaccination, eight ewes aborted within a period of 20 days and three gave birth to weak, full-term lambs which died within 24 h (Table 1). In the Mafraq and Zerka regions, eight B. melitensis strains all of biovar 3 were isolated from sheep. One strain each of B. melitensis biovar 1 and 3 were isolated from a bovine calf and fetus, respectively in Amman. B. melitensis biovar 1 was also isolated from an aborted goat fetus in Amman (Table 1). The Brucella melitensis biovars had the following characteristics: CO, independent; agglutination by type M monospecific antisera (biovar I), agglutination by types A and M monospecific antisera (biovar 3); did not produce H,S; grew in the presence of basic fuchsin (20 pg per ml), thionin (20 pg per ml) and safranin 0 ( 100 pg per ml); sometimes lysed by bacteriophages Bk2, Iz and Wb but not by Tb. Rev 1 was differentiated from Brucella melitensis biovar 1 by the following characteristics: it did not grow in the presence of basic fuchsin (20 pg per ml), thionin (20 pg per ml) and safranin 0 (100 pg per ml) in air, or in the presence of 5 units of penicillin per ml, but grew in the presence of 2*4pg per ml of streptomycin.
Brucella
melitensis
241
in Jordan
Serology Eleven ranging fetus of (Table
of 12 sera from aborted ewes from six farms in Amman gave CFT titres from 1 in 40 to 1 in 320. B. melitensis biovar 3 was isolated from the one sheep (ewe number 11)) which was negative to the RBPT and CFT 2).
Brucella
No. of isolates
melitensis
biovars
Table isolated
1 from
ruminants
in Jordan
No. offarms
Location
Source of isolations
Biovars isolated
17 3 2 1
17 2 1 1
Amman Amman Amman Amman
ovine ovine ovine ovine
3 (3) 3 and 1 (Rev 1
5 3
2 1
Mafraq Zerka
ovine lambs* ovine (2 vaginal
1
1
Amman
caprine
1 1
1 1
Amman Amman
bovine bovine
* Died within minutes of birth. (3) The isolates from these animals aborted fetuses.
Serology
Farm
and
clinical
history
were not biotyped
of 14 aborted melitensis
Ewe no.
RBPT
Table ewes biovar
fetuses/lambs* vaginal swabs fetuses fetus
swabs, 1 lamb*)
3 3
fetus
1
calf* fetus
since biovar
2 from six f-s 3 was isolated
CFT
1 3
3 had been isolated
in Amman
from
from
their
which
Clinical his&y
1 2 3 4 5
+ + + + +
l/320 l/l60 l/40 l/320 l/80
2
6
NK
l/40
15 of 300 ewes aborted
3
7 8 9
+ +
l/l60 l/320 l/40
14 of 220 ewes aborted
4
10
NK
l/40
Farmer’s wife contracted brucellosis
5 6
11 12
+
1
f positive;
- negative;
NK=
+
not known.
l/80
8 of46 ewes aborted
B.
1)
242
F. M. M. Aldomy
et al.
Discussion The prevalence of human brucellosis in Jordan has risen dramatically during the past 5 years. The number of casesreported during the years 1985 to 1990 was 17, 521, 503, 463, 628 and 729, respectively (Directorate of Primary Health Care, Ministry of Health, 1991). Eight randomly selected B. m&ten&s isolates from 106 casesof human brucellosis studied at the Jordan University hospital were identified as either biovar 1 or 3 (Shehabi, Shakir, El-Khateeb, Qubain, Fararjeh and Shamat, 1990). The present study is the first record of these biovars in ruminants in Jordan and shows that the majority of cases of Brucetta abortion in sheep in the three districts studied are due to B. melitensis biovar 3. The isolation of Rev 1 from aborted fetuses following vaccination with Rev 1 vaccine (Pendik, Turkey) containing 5 X lo8 organisms (half the manufacturer’s recommended dose) illustrates the known danger of vaccinating pregnant sheep. This dose vastly exceeds the recommendation that “a lower dose of Rev 1 vaccine (5 to 10 x lo4 cells) given to adult sheep and goats might play a useful role at the beginning of a vaccination campaign where some of the animals in a herd may be pregnant” Woint FAO/WHO Expert Committee on Brucellosis, 1986). Eleven of 12 sera examined by RBPT and CFT showed diagnostically significant titres. One (ewe 1l), from which B. melitensis was isolated, was negative to both tests providing further illustration of the limitations of serology. On a larger scale, MacMillan (1990) states that of 300 sera from bacteriologically confirmed brucella abortion, 91-4 per cent were positive to the RBPT and 92-7 per cent positive to the CFT. Acknowledgments
The authors wish to thank the Ministry of Agriculture, Government of the Hashimite Kingdom of Jordan, the British Council and the GTZ VEEP Project in Jordan for supporting and financing this study. We also thank Dr Safi Hussien for assistance with the serology and Dr E. S. Broughton for comments on the manuscript. References
Alton, G. G. and Jones, L. M. (1967). Laboratov Organisation, Geneva.
Techniques in Brucellosis. World Health
Corbel, M. J. and Hendry, D. McL. F. D. (1983). Methodsfor the IdentiJication of Brucella. Ministry of Agriculture, Fisheries and Food, Alnwick. Department of Animal Production and Health, Ministry of Agriculture, Jordan ( 1989). Annual Report. Directorate of Primary Health Care,’ Ministry of Health, Jordan ( 1991). Report on Brucellosis in Jordan. Joint FAO/WHO Expert Committee on Brucellosis (1986). Technical Report Series 740. World Health Organisation, Geneva. Macmillan, A. P. (1990). Conventional serological tests. In Animal Brucellosis. Nielsen and Duncan, Eds, CRC Press, Boca Raton, Florida. Shehabi, A., Shakir, K., El-Khateeb, M., Q u b ain, H., Fararjeh, N. and Shamat, A. R. A. (1990). Diagnosis and treatment of 106 cases of human brucellosis. Journal of Infection, 20, 5-l 0. Received, February 5th, 1992 Accepted, May 23rd, 1992
1
