Veterinary Microbiology, 26 ( 1991 ) 103-105 Elsevier Science Publishers B.V., Amsterdam
103
Serological evaluation of Ehrlichia canis infections in military dogs in Africa and Reunion Island P. Brouqui a, B. Davoust b, S. Hadda&, E. Vidor d and D.
Raoult a
aCentre National de Reference des Rickettsioses, HOpital Timone, 13385 Marseille Cedex 5, France bService vktOrinaire des Armkes, 48 rue du capitaine Galinat, 13998 Marseille Armkes, France cService vktOrinaire des Armkes, Nouvelle plage II C12, Side Salem, 7000 Bizerte, Tunisia dlFFA Laboratories, Institut Merieux France, 254 rue Marcel Merieux, B.P. 7009, 69342 Lyon Cedex 07, France (Accepted 14 June 1990)
ABSTRACT Brouqui, P., Davoust, B., Haddad, S., Vidor, E. and Raoult, D., 1991. Serological evaluation of Ehrlichia canis infections in military dogs in Africa and Reunion Island. Vet. Microbiol., 26: 103-105. Sixty-eight dogs from four African countries and Reunion Island were tested for antibodies against Ehrlichia canis. Twenty-six dogs (50%) in Tunisia, Senegal and Chad were found positive using the indirect fluorescence antibody test. Dogs from both the Central African Republic and Reunion Island were all negative. Thus, this preliminary report confirms the presence of E. canis in Africa. Larger studies will be necessary to evaluate the current epidemiologic situation of canine ehrlichiosis in these countries.
INTRODUCTION
Canine ehrlichiosis is caused by the rickettsia Ehrlichia canis and is transmitted by the brown dog tick Rhipicephalus sanguineus. This disease was first described in 1935 in Algeria (Donatien and Lestoquard, 1936). It was later reported in Tunisia (Chabassol and Michel, 1972), Chad (Receveur and Hugaud, 1949 ), and the Central African Republic (Itard, 1957 ). Canine ehrlichiosis is also known as tropical canine pancytopenia. Diseased dogs present with fever, anorexia, asthenia, and epistaxis. These manifestations are associated with thrombocytopenia, leucopenia, and anemia. German Shepherds are more susceptible to infection than other dogs. Diagnosis is either by direct examination of the monocytes in blood smears, or serologically by the use of the indirect fluorescence antibody test (Ristic et al., 1972). The disease appears to be enzootic in parts of Africa. Several severe cases have been diag0378-1135/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
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P. BROUQUI ET AL.
nosed in the last three years in African military dogs (Davoust, unpublished data). MATERIALS AND METHODS
Serological assessment was made in 68 German Shepherd Dogs aged 2-10 years, living in kennels in Africa: Tunisia ( 19 dogs ), Chad ( 18 dogs), Senegal ( 15 dogs), the Central African Republic (8 dogs), and Reunion Island (8 dogs ). In Senegal, Chad, and the Central African Republic, the dogs had lived in these countries for between 4 months and 2 years, and were then brought back to France. The dogs taken to Reunion Island and Tunisia were not returned to France. No clinical symptoms of ehrlichiosis were noted on the day of sampling or during the previous 15 days. Blood samples were centrifuged, and the plasma was removed and frozen at - 8 0 °C until tested. In Senegal, Chad, and the Central African Republic, a preventive program for the disease has been in effect for several years; this consists of clearing undergrowth and removal of ticks from the dogs. In Senegal, 6 mg/kg doxycyline (Pfizer) was added daily to the food of the dogs. On returning to France, all the dogs from these three countries were treated with doxycyline at 10 mg/kg per day for 10 days. The dogs were tested before going from France to Africa, and were serologically negative for E. canis antibodies. For serological diagnosis, we used the indirect fluorescence antibody test (Ristic et al., 1972). The E. canis antigen was grown in mouse-dog hybridoma ( M D H ) cells (D. Vidor) and fixed in formalin. 25% of the cells were infected with the antigen. It was placed on Dynatech 30-spot slides, then fixed in cold acetone for 20 min. The slides were dried and the sera were placed on spots after dilution in 3% non-fat dried milk in phosphate buffered saline (PBS). After 30 min incubation at 37°C, the slides were washed twice for 10 min in 2% Tween-PBS, and once in sterile water for 10 min. Fluorescence was obtained using a rabbit anti-dog fluorescein conjugate (Bioyeda 3257-1 ) at 1/100 in 3% non-fat dried milk, 0.25% Evans blue (Bio-Merieux 7549-1 ) and PBS. It was placed on the slides and incubated for 30 min at 37°C, then washed three times, dried and mounted with Fluoprep (Bio-Merieux 7552-1 ). The slides were observed with a Nikon epifluorescence microscope. Sera were considered positive if the titer was 1/ 25 or higher. RESULTS AND DISCUSSION
Results are summarized in Table 1. The prevalence of the disease was highest in Tunisia; in 1985 a very high prevalence was noted (Haddad, personal communication). In the Tunisian kennel, 13 of the 19 dogs tested were found to be positive, with high titers. This may be explained by the presence of chronically infected dogs that are reservoirs for E. canis (Smith et al., 1976).
105
E I l R L I C f f I 4 (~,tlX,'lS INFECTIONS IN DOGS
TABLE 1 Serological evaluation ofE. canis in Africa and Reunion Island by IFA Central African Republic
Reunion Island
Total
Country
Tunisia
Senegal
Chad
Number of dogs tested
19
15
18
68
Number of dogs positive
13
8
5
26
3 2
4
1
1
-Fiter 1/25 1/50 1/100 1/200 1/400 1/800 1/1600
5 2 2 4
1 1
E. canis is present in Senegal and Chad, but the low titers of antibodies and the lack of subsequent cases of ehrlichiosis are probably due to the preventive measures instituted there. In the Central African Republic, as well as in Reunion Island, no dog was found to be positive, but the number of sera tested was not large enough to be significant. This preliminary study confirms the presence ofE. canis in Africa. Larger studies will be necessary to evaluate the epidemiologic status of canine ehrlichiosis.
REFERENCES Chabassol, C. and Michel, C., 1972. Syndrome h6morragique thrombotique thrombocytopenique du chien en Tunisie: comparaison avec la pancytopenie canine tropicale r6cemment d6crit6 au Sud-Vietnam. Soc. Med. Chir. H6p. For. San. Arm., 3:189-195. Donatien, A. and Lestoquard, F., 1936. Existence d'une rickettsia du chien. Bull. Soc. Pathol. Exot., 26: 418. itard, J., 1957. Sur un cas de rickettsiose canine a Rickettsia canis en Quabangui Chari. Rev. l~lev. M6d. V61. Pays. Trop., 10: 219. Receveur, P. and Hugaud, G., 1949. Existence de Rickettsia canis au Tchad. Rev. l~lev. M6d. V6t. Pays. Trop., 3: 45. Ristic. M., Huxsoll, D.L., Weisiger, R.M., Hildebrandt, P.K. and Nyindo, M.B.A., 1972. Serological diagnosis of tropical canine pancytopenia by indirect immunofluorescence. Infect. Immun., 6: 226-231. Smith, R.D., Sells, D.M., Stephenson, E.H., Ristic, M. and Huxsoll, D.C., 1976. Development of Ehrlichia canis, causalive agent of canine ehrlichiosis, in the tick Rhipicephalus sanguineus and its differentiation from a symbiotic rickettsia. Am. J. Vet. Res., 37:119-126.
103
Serological evaluation of Ehrlichia canis infections in military dogs in Africa and Reunion Island P. Brouqui a, B. Davoust b, S. Hadda&, E. Vidor d and D.
Raoult a
aCentre National de Reference des Rickettsioses, HOpital Timone, 13385 Marseille Cedex 5, France bService vktOrinaire des Armkes, 48 rue du capitaine Galinat, 13998 Marseille Armkes, France cService vktOrinaire des Armkes, Nouvelle plage II C12, Side Salem, 7000 Bizerte, Tunisia dlFFA Laboratories, Institut Merieux France, 254 rue Marcel Merieux, B.P. 7009, 69342 Lyon Cedex 07, France (Accepted 14 June 1990)
ABSTRACT Brouqui, P., Davoust, B., Haddad, S., Vidor, E. and Raoult, D., 1991. Serological evaluation of Ehrlichia canis infections in military dogs in Africa and Reunion Island. Vet. Microbiol., 26: 103-105. Sixty-eight dogs from four African countries and Reunion Island were tested for antibodies against Ehrlichia canis. Twenty-six dogs (50%) in Tunisia, Senegal and Chad were found positive using the indirect fluorescence antibody test. Dogs from both the Central African Republic and Reunion Island were all negative. Thus, this preliminary report confirms the presence of E. canis in Africa. Larger studies will be necessary to evaluate the current epidemiologic situation of canine ehrlichiosis in these countries.
INTRODUCTION
Canine ehrlichiosis is caused by the rickettsia Ehrlichia canis and is transmitted by the brown dog tick Rhipicephalus sanguineus. This disease was first described in 1935 in Algeria (Donatien and Lestoquard, 1936). It was later reported in Tunisia (Chabassol and Michel, 1972), Chad (Receveur and Hugaud, 1949 ), and the Central African Republic (Itard, 1957 ). Canine ehrlichiosis is also known as tropical canine pancytopenia. Diseased dogs present with fever, anorexia, asthenia, and epistaxis. These manifestations are associated with thrombocytopenia, leucopenia, and anemia. German Shepherds are more susceptible to infection than other dogs. Diagnosis is either by direct examination of the monocytes in blood smears, or serologically by the use of the indirect fluorescence antibody test (Ristic et al., 1972). The disease appears to be enzootic in parts of Africa. Several severe cases have been diag0378-1135/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
104
P. BROUQUI ET AL.
nosed in the last three years in African military dogs (Davoust, unpublished data). MATERIALS AND METHODS
Serological assessment was made in 68 German Shepherd Dogs aged 2-10 years, living in kennels in Africa: Tunisia ( 19 dogs ), Chad ( 18 dogs), Senegal ( 15 dogs), the Central African Republic (8 dogs), and Reunion Island (8 dogs ). In Senegal, Chad, and the Central African Republic, the dogs had lived in these countries for between 4 months and 2 years, and were then brought back to France. The dogs taken to Reunion Island and Tunisia were not returned to France. No clinical symptoms of ehrlichiosis were noted on the day of sampling or during the previous 15 days. Blood samples were centrifuged, and the plasma was removed and frozen at - 8 0 °C until tested. In Senegal, Chad, and the Central African Republic, a preventive program for the disease has been in effect for several years; this consists of clearing undergrowth and removal of ticks from the dogs. In Senegal, 6 mg/kg doxycyline (Pfizer) was added daily to the food of the dogs. On returning to France, all the dogs from these three countries were treated with doxycyline at 10 mg/kg per day for 10 days. The dogs were tested before going from France to Africa, and were serologically negative for E. canis antibodies. For serological diagnosis, we used the indirect fluorescence antibody test (Ristic et al., 1972). The E. canis antigen was grown in mouse-dog hybridoma ( M D H ) cells (D. Vidor) and fixed in formalin. 25% of the cells were infected with the antigen. It was placed on Dynatech 30-spot slides, then fixed in cold acetone for 20 min. The slides were dried and the sera were placed on spots after dilution in 3% non-fat dried milk in phosphate buffered saline (PBS). After 30 min incubation at 37°C, the slides were washed twice for 10 min in 2% Tween-PBS, and once in sterile water for 10 min. Fluorescence was obtained using a rabbit anti-dog fluorescein conjugate (Bioyeda 3257-1 ) at 1/100 in 3% non-fat dried milk, 0.25% Evans blue (Bio-Merieux 7549-1 ) and PBS. It was placed on the slides and incubated for 30 min at 37°C, then washed three times, dried and mounted with Fluoprep (Bio-Merieux 7552-1 ). The slides were observed with a Nikon epifluorescence microscope. Sera were considered positive if the titer was 1/ 25 or higher. RESULTS AND DISCUSSION
Results are summarized in Table 1. The prevalence of the disease was highest in Tunisia; in 1985 a very high prevalence was noted (Haddad, personal communication). In the Tunisian kennel, 13 of the 19 dogs tested were found to be positive, with high titers. This may be explained by the presence of chronically infected dogs that are reservoirs for E. canis (Smith et al., 1976).
105
E I l R L I C f f I 4 (~,tlX,'lS INFECTIONS IN DOGS
TABLE 1 Serological evaluation ofE. canis in Africa and Reunion Island by IFA Central African Republic
Reunion Island
Total
Country
Tunisia
Senegal
Chad
Number of dogs tested
19
15
18
68
Number of dogs positive
13
8
5
26
3 2
4
1
1
-Fiter 1/25 1/50 1/100 1/200 1/400 1/800 1/1600
5 2 2 4
1 1
E. canis is present in Senegal and Chad, but the low titers of antibodies and the lack of subsequent cases of ehrlichiosis are probably due to the preventive measures instituted there. In the Central African Republic, as well as in Reunion Island, no dog was found to be positive, but the number of sera tested was not large enough to be significant. This preliminary study confirms the presence ofE. canis in Africa. Larger studies will be necessary to evaluate the epidemiologic status of canine ehrlichiosis.
REFERENCES Chabassol, C. and Michel, C., 1972. Syndrome h6morragique thrombotique thrombocytopenique du chien en Tunisie: comparaison avec la pancytopenie canine tropicale r6cemment d6crit6 au Sud-Vietnam. Soc. Med. Chir. H6p. For. San. Arm., 3:189-195. Donatien, A. and Lestoquard, F., 1936. Existence d'une rickettsia du chien. Bull. Soc. Pathol. Exot., 26: 418. itard, J., 1957. Sur un cas de rickettsiose canine a Rickettsia canis en Quabangui Chari. Rev. l~lev. M6d. V61. Pays. Trop., 10: 219. Receveur, P. and Hugaud, G., 1949. Existence de Rickettsia canis au Tchad. Rev. l~lev. M6d. V6t. Pays. Trop., 3: 45. Ristic. M., Huxsoll, D.L., Weisiger, R.M., Hildebrandt, P.K. and Nyindo, M.B.A., 1972. Serological diagnosis of tropical canine pancytopenia by indirect immunofluorescence. Infect. Immun., 6: 226-231. Smith, R.D., Sells, D.M., Stephenson, E.H., Ristic, M. and Huxsoll, D.C., 1976. Development of Ehrlichia canis, causalive agent of canine ehrlichiosis, in the tick Rhipicephalus sanguineus and its differentiation from a symbiotic rickettsia. Am. J. Vet. Res., 37:119-126.
