842 TRANSACTIONS OF THE ROYALSOCIETY OF TROPICAL MEDICINEAND HYGIENE(1990)84, 842-845
Serological evidence of infection (family Rhabdoviridae)
of dogs and man in Nigeria by lyssaviruses
‘Department of A. B. Ogunkoya’, G. W. Beran4, J. U. Urnoh’, N. E. Gomwalk3 and I. A. Abdulkadir’ Surgery and Medicine, Faculty of Veterinary Medicine, *Department of Veterinary Public Health, and 3Department of Microbiology, Ahmadu Belle University, Zaria, Nigeria; 4Department of Microbiology, Public Health and Preventive Medicine, Veterinay Faculty, Iowa State University, Ames, Iowa, USA
Abstract A survey was conducted for serum neutralizing antibodies against 3 members of the Lyssavirus group (rabies, Lagos bat and Mokola viruses) in Nigerian dogs and humans. Of 463 unvaccinated dogs sampled, 142 (30.7%) had antibodies against rabies; 39.2% of the stray dogs were positive. Of 241 dogs tested, 17.4% had antibodies against Mokola virus and 5.8% against Lagos bat virus. 28.6% of human samples tested had antibodies against rabies and, of 158 human samples tested, 12 (7.5%) had antibodies against Mokola virus and 4 (2.5%) had antibodies against Lagos bat virus. Introduction Rabies virus is the type species of the genus Lyssavirus in the familv Rhabdoviridae: the aenus also contains a number of rabies-related viruses Lolated in Africa @HOPE,1982). Mokola virus was isolated from the viscera of shrews (Crocidura sp.) in Ibadan in 1968@HOPEet al., 1970) and Lagos bat virus was isolated in Lagos from the vooled brains of frueivorous bats (Eidolon helvum) by BOULGER& PORTERFIELD(1958):The presenceof these viruses complicates the rabies control and eradication programmes because vaccination against rabies does not necessarily protect against rabiesrelated viruses (WIKTOR et al., 1983). In rabies enzootic areas of Nigeria and elsewhere, serum neutralizing antibodies against rabies and rabies-related viruses have been detected in annarently healthy animals (KEMPet al, 1972; EVERA~~et al., 1981; YASMUTH et al.. 1974). RLJEGSECGERet al. (1961) found heat-stable antibodies in 6% of 280 unvaccinated veterinary students in the USA. It appears, therefore, that rabies is not necessarily fatal, and that there may be subclinical infections probably due to attenuated strains of rabies virus (RUEGSEGGER et al.. 1961) or as vet unidentified related viruses (SHAPEet al, 1970s The aim of this study was to seekevidence of latent infections with rabies or rabies-related viruses in dogs and humans in Kaduna, Oyo and Lagos states of Nigeria. Materials and Methods Canine samples
Blood for serum sampleswas obtained from dogs at the veterinary clinics in Zaria, Kaduna and Kafanthan in Kaduna state, Ibadan in Oyo state, and Lagos and Badagry in Lagos state, Nigeria. Information on age, sex, breed and reasons for presentation at the clinic were recorded for each dog.
Blood samples were not collected from severely ill or anaemic dogs or those less than 3 months old. Samples were also obtained from rural dogs. The ages, sexes and breeds of these dogs were also recorded. Human samples
Serum samples were collected from (i) 5 persons nreviouslv bitten bv dogs but who had received no post-exposure vaccination; and (ii) persons who could not recollect any history of exposure and had never been given any anti-rabies prophylaxis. A questionnaire was prepared to record information relating to exposure of each person whose blood sample was taken. All serum samples were stored at -20°C until used. Treatment of serum samples
Both human and dog-sera were heat-inactivated at 56°C for 30 min. All doa sera were treated with 25% kaolin to absorb non-speiific inhibitors (HIERHOLZER et al., 1969). Purification and titration of immunoglobulin
The y-globulin fraction was separated from some selected serum samplesby adding an equal volume of saturated ammonium sulphate solution (HERBERT, 1974). Selectedsamnlesincluded those known to have given positive resuhs in the fluorescent antibody test, and a few negative sera. The precipitate recovered after centrifugation was dissolved in distilled water and reprecipitated by addition of a half-volume of saturated ammonium sulphate (33% final saturation). The final precipitate was dissolved in water and dialysed against several changes of O*15 N NaCl (pH 8.0) until the dialysate solution failed to form a precipitate with 1% barium chloride solution. The globulin fractions thus obtained were titrated and the results were compared with those obtained by titrating the corresponding unfractionated samples. Serum neutralizing antibody test against rabies, Mokola and Lagos bat viruses
The test used was the rapid fluorescent focus inhibition test (RFFIT), as described by SMITH et al. (1973). After screening at 1:16final dilution, 35 of the positive human sera and 90 nositive doa sera were &rated against rabies virus. detection oi‘ antibodies to Mokola and Lagos bat viruses was also bv RFFIT. but the staining was an indirect fluorescent antibody
843 method using monoclonal antibody 422-5* prepared as mouse ascitic fluid (FLAMAND et al., 1980). Briefly, an equal amount of the test serum and the appropriate dilution of the corresponding challenge viruses (Mokola or Lagos bat) were incubated in a 96-well microtitre plate for one hour. 0.05 ml of the serum-virus mixture was adsorbed and grown on a monolayer of BHK-21 cells in the wells of Terasaki histoplates and incubated at 37°C in 5% Cq2. After 24 h the histoplates were removed, washed in phosphate-buffered saline (pH 7.4), and fixed in cold 80% acetonefor 30 min. After drying, 5 pl of monoclonal antibody 422-5 were dispensed in each well and incubated for 30 min at 37°C. The plates were again washed twice and 5 ~1of anti-mouse globulin conjugate were placed in each well and incubated for 30 min. The plates were washed and read while still wet. After the initial screening, 12 positive human sera and 11 positive dog sera were titrated against Mokola virus. Four positive human and 12 positive dog sera were titrated against Lagos bat virus. Known positive and negative sera against Mokola and Lagos bat viruses (obtained from Dr Wiktor) were used as controls.
Dr T. J. Wiktor of the Wistar Institute for Anatomy and Biology (Philadelphia, Pennsylvania, USA) for confirmatory testing. The samples sent were from all the categories of dogs. Results Eight hundred and fifty-seven serum samples were collected from dogs. 463 dogs were owned and unvaccinated, 212 were strays, 74 were owned and vaccinated, and 108 were owned dogs of unknown vaccination status. 311 samples (36%) were positive. The National Veterinary Services Laboratories found 39% (150/386) to be positive, using 1:16 final dilution, and the Wistar Institute of Anatomy and Biology in Philadelphia, using 1:50 final dilution, found 25% (47/187) to be positive. The concordance between our results (obtained at the College of Veterinary Medicine, Iowa State University) and those of the National Veterinary Services Laboratories was 97%. The results obtained from the Wistar Institute could not be compared because a 1:50 dilution was used. Of the 463 owned. unvaccinated dogs, 142 (31%) had serum neutralizing antibody deiected ai 1:8 screening dilution, as did 83 (39%) of the 212 stray dogs and 42 (39%) of the 108 dogs owned but with unknown vaccination history. Overall, 265 (34%) of the 783 dogs not known to have been vaccinated had serum neutralizing antibody titres of at least 1:8. The titres obtained by testing y-globulin precipitated from dog sera were similar to those obtained with the corresponding whole sera. The results of titrating the canine sera are shown in Table 1.
Rabies serum mouse neutralization test
The neutralizing activity of some positive sera already titrated by RFFIT was investigated by means of a mouse neutralization test (MNT) in three-weekold white mice as previously described (CDC, 1981). Two-fold serial dilutions of the samples were mixed with equal volumes of virus dilution in test tubes and incubated for 90 min at 37°C. 0.03 ml of each virus
Table 1. Antibody titres in canine serum samples against rabies, Mokola and Lagos bat viruses Rabies Titre
No. of sera positive
Percentage
No. of sera positive
;:;6
::,
24.4 22.2
4
I;32 1:64 1:128
19 17 9
21.1 18.9 10.0
i 0 1
1:512 1:256
ii
3.3
i
serum dilution was inoculated intracerebrally into 5 mice per dilution. Controls were also set up. The mice were observed for 14 days for clinical signs of rabies or death. The antibody titres of the serum samples were calculated by the method of Reed & Muench (WHO, 1984). Validation
tests
The first batch of serum samplestested was sent to Dr T. 0. Bunn of the National Veterinary Services Laboratories (Ames, Iowa, USA) and the second to ‘Monoclonal antibody 422-5is known to react with Lagos bat andMokolavirusesandwaskindly suppliedby Dr T. J. Wiktor of the Wistar Institute, Philadelphia, USA. The antigens used (BHK-21 adapted Mokola and Lagos bat viruses) were also obtained from Dr Wiktor.
Mokola Percentage
No. of sera positive
36.4 27.3 27,3
; 0
61 -
: :
Lagos bat Percentage f: -
Human sera
A total of 350 human serawas examined, 100 (29%) of which contained anti-rabies antibody (Table 2). The incidence was higher in males (36%) than in females (24%). The results of titrating 35 human sera are shown in Table 3. Onlv 5 oersons indicated that thev had been previously bitted by dogs; 2 of these had antibody against rabies. Four of the 6 dog catchers who had worked at the Onireke health centre in Ibadan for several years had antibodies against rabies and one had antibodies against Mokola virus. They claimed that they had never been bitten by a dog or cat. Serological findings against rabies-related viruses
241 canine sera and 158 human sera were tested for antibodies to the rabies-related Mokola and Lagos bat
844 Table 2. Rabies seropositivity
someapparently healthy dogs and people. There are 2 possible explanations for this result: either the seropositive dogs and people were exposed to the virus, becamesick and then recovered, or they were exposed and developed no apparent sickness but produced detectable antibodies. It is likely that infection was halted before entering nerve pathways. BAER & OLSON (1972) reported that, following apparent recovery from clinical rabies, 4 pigs developed antibodies within 2 months. In Grenada, 2 goats and a donkey known to have been bitten by mongoosesshowed rabies antibodies at a low titre 30 and 54 days after exposure (EVERARDet al., 1981). FEKADU & BAER (1980) and FEKADU et al. (1981) found neutralizing antibody in serum and cerebrospinal fluid of dogs that had recovered from clinical rabies.
in Nigeria”
Age group (years) la-14 E-19 20-24 25-29 30-34 35-39 4a-44 45-49 50-54 55-59 560 Total
M&S 216 2115 9128 lo/23 8114 419 217 115 4110 O/l 6115 48/133
“Rapid fluorescent
Table
Females
(33.3%) (13.3%) (32.1%) (43.5%) (57.1%) (44.4%) (28.6%) (20%) (40%) 6) (40%) (36.1%)
focus inhibition
3. Antibody
Titre
3112 6121 18154 8152 3124 3/18 8/20 117 O/l 013 2/S 521217
Total
(25.0%) (28.6%) (33.3%) (15.5%) (12.5%) (16.7%) (40%) (14.4%) 6) 6) (40%) (24%)
5118 8136 27182 18175 11138 7127 10127 2112 4111 o/4 8/20 100/350
(27.8%) (22%) (329%) (24%) (28.9%) (25.9%) (37%) (36.7%) (36.4%) (6) (40%) (28.6%)
test, 1:16 dilution.
titres in human serum samples against rabies, Mokola
No. of sera positive
Rabies Percentage
18
51.4
9 5 3
25.7 14.3 8.6
iif6
1:32 1:64”
No. of sera positive
Mokolo Percentage
and Lagos bat viruses
No. of sera positive 2
2 2
1 1
:i 16.6 8.3
1
Lagos bat Percentage 50
11.1 11.1 -
“No sera were positive at higher titres. Table 4. Seropositivity of serum samples from Nig-erian dogs and humans against Mokola and Lagos bat virusesa
Dog sera (n=241) Mokola virus Lagos bat virus Human sera (n=158) Mokola virus Lagos bat virus
Number positive
Percentage positive
44
17.4 5.8
14 12 4
;:;
test, 1:16 dil-
viruses (Table 4). The results of titrating some of these sera are shown in Tables 1 and 3. Cross reactions between rabies, Mokola and Lagos bat viruses were not observed. Sera which were positive for Mokola were not positive for rabies, and vice versa. test.
In general, titres obtained correlated well with the MNT results (Table 5). Usually, a higher titre was observed in the MNT compared with that obtained by the RFFIT. Discussion
RFFIT
MNT
1:128 1:8 1:8
1:128 1:91 1:32
Canine sera
“Rapid fluorescent focus inhibition ution.
Mouse neutralization
Table 5. Comparison between titres obtained by rapid fluorescent focus inhibition test (RFFIT) and mouse neutralization test (MNT) with canine and human sera
This study has provided serological evidence of infections with members of the Lyssavirus group in
746a 703a 303b
Human sera 107c 99d
1OOd
1:64
1:64
1:8
1:32
1:32
1:50
“Unvaccinated domestic dog. bUnvaccinated stray dog. ‘Previous dog bite, no treatment given. dNo known previous exposure or anti-rabies treatment. Non-fatal infection in dogs could have been overlooked, especially in the stray animals. The detection of antibodies in the human population, however, has posed serious questions of how these people came in contact with the rabies antigen. If the stimulating antigen was rabies virus, how were the people exposed? Only 5% of the 350 people examined reported that they had previously been bitten by a dog; 2 of these had antibody titres. Four of the 6 dog catchers had antibodies against rabies, and one had antibodies against Mokola virus; none had any history
845
of having been bitten by a rabid dog or of receiving prophylactic rabies vaccinations. None of the other people tested had a history of any dog bite or of antirabies vaccinations. As only 1.4% of the people provided a history of dog bites, it is difficult to attribute a 28% prevalence of antibody titre to forgotten dog bites. Further possible explanations must be sought. The antigens inducing the detected antibodies could be viruses belonging to the Lyssavirus group which are yet to be identified, or some unidentified infectious agents cross-reacting with lyssaviruses, possibly arthropodborne. Acknowledgements We thank Dr T. 0. Bunn of the United States Deoartment of Agriculture Animal and Plant Health Inspettion Services, and Dr T. J. Wiktor of the Wistar Institute, Philadelphia, for confirmatory testing of the sera. Dr Wiktor also supplied the monoclonal antibody, specific sera, and tissue culture-adapted viruses. Special and sincere thanks go to Dr T. T. Kramer, chairman of the department of Veterinary Microbiology and Preventive Medicine, Drs Kenneth B. Platt, Martin L. Kaeberla and Loren A. Will, all of the Department of Veterinary Microbiology and Preventive Medicine, for their enthusiastic cooperation and support during the work. We also thank the Veterinary Officers in Nigeria for their cooperation. The techincal assistance given by Mr Daniel Imoru, Dr S. Baba, Mrs Dorothy Murphy, Dr M. A. Mushin, Dr Lekan Ayanwale and Dr (Mrs) Y. 0. Ogunkoya was highly appreciated. References Baer, 0. M. & Olson, H. R. (1972). Recovery of pigs from rabies. Journal of rhe American Veten’nary Medical Association, 160, 1127-l 132. Boulger, L. K. & Porterfield, J. (1958). Isolation of a virus from Nigerian fruit bats. Transactions of rhe Royal Society of Tropical Medicine and Hygiene, 52, 421-424. CDC (1981). Laboratory Methods for Detecting Rabies. Willis, M., Velleca, & Forrester, F. T. (editors). Atlanta, GA 30333, USA: US Department of Health and Human Services, Public Health Services. Everard, C. 0. R., Baer, G. M., Allis, M. E. & Salley, A. M. (1981). Rabies serum neutralizing antibodies in mongoose from Grenada. Transactions of the Royal Society of Tropical Medicine and Hygiene, 75, 654-666. Fekadu, M. & Baer, G. M. (1980). Recovery from clinical rabies of two dogs inoculated with a rabies virus strain from Ethiopia. American Journal of Veterinary Research, 41, 1632-1634.
Fekadu, N., Shaddock, J. H. & Baer, G. M. (1981). Intermittent excretion of rabies virus in the saliva of a dog two and six months after it had recovered from exuerimental rabies. American youma of Trobical Medi* 1 c&e and Hygiene, 3, 1113-11~5. Flamand, A., Wiktor, T. J. & Koprowski, H. (1980). Use of hvbridoma monoclonal antibodies in the detection of antigenic differences between rabies and rabies-related virus proteins. I. The nucleocapsid protein. Journal of General Virology, 48, 97-102. Herbert, G. A. (1974). Ammonium sulphate fractionation of sera from mouse, hamster, guinea pig, monkey, chimpanzee, swine, chicken and cattle. Applied Microbiology, 27, 389-393. Hierholzer, J. C., Suggs, M. T. & Hall, E. C. (1969). Standardized viral haemagglutination and haemagglutination-inhibition test. II. Description and statistical evalution. Applied Microbiology, 18, 824833. Kemp, C. D., Causey, 0. R., Dorothy, L., Odelolar, A. & Fabiti, A. (1972). Mokola virus, further studies on IBAN 27377. A new rabies-related etiological agent of zoonoses in Nigeria. American Journal of Tropical Medicine and Hygiene, 21, 356-359. Ruegsegger, J. M., Black, J. & Sharpless, G. R. (1961). Primary antirabies immunization of man with the Flury virus vaccine.American Journal of Tropical Medicine and Hygiene, 51, 706-716. Shope, R. E. (1982). Rabies related viruses. YaleJournal of Biology and Medicine, 55, 271-275. Shope, R. C., Murphy, F. A., Harrison, A. K., Causey, 0. R., Hemp, G. E., Simpson, D. T. H. & Moore, D. I. (1970). Two African viruses serologically and morphologically related to the rabies virus. Journal of Virology, 6, 690-692. Smith, J. S., Yager, I’. A. & Baer, G. N. (1973). A rapid tissue culture test for determining rabies neutralizing antibody. In: Laboratoty Techniques in Rabies, Kaplan, M. M. & Kaprowski, E. (editors). Geneva: World Health Organization, pp. 35+357. WHO (1984). Guidelines for dog rabies control. Geneva: World Health Organization, mimeographed document no. VPHl83.43. Wiktor, J., Macfaran, R. I., Foggin, C. M. & Kowprowski, H. (1983). Antigenic analysis of rabies and Mokola virus. (Symposium on monoclonal antibodies. Standardization of their characterization and use, Paris, France.) Develogments in Bioloaical Standardisation. 57. 199-211. Yasmuth, C., Evans, R. C. & Doege, T. 6. (1474). Rabies antibody in healthy dogs and vaccine response after MPA. Journal of the Medical Association of Thailand, 57, 131-134.
Received 22 December 1987; revised 20 February 1990; accepted for publication
20 February
1990
Serological evidence of infection (family Rhabdoviridae)
of dogs and man in Nigeria by lyssaviruses
‘Department of A. B. Ogunkoya’, G. W. Beran4, J. U. Urnoh’, N. E. Gomwalk3 and I. A. Abdulkadir’ Surgery and Medicine, Faculty of Veterinary Medicine, *Department of Veterinary Public Health, and 3Department of Microbiology, Ahmadu Belle University, Zaria, Nigeria; 4Department of Microbiology, Public Health and Preventive Medicine, Veterinay Faculty, Iowa State University, Ames, Iowa, USA
Abstract A survey was conducted for serum neutralizing antibodies against 3 members of the Lyssavirus group (rabies, Lagos bat and Mokola viruses) in Nigerian dogs and humans. Of 463 unvaccinated dogs sampled, 142 (30.7%) had antibodies against rabies; 39.2% of the stray dogs were positive. Of 241 dogs tested, 17.4% had antibodies against Mokola virus and 5.8% against Lagos bat virus. 28.6% of human samples tested had antibodies against rabies and, of 158 human samples tested, 12 (7.5%) had antibodies against Mokola virus and 4 (2.5%) had antibodies against Lagos bat virus. Introduction Rabies virus is the type species of the genus Lyssavirus in the familv Rhabdoviridae: the aenus also contains a number of rabies-related viruses Lolated in Africa @HOPE,1982). Mokola virus was isolated from the viscera of shrews (Crocidura sp.) in Ibadan in 1968@HOPEet al., 1970) and Lagos bat virus was isolated in Lagos from the vooled brains of frueivorous bats (Eidolon helvum) by BOULGER& PORTERFIELD(1958):The presenceof these viruses complicates the rabies control and eradication programmes because vaccination against rabies does not necessarily protect against rabiesrelated viruses (WIKTOR et al., 1983). In rabies enzootic areas of Nigeria and elsewhere, serum neutralizing antibodies against rabies and rabies-related viruses have been detected in annarently healthy animals (KEMPet al, 1972; EVERA~~et al., 1981; YASMUTH et al.. 1974). RLJEGSECGERet al. (1961) found heat-stable antibodies in 6% of 280 unvaccinated veterinary students in the USA. It appears, therefore, that rabies is not necessarily fatal, and that there may be subclinical infections probably due to attenuated strains of rabies virus (RUEGSEGGER et al.. 1961) or as vet unidentified related viruses (SHAPEet al, 1970s The aim of this study was to seekevidence of latent infections with rabies or rabies-related viruses in dogs and humans in Kaduna, Oyo and Lagos states of Nigeria. Materials and Methods Canine samples
Blood for serum sampleswas obtained from dogs at the veterinary clinics in Zaria, Kaduna and Kafanthan in Kaduna state, Ibadan in Oyo state, and Lagos and Badagry in Lagos state, Nigeria. Information on age, sex, breed and reasons for presentation at the clinic were recorded for each dog.
Blood samples were not collected from severely ill or anaemic dogs or those less than 3 months old. Samples were also obtained from rural dogs. The ages, sexes and breeds of these dogs were also recorded. Human samples
Serum samples were collected from (i) 5 persons nreviouslv bitten bv dogs but who had received no post-exposure vaccination; and (ii) persons who could not recollect any history of exposure and had never been given any anti-rabies prophylaxis. A questionnaire was prepared to record information relating to exposure of each person whose blood sample was taken. All serum samples were stored at -20°C until used. Treatment of serum samples
Both human and dog-sera were heat-inactivated at 56°C for 30 min. All doa sera were treated with 25% kaolin to absorb non-speiific inhibitors (HIERHOLZER et al., 1969). Purification and titration of immunoglobulin
The y-globulin fraction was separated from some selected serum samplesby adding an equal volume of saturated ammonium sulphate solution (HERBERT, 1974). Selectedsamnlesincluded those known to have given positive resuhs in the fluorescent antibody test, and a few negative sera. The precipitate recovered after centrifugation was dissolved in distilled water and reprecipitated by addition of a half-volume of saturated ammonium sulphate (33% final saturation). The final precipitate was dissolved in water and dialysed against several changes of O*15 N NaCl (pH 8.0) until the dialysate solution failed to form a precipitate with 1% barium chloride solution. The globulin fractions thus obtained were titrated and the results were compared with those obtained by titrating the corresponding unfractionated samples. Serum neutralizing antibody test against rabies, Mokola and Lagos bat viruses
The test used was the rapid fluorescent focus inhibition test (RFFIT), as described by SMITH et al. (1973). After screening at 1:16final dilution, 35 of the positive human sera and 90 nositive doa sera were &rated against rabies virus. detection oi‘ antibodies to Mokola and Lagos bat viruses was also bv RFFIT. but the staining was an indirect fluorescent antibody
843 method using monoclonal antibody 422-5* prepared as mouse ascitic fluid (FLAMAND et al., 1980). Briefly, an equal amount of the test serum and the appropriate dilution of the corresponding challenge viruses (Mokola or Lagos bat) were incubated in a 96-well microtitre plate for one hour. 0.05 ml of the serum-virus mixture was adsorbed and grown on a monolayer of BHK-21 cells in the wells of Terasaki histoplates and incubated at 37°C in 5% Cq2. After 24 h the histoplates were removed, washed in phosphate-buffered saline (pH 7.4), and fixed in cold 80% acetonefor 30 min. After drying, 5 pl of monoclonal antibody 422-5 were dispensed in each well and incubated for 30 min at 37°C. The plates were again washed twice and 5 ~1of anti-mouse globulin conjugate were placed in each well and incubated for 30 min. The plates were washed and read while still wet. After the initial screening, 12 positive human sera and 11 positive dog sera were titrated against Mokola virus. Four positive human and 12 positive dog sera were titrated against Lagos bat virus. Known positive and negative sera against Mokola and Lagos bat viruses (obtained from Dr Wiktor) were used as controls.
Dr T. J. Wiktor of the Wistar Institute for Anatomy and Biology (Philadelphia, Pennsylvania, USA) for confirmatory testing. The samples sent were from all the categories of dogs. Results Eight hundred and fifty-seven serum samples were collected from dogs. 463 dogs were owned and unvaccinated, 212 were strays, 74 were owned and vaccinated, and 108 were owned dogs of unknown vaccination status. 311 samples (36%) were positive. The National Veterinary Services Laboratories found 39% (150/386) to be positive, using 1:16 final dilution, and the Wistar Institute of Anatomy and Biology in Philadelphia, using 1:50 final dilution, found 25% (47/187) to be positive. The concordance between our results (obtained at the College of Veterinary Medicine, Iowa State University) and those of the National Veterinary Services Laboratories was 97%. The results obtained from the Wistar Institute could not be compared because a 1:50 dilution was used. Of the 463 owned. unvaccinated dogs, 142 (31%) had serum neutralizing antibody deiected ai 1:8 screening dilution, as did 83 (39%) of the 212 stray dogs and 42 (39%) of the 108 dogs owned but with unknown vaccination history. Overall, 265 (34%) of the 783 dogs not known to have been vaccinated had serum neutralizing antibody titres of at least 1:8. The titres obtained by testing y-globulin precipitated from dog sera were similar to those obtained with the corresponding whole sera. The results of titrating the canine sera are shown in Table 1.
Rabies serum mouse neutralization test
The neutralizing activity of some positive sera already titrated by RFFIT was investigated by means of a mouse neutralization test (MNT) in three-weekold white mice as previously described (CDC, 1981). Two-fold serial dilutions of the samples were mixed with equal volumes of virus dilution in test tubes and incubated for 90 min at 37°C. 0.03 ml of each virus
Table 1. Antibody titres in canine serum samples against rabies, Mokola and Lagos bat viruses Rabies Titre
No. of sera positive
Percentage
No. of sera positive
;:;6
::,
24.4 22.2
4
I;32 1:64 1:128
19 17 9
21.1 18.9 10.0
i 0 1
1:512 1:256
ii
3.3
i
serum dilution was inoculated intracerebrally into 5 mice per dilution. Controls were also set up. The mice were observed for 14 days for clinical signs of rabies or death. The antibody titres of the serum samples were calculated by the method of Reed & Muench (WHO, 1984). Validation
tests
The first batch of serum samplestested was sent to Dr T. 0. Bunn of the National Veterinary Services Laboratories (Ames, Iowa, USA) and the second to ‘Monoclonal antibody 422-5is known to react with Lagos bat andMokolavirusesandwaskindly suppliedby Dr T. J. Wiktor of the Wistar Institute, Philadelphia, USA. The antigens used (BHK-21 adapted Mokola and Lagos bat viruses) were also obtained from Dr Wiktor.
Mokola Percentage
No. of sera positive
36.4 27.3 27,3
; 0
61 -
: :
Lagos bat Percentage f: -
Human sera
A total of 350 human serawas examined, 100 (29%) of which contained anti-rabies antibody (Table 2). The incidence was higher in males (36%) than in females (24%). The results of titrating 35 human sera are shown in Table 3. Onlv 5 oersons indicated that thev had been previously bitted by dogs; 2 of these had antibody against rabies. Four of the 6 dog catchers who had worked at the Onireke health centre in Ibadan for several years had antibodies against rabies and one had antibodies against Mokola virus. They claimed that they had never been bitten by a dog or cat. Serological findings against rabies-related viruses
241 canine sera and 158 human sera were tested for antibodies to the rabies-related Mokola and Lagos bat
844 Table 2. Rabies seropositivity
someapparently healthy dogs and people. There are 2 possible explanations for this result: either the seropositive dogs and people were exposed to the virus, becamesick and then recovered, or they were exposed and developed no apparent sickness but produced detectable antibodies. It is likely that infection was halted before entering nerve pathways. BAER & OLSON (1972) reported that, following apparent recovery from clinical rabies, 4 pigs developed antibodies within 2 months. In Grenada, 2 goats and a donkey known to have been bitten by mongoosesshowed rabies antibodies at a low titre 30 and 54 days after exposure (EVERARDet al., 1981). FEKADU & BAER (1980) and FEKADU et al. (1981) found neutralizing antibody in serum and cerebrospinal fluid of dogs that had recovered from clinical rabies.
in Nigeria”
Age group (years) la-14 E-19 20-24 25-29 30-34 35-39 4a-44 45-49 50-54 55-59 560 Total
M&S 216 2115 9128 lo/23 8114 419 217 115 4110 O/l 6115 48/133
“Rapid fluorescent
Table
Females
(33.3%) (13.3%) (32.1%) (43.5%) (57.1%) (44.4%) (28.6%) (20%) (40%) 6) (40%) (36.1%)
focus inhibition
3. Antibody
Titre
3112 6121 18154 8152 3124 3/18 8/20 117 O/l 013 2/S 521217
Total
(25.0%) (28.6%) (33.3%) (15.5%) (12.5%) (16.7%) (40%) (14.4%) 6) 6) (40%) (24%)
5118 8136 27182 18175 11138 7127 10127 2112 4111 o/4 8/20 100/350
(27.8%) (22%) (329%) (24%) (28.9%) (25.9%) (37%) (36.7%) (36.4%) (6) (40%) (28.6%)
test, 1:16 dilution.
titres in human serum samples against rabies, Mokola
No. of sera positive
Rabies Percentage
18
51.4
9 5 3
25.7 14.3 8.6
iif6
1:32 1:64”
No. of sera positive
Mokolo Percentage
and Lagos bat viruses
No. of sera positive 2
2 2
1 1
:i 16.6 8.3
1
Lagos bat Percentage 50
11.1 11.1 -
“No sera were positive at higher titres. Table 4. Seropositivity of serum samples from Nig-erian dogs and humans against Mokola and Lagos bat virusesa
Dog sera (n=241) Mokola virus Lagos bat virus Human sera (n=158) Mokola virus Lagos bat virus
Number positive
Percentage positive
44
17.4 5.8
14 12 4
;:;
test, 1:16 dil-
viruses (Table 4). The results of titrating some of these sera are shown in Tables 1 and 3. Cross reactions between rabies, Mokola and Lagos bat viruses were not observed. Sera which were positive for Mokola were not positive for rabies, and vice versa. test.
In general, titres obtained correlated well with the MNT results (Table 5). Usually, a higher titre was observed in the MNT compared with that obtained by the RFFIT. Discussion
RFFIT
MNT
1:128 1:8 1:8
1:128 1:91 1:32
Canine sera
“Rapid fluorescent focus inhibition ution.
Mouse neutralization
Table 5. Comparison between titres obtained by rapid fluorescent focus inhibition test (RFFIT) and mouse neutralization test (MNT) with canine and human sera
This study has provided serological evidence of infections with members of the Lyssavirus group in
746a 703a 303b
Human sera 107c 99d
1OOd
1:64
1:64
1:8
1:32
1:32
1:50
“Unvaccinated domestic dog. bUnvaccinated stray dog. ‘Previous dog bite, no treatment given. dNo known previous exposure or anti-rabies treatment. Non-fatal infection in dogs could have been overlooked, especially in the stray animals. The detection of antibodies in the human population, however, has posed serious questions of how these people came in contact with the rabies antigen. If the stimulating antigen was rabies virus, how were the people exposed? Only 5% of the 350 people examined reported that they had previously been bitten by a dog; 2 of these had antibody titres. Four of the 6 dog catchers had antibodies against rabies, and one had antibodies against Mokola virus; none had any history
845
of having been bitten by a rabid dog or of receiving prophylactic rabies vaccinations. None of the other people tested had a history of any dog bite or of antirabies vaccinations. As only 1.4% of the people provided a history of dog bites, it is difficult to attribute a 28% prevalence of antibody titre to forgotten dog bites. Further possible explanations must be sought. The antigens inducing the detected antibodies could be viruses belonging to the Lyssavirus group which are yet to be identified, or some unidentified infectious agents cross-reacting with lyssaviruses, possibly arthropodborne. Acknowledgements We thank Dr T. 0. Bunn of the United States Deoartment of Agriculture Animal and Plant Health Inspettion Services, and Dr T. J. Wiktor of the Wistar Institute, Philadelphia, for confirmatory testing of the sera. Dr Wiktor also supplied the monoclonal antibody, specific sera, and tissue culture-adapted viruses. Special and sincere thanks go to Dr T. T. Kramer, chairman of the department of Veterinary Microbiology and Preventive Medicine, Drs Kenneth B. Platt, Martin L. Kaeberla and Loren A. Will, all of the Department of Veterinary Microbiology and Preventive Medicine, for their enthusiastic cooperation and support during the work. We also thank the Veterinary Officers in Nigeria for their cooperation. The techincal assistance given by Mr Daniel Imoru, Dr S. Baba, Mrs Dorothy Murphy, Dr M. A. Mushin, Dr Lekan Ayanwale and Dr (Mrs) Y. 0. Ogunkoya was highly appreciated. References Baer, 0. M. & Olson, H. R. (1972). Recovery of pigs from rabies. Journal of rhe American Veten’nary Medical Association, 160, 1127-l 132. Boulger, L. K. & Porterfield, J. (1958). Isolation of a virus from Nigerian fruit bats. Transactions of rhe Royal Society of Tropical Medicine and Hygiene, 52, 421-424. CDC (1981). Laboratory Methods for Detecting Rabies. Willis, M., Velleca, & Forrester, F. T. (editors). Atlanta, GA 30333, USA: US Department of Health and Human Services, Public Health Services. Everard, C. 0. R., Baer, G. M., Allis, M. E. & Salley, A. M. (1981). Rabies serum neutralizing antibodies in mongoose from Grenada. Transactions of the Royal Society of Tropical Medicine and Hygiene, 75, 654-666. Fekadu, M. & Baer, G. M. (1980). Recovery from clinical rabies of two dogs inoculated with a rabies virus strain from Ethiopia. American Journal of Veterinary Research, 41, 1632-1634.
Fekadu, N., Shaddock, J. H. & Baer, G. M. (1981). Intermittent excretion of rabies virus in the saliva of a dog two and six months after it had recovered from exuerimental rabies. American youma of Trobical Medi* 1 c&e and Hygiene, 3, 1113-11~5. Flamand, A., Wiktor, T. J. & Koprowski, H. (1980). Use of hvbridoma monoclonal antibodies in the detection of antigenic differences between rabies and rabies-related virus proteins. I. The nucleocapsid protein. Journal of General Virology, 48, 97-102. Herbert, G. A. (1974). Ammonium sulphate fractionation of sera from mouse, hamster, guinea pig, monkey, chimpanzee, swine, chicken and cattle. Applied Microbiology, 27, 389-393. Hierholzer, J. C., Suggs, M. T. & Hall, E. C. (1969). Standardized viral haemagglutination and haemagglutination-inhibition test. II. Description and statistical evalution. Applied Microbiology, 18, 824833. Kemp, C. D., Causey, 0. R., Dorothy, L., Odelolar, A. & Fabiti, A. (1972). Mokola virus, further studies on IBAN 27377. A new rabies-related etiological agent of zoonoses in Nigeria. American Journal of Tropical Medicine and Hygiene, 21, 356-359. Ruegsegger, J. M., Black, J. & Sharpless, G. R. (1961). Primary antirabies immunization of man with the Flury virus vaccine.American Journal of Tropical Medicine and Hygiene, 51, 706-716. Shope, R. E. (1982). Rabies related viruses. YaleJournal of Biology and Medicine, 55, 271-275. Shope, R. C., Murphy, F. A., Harrison, A. K., Causey, 0. R., Hemp, G. E., Simpson, D. T. H. & Moore, D. I. (1970). Two African viruses serologically and morphologically related to the rabies virus. Journal of Virology, 6, 690-692. Smith, J. S., Yager, I’. A. & Baer, G. N. (1973). A rapid tissue culture test for determining rabies neutralizing antibody. In: Laboratoty Techniques in Rabies, Kaplan, M. M. & Kaprowski, E. (editors). Geneva: World Health Organization, pp. 35+357. WHO (1984). Guidelines for dog rabies control. Geneva: World Health Organization, mimeographed document no. VPHl83.43. Wiktor, J., Macfaran, R. I., Foggin, C. M. & Kowprowski, H. (1983). Antigenic analysis of rabies and Mokola virus. (Symposium on monoclonal antibodies. Standardization of their characterization and use, Paris, France.) Develogments in Bioloaical Standardisation. 57. 199-211. Yasmuth, C., Evans, R. C. & Doege, T. 6. (1474). Rabies antibody in healthy dogs and vaccine response after MPA. Journal of the Medical Association of Thailand, 57, 131-134.
Received 22 December 1987; revised 20 February 1990; accepted for publication
20 February
1990
