Article — Artikel

Clinical and serological response of wild (Lycaon pictus) to vaccination against canine distemper, canine parvovirus infection and rabies

J van Heerdena, J Binghamb, M van Vuurenc , R E J Burroughsd and E Stylianidesc

(Canis familiaris)14 or jackals (Canis ABSTRACT mesomelas)17 although the source of infec- Wild dogs Lycaon pictus (n = 8) were vaccinated 4 times against canine distemper (n =8) tion cannot always be confirmed. It is not (initially with inactivated and subsequently with live attenuated strains of canine known whether commercial rabies vac- distemper) and canine parvovirus infection (n = 8) over a period of 360 days. Four of the cine (oral or injectable) intended for the wild dogs were also vaccinated 3 times against rabies using a live oral and 4 with vaccination of domestic dogs, given at an inactivated parenteral vaccine. Commercially-available canine distemper, canine appropriate intervals, would generate a parvovirus and parenteral rabies , intended for use in domestic dogs, were used. protective antibody response in wild None of the vaccinated dogs showed any untoward clinical signs. The inactivated canine distemper vaccine did not result in seroconversion whereas the attenuated live vaccine dogs. Seroconversion after vaccination of resulted in seroconversion in all wild dogs. Presumably protective concentrations of wild dogs with commercial vaccines, 14,28 antibodies to canine distemper were present in all wild dogs for at least 451 days. has, however been reported (J. van Canine parvovirus haemagglutination inhibition titres were present in all wild dogs prior Heerden, unpubl. obs., 1994; D.G.A. to the administration of vaccine and protective concentrations persisted for at least 451 Meltzer, unpubl. obs., 1994). days. Vaccination against parvovirus infection resulted in a temporary increase in canine Despite the fact that canine distemper parvovirus haemagglutination inhibition titres in most dogs. Administration of both virus has never been isolated from either inactivated parenteral and live oral rabies vaccine initially resulted in seroconversion in 7 of free-ranging or captive wild dogs, clinical, 8 dogs. These titres, however, dropped to very low concentrations within 100 days. Booster histopathological and serological evidence administrations resulted in increased antibody concentrations in all dogs. It was concluded indicates that wild dogs are susceptible to that the vaccines were safe to use in healthy subadult wild dogs and that a vaccination pro- canine distemper virus infection1–3,19,21,30. tocol in free-ranging wild dogs should at least incorporate booster vaccinations against ra- Clinical signs suggestive of canine distem- bies 3–6 months after the first inoculation. per have been observed in free-ranging Key words: canine distemper, canine parvovirus, Lycaon pictus, rabies, vaccination, wild wild dogs in the Serengeti24 , the Kruger dogs. National Park23, and the Hluhluwe-Umfo- Van Heerden J, Bingham J, Van Vuuren M, BurroughsREJ,Stylianides E Clinical and lozi Park (G Andreka and J van Heerden, serological response of wild dogs (Lycaon pictus) to vaccination against canine distemper, unpubl. obs., 1994). Ten free-ranging wild canine parvovirus infection and rabies. Journal of the South African Veterinary Association dogs died of canine distemper in north- (2002) 73(1): 8–12 (En.). 16 Dalham Road, Kimberley, 8301 South Africa. ern Botswana3. Serological evidence of exposure of free-ranging wild dogs to the distemper virus has been demon- INTRODUCTION myxoviridae, are possibly the 2 most strated in wild dogs in the Hluhluwe- The wild dog (Lycaon pictus) is a highly important viral diseases of carnivores that Umfolozi Park (G Andreka and J van endangered canid species that occurs may impact significantly on free-ranging Heerden, unpubl. obs., 1994), in Northern in fragmented pockets throughout its populations1. Botswana3, in the Selous Game Reserve former range12. Various reasons for its Rabies was responsible for the death of and in the Tsumkwe District of Namibia19. decline have been postulated and include 21 of 23 wild dogs, and probably responsi- Vaccination of captive wild dogs, espe- habitat destruction, persecution, compe- ble for the subsequent disappearance of 8 cially pups, with live attenuated vaccines tition with other predators, lack of genetic wild dog packs in Kenya18. Rabies in wild intended for use in domestic dogs, in- heterozygosity and disease11,12. dogs has also been reported from Tanza- duced distemper-like disease in a number The potential threat of infectious disease nia14, Namibia25 and from a captive pack in of animals10,21,30. Captive wild dogs at the to free-ranging populations of carnivores, Zimbabwe (Veterinary Research Labora- De Wildt Cheetah Research Centre have, especially metapopulations, is increas- tory, Harare, unpubl. data). It is also sus- however, been vaccinated with live canine ingly recognised33. Rabies, caused by a pected to have occurred in the Central distemper commercial dog vaccines virus in the family Rhabdoviridae, and African Republic and Zambia33.In (Pfizer Animal Health) on numerous canine distemper, caused by a virus of the Madikwe, South Africa, Hofmeyr et al.17 occasions, without any untoward effects27 genus in the family Para- witnessed the decimation of a pack of (J van Heerden, unpubl. obs., 1993). Re- wild dogs from 24 to 3, directly or indi- cently, vaccine-induced distemper was a 16 Dalham Road, Kimberley, 8301 South Africa. rectly as a result of rabies. Some of the observed in 3-and-a-half month old wild bARC-Onderstepoort Veterinary Institute, Private Bag X5, Onderstepoort, 0110 South Africa. dogs that died were vaccinated against dog puppies vaccinated with canine dis- cDepartment of Veterinary Tropical Diseases, Faculty of rabies (Pfizer Animal Health) more than 2 temper vaccine (Vanguard Puppy 5, Veterinary Science, Private Bag X4, Onderstepoort, years before the outbreak. Wild dog packs Pfizer Animal Health) (R E J Burroughs, 0110 South Africa. dPrivate practitioner, Pretoria. appear to acquire infectious disease unpubl. obs., 2001). Limited investiga- Received: May 2001. Accepted: February 2002. through contact with rabid domestic dogs tions into the use of domestic dog distem-

8 0038-2809 Tydskr.S.Afr.vet.Ver. (2002) 73(1): 8–12 Table 1: Days on which wild dogs were immobilised and blood specimens collected as well as the vaccination regimens for the three experimental groups.

Day Group 1 (n = 2) Group 2 (n = 4) Group 3 (n = 4)

0 Inactivated canine distemper vaccine Inactivated canine distemper vaccine Inactivated canine distemper vaccine subcutaneously subcutaneously. subcutaneously. Rabies vaccine intramuscularly Oral rabies vaccine 27 No vaccines administered Live attenuated canine distemper and Live attenuated canine distemper and parvovirus vaccine subcutaneously parvovirus vaccine subcutaneously 48 No vaccines administered Live attenuated canine distemper vaccine Live attenuated canine distemper and parvovirus subcutaneouslyvaccine and parvovirus subcutaneously 109 No vaccines administered No vaccines administered No vaccines administered 167 No vaccines administered Rabies vaccine intramuscularly Oral rabies vaccine 200 No vaccines administered No vaccines administered No vaccines administered 363 No vaccines administered Live attenuated canine distemper and Live attenuated canine distemper and parvovirus vaccine subcutaneously. parvovirus vaccine subcutaneously. Rabies vaccine intramuscularly Oral rabies vaccine 391 No vaccines administered No vaccines administered No vaccines administered 451 No vaccines administered No vaccines administered No vaccines administered 634 No vaccines administered No vaccines administered No vaccines administered per vaccines in wild dogs have been own facilities for capturing, handling and cell culture origin (Paris strain (PV-4)); 28,32 7.25 conducted . feeding of the animals. each dose contains at least 10 FID50 of Canine parvovirus infection is a poten- The body masses of the 2 males were rabies virus before inactivation) (Defen- tially important cause of mortality in respectively 20 and 22 kg at the beginning sor®, Pfizer Animal Health) intended for domestic dog puppies4. Antibodies to the of the experiment and 25 and 31 kg at the use in domestic animals, and a live oral virus have been found in some free- end of the experiment. The body masses vaccine (an escape mutant live rabies ranging wild dog populations31. It is, how- of the females ranged from 17 to 19 kg at vaccine20, Virbac Laboratories, France) ever, not known whether parvovirus the beginning of the experiment and diluted in tissue culture medium with persists in wild dog populations or ranged from 21 to 25 kg at the end of the 10 % foetal calf serum to give a titre of 8.0 whether wild dogs are infected through experiment. Dogs were immobilised by log10 median tissue culture infectious contact with infected domestic dogs. intramuscular administration of a total doses per m (TCID 50/m ). The titre of Seroconversion following the administra- dose of medetomidine (Domitor, Novartis) the oral vaccine was determined by titra- tion of canine parvovirus vaccine has ranging from 80 to 90 µg in combination tion in BHK-21 cells. Live attenuated been described in wild dogs27. with a total dose of ketamine hydrochlo- distemper vaccine (given subcutaneously The aim of this investigation was to ride (Anaket-V, Centaur Labs) ranging in the neck area) and rabies vaccine were monitor the safety and efficacy (in induc- from 20 to 25 mg. The anaesthetic drugs administered on 3 occasions to Group 2 ing antibodies) of commercial rabies, were administered with a pole syringe and 3 experimental wild dogs (Table 1). canine distemper and canine parvovirus while the wild dogs were temporarily The inactivated rabies vaccine was given domestic dog vaccines in wild dogs. restrained in a crush. Following collection intramuscularly in the gluteal muscle and of specimens, weighing of the dogs and the oral vaccine was administered by MATERIALS AND METHODS administration of the respective vaccines, deposition of 1 m of SAG-2 vaccine into Ten captive-bred wild dogs (Numbers 1 anaesthesia was reversed with the intra- the oral cavity by syringe. to 10), individually identified by inserted muscular administration of atipamezole Following administration of vaccines, transponders, 2 males and 8 females 11 hydrochloride (Antisedan, Novartis) at a the wild dogs were observed daily for months of age were randomly allocated dose ranging from 0.8 to 0.9 m . clinical signs of disease. to 1 of 3 treatment groups (Table 1). The inactivated vaccine used was a Blood specimens were collected on 10 Group 1 consisted of 2 control wild dogs formaldehyde-inactivated Rockborn occasions (Table 1) from all experimental that only received inactivated canine strain of canine distemper virus, manu- animals. Clotted specimens were trans- distemper vaccine, Group 2 consisted of 4 factured for non-commercial purposes ported to the laboratory within 2 hours, animals that received live attenuated (Lot Number 930601, Akzo Nobel, centrifuged and stored frozen until tested canine distemper vaccine, canine par- Intervet South Africa). The attenuated for antibodies to canine distemper, canine vovirus vaccine, and rabies vaccine intra- distemper vaccine used was a commercial parvovirus, and rabies at the end muscularly, and Group 3 consisted of 4 vaccine intended for use in dogs, contain- of the experiment. Rabies antibodies were animals that received live canine distem- ing live strains of canine distemper virus detected using the neutralisation test as per vaccine, canine parvovirus vaccine, (Snyder Hill strain, modified by adapta- described by Cliquet et al.8 Antibodies to and oral rabies vaccine (Table 1). Before tion to NL-DK-1 cells) and canine par- canine distemper virus were detected by administration of the live distemper and vovirus (NL-35-D strain) (Vanguard means of a serum neutralisation test5. parvovirus vaccine, all dogs were vacci- puppy 5, Pfizer Animal Health). This Serum antibodies against canine parvovi- nated with an inactivated canine distem- vaccine also contains the Manhattan rus were determined by means of a per vaccine. All animals were in an strain of canine adenovirus type 2 and the haemagglutination inhibition test. The apparently healthy physical condition. NL-CPI-5 strain of canine parainfluenza procedure was briefly as follows: sera The dogs were held in 2 adjoining camps, virus. The rabies vaccines used were a were diluted 1:2 in phosphate buffered approximately 50 m2 in size, each with its commercial inactivated rabies vaccine of saline (pH 7.4) and heat-inactivated at

0038-2809 Jl S.Afr.vet.Ass. (2002) 73(1): 8–12 9 Table 2: Rabies serum neutralising antibody titres (in international units per millilitre) in each of three experimental groups of wild dogs. A titre of 0.5 IU/m is considered significant.

Id No. Group Day 0 Day 27 Day 48 Day 109 Day 167 Day 200 Day 363 Day 391 Day 451 Day 634

10 1 <0.02 <0.06 <0.06 <0.1 <0.03 <0.03 <0.04 <0.02 <0.02 <0.04 2 1 <0.02 <0.13 1 2 <0.02 18.2 1.51 <0.1 0.3 41.7 18.2 13.8 7.94 31.6 3 2 <0.02 3.5 2.63 0.87 0.87 380 41.7 75.9 42.7 41.7 5 2 <0.02 <0.13 <0.06 <0.1 <0.03 4.6 6 302 42.7 31.6 6 2 <0.02 10.5 1.51 0.87 0.29 380 126 381.9 166 218.8 4 3 <0.02 3.5 1.51 0.17 0.06 7.9 6 2.63 2.63 2 7 3 <0.02 6 4.57 0.17 0.06 4.6 2 4.57 2.63 3.5 8 3 <0.02 10.5 4.57 0.17 <0.03 24 7.9 13.8 13.8 31.6 9 3 <0.02 13.8 13.8 1.51 0.87 7.9 10.5 7.94 4.57 3.5

56 °C for 30 min. Duplicate serial 2-fold the intestines. The intestinal wall was ulation was based on the fact that attenu- dilutions of the sera were made in a barbi- congested and flaccid. Histopathological ated vaccines have been reported to yield tal-acetate buffer (pH 6.2). To the first examination of the small intestines adverse effects in wild carnivores22. dilution series, 8 HA units of CPV (refer- demonstrated severe, acute, multifocal Whereas recommendations on vaccina- ence strain CPV-2) was added, while the bacterial necrotic enteritis with areas of tion of wild carnivores with live canine second set of serum dilutions contained pseudomembrane formation. Small distemper virus-containing vaccines buffer and therefore served as a control Gram-positive rods were present on the should be based on clinical trials and not for nonspecific inhibition. After the addi- surface of the villi and Gram-negative extrapolation, the use of a live vaccine tion of the antigen, the plates were held at rods in the crypts. Marked acute necrosis was not deemed to be without risk, and room temperature for 80 min. This was of the follicles in the spleen and lymph therefore raised ethical concerns when followed by the addition of a 1 % porcine nodes was evident. The alveolar walls in considering the current status of wild red blood cell suspension (prepared in the lung were thickened due to conges- dogs. Although the wild dogs in this barbital-acetate buffer). Plates were tion and leukostasis. A few large bronchi trial did not seroconvert following the placed at 4 °C overnight and the titre was showed a mild acute neutrophil infiltra- use of the inactivated vaccine, a cellular read as the highest dilution at which 50 % tion. The liver and kidney were severely response was most probably generated, inhibition occurred. The positive control congested. The brain and spinal cord and may have contributed to the strong serum of a known antibody titre was showed severe oedema. There were no anamnestic response after the first inocu- included with each batch of tests. histopathological indications of canine lation with an attenuated vaccine. Full autopsies, including histopatho- distemper infection. The fluorescent anti- During the trial there was no indication logical examination, and direct fluores- body test for the detection of rabies virus of vaccine-induced disease caused by the cent antibody staining for rabies virus, yielded negative results. Electron micro- live distemper, parvovirus or rabies were conducted on all animals that died scopic examination of intestinal contents vaccines. The continued healthy state of during the trial. for the presence of parvovirus was nega- the experimental animals throughout the tive. Aerobic and anaerobic culturing of duration of the trial is supported by their RESULTS tissue specimens yielded growth of gains in body mass. Although none of the All wild dogs remained healthy Streptococcus canis, Klebsiella pneumoniae, experimental wild dogs in this investiga- throughout the experiment with the Flavobacterium sp. and Citrobacter freundii. tion that received distemper vaccine exception of 1 of the control dogs (Num- Rabies serum neutralising antibody developed any untoward side-effects, the ber 2), which developed an acute and titres are given in Table 2, canine distem- vaccine cannot be recommended without severe watery diarrhoea on Day 50. This per serum neutralising antibody titres in reservation as safe for use in wild dogs. wild dog was anorexic, depressed, the Table 3, and canine parvovirus haemag- Most reports on vaccine-induced distem- diarrhoea progressed and it died within glutination inhibition titres in Table 4. per and/or distemper-like disease in wild 24 hours. An autopsy revealed an animal dogs occurred in puppies, especially in in fair condition with an empty stomach, DISCUSSION those infected with a concomitant patho- enlarged adrenal cortices and a large The decision to use an inactivated gen. Vaccination of wild dogs with atten- amount of pink to dark red watery fluid in canine distemper vaccine as the first inoc- uated canine distemper vaccines should

Table 3: Canine distemper serum-neutralising antibody titres in three experimental groups of wild dogs. Titres were determined in the range 1:5 to 1:160. Where no end point of neutralisation was reached at a titre of 1:160, the titre was recorded as >1:160.

Id No. Group Day 0 Day 27 Day 48 Day 109 Day 167 Day 200 Day 363 Day 391 Day 451

10100000 0000 21000 1 2 0 0 1:40 >1:160 1:80 1:80 1:40 1:40 1:80 3 2 0 0 >1:160 1:80 1:80 1:80 1:80 1:40 1:40 5 2 0 0 >1:160 1:80 1:80 1:40 1:80 1:40 1:160 6 2 0 0 1:160 1:160 >1:160 1:80 1:80 1:80 1:160 4 3 0 0 >1:160 1:160 >1:160 >1:160 >1:160 >1:160 >1:160 7 3 0 0 1:160 1:80 1:80 1:40 1:80 1:40 1:20 8 3 0 0 >1:160 1:160 >1:160 1:80 >1:160 >1:160 >1:160 9 3 0 0 1:40 1:80 1:10 1:10 1:10 1:10 1:10

10 0038-2809 Tydskr.S.Afr.vet.Ver. (2002) 73(1): 8–12 Table 4: Canine parvovirus haemagglutination inhibition titres in three experimental groups of wild dogs.

Id No. Day 0 Day 27 Day 48 Day 109 Day 167 Day 200 Day 363 Day 391 Day 451

1 1:1024 1:1024 1:2048 1:512 1:512 1:256 1:256 1:512 1:256 2 1:1024 1:1024 1:1024 3 1:512 1:1024 1:2048 1:1024 1:512 1:512 1:512 1:1024 1:1024 4 1:1024 1:1024 1:2048 1:1024 1:1024 1:1024 1:512 1:1024 1:1024 5 1:512 1:512 1:2048 1:256 1:512 1;256 1:256 1:512 1:512 6 1:512 1:1024 >1:8192 1:2048 1:1024 1:512 1:2048 1:8192 1:2048 7 1:512 1:2048 1:4096 1:1024 1:512 1:512 1:1024 1:1024 1:1024 8 1:1024 1:2048 1:8192 1:2048 1:1024 1:1024 1:1024 1:8192 1:2048 9 1:1024 1:1024 1:8192 1:1024 1:1024 1:1024 1:1024 1:1024 1:2048 10 1:2048 1:1024 1:2048 1:2048 1:1024 1:512 1:1024 1:1024 1:2048 therefore preferably be undertaken in primary vaccination are short-lived and that serum-neutralising titres of 1:20 are healthy wild dogs that are free from other booster vaccinations should be given protective for canine distemper7. Evalua- infections. The exact age at which pups within about 3 months. Booster vaccina- tion of vaccines on the basis of antibody can safely be vaccinated has not been tions were effective for stimulating high response alone was the most practical established, but it is possible that their and more persistent neutralising anti- method of assessing efficacy. Although state of health may be more important body concentrations. Although both there may be unquantifiable protection in than their age. types of vaccine demonstrated a booster the absence of neutralising antibody, it is The death of one of the control dogs effect, it appeared that parenteral vacci- most likely that such protection would from severe bacterial enteritis shortly nation stimulated higher concentrations not be of long duration and therefore after the third immobilisation of the wild of neutralising antibodies than oral vacci- could, for practical purposes, be consid- dogs might have been precipitated by the nation at the evaluated dose rate. This, ered inadequate. We therefore consid- stress of capturing and darting. Darting of however, does not necessarily imply that ered a significant antibody titre as captive wild dogs is frequently associated immunity was more protective or of indicating protection and the lack thereof with excitement, running, panting and longer duration with parenteral vaccina- as an indication of probably inadequate vocalisation. There was also a marked tion. Higher oral doses within safe protection. variation in behaviour between the differ- concentration limits may well induce This trial has attempted to demonstrate ent animals. A detailed autopsy and micro- higher antibody concentrations. efficacy and safety of commercial dog biological investigation was undertaken Canine parvovirus haemagglutination vaccines against 3 important canid to exclude the possibility of death due to inhibition titres were present in the exper- diseases in wild dogs. Unlike the inacti- any of the 3 infectious diseases under in- imental wild dogs before the administra- vated vaccine, live distemper vaccine vestigation. tion of vaccine. The experimental dogs appears to be effective, but it should be Failure to seroconvert following the use were housed on a facility where domestic used with care, as the risk of vaccine-in- of an inactivated distemper vaccine is in dogs were held in close proximity, which duced disease with the currently avail- accordance with the findings of other could possibly explain the positive able vaccines registered for domestic dogs workers who found little serological evi- haemagglutination inhibition titres be- remains. Although all wild dogs had dence of protection in follow-up studies fore vaccination. An anamnestic response parvovirus antibody through natural in several species9,15. Seroconversion was was observed after administration of the exposure at the start of the trial, the par- obtained following administration of the first vaccine in 4 of the 8 dogs, while 5 out vovirus vaccine induced an anamnestic attenuated vaccine and protective titres of 8 dogs demonstrated an increase after a response in Dogs 5, 6, 8 and 9 following remained throughout the observation booster injection on Day 360. Antibodies the first vaccination. Immunity to canine period. Booster injections in general did to canine parvovirus have been demon- parvovirus is believed to be antibody-me- not result in increased antibody titres, but strated in samples from some free- diated, and haemagglutination inhibition the frequency of sampling may have been ranging populations, whereas samples titres equal to or more than 1:80 are con- inadequate to detect booster effects. from other free-ranging populations have sidered protective7. Both partenteral and All wild dogs given oral rabies vaccine tested negative31. SAG-2 oral vaccine caused sero- and all but one given the parenteral No challenge studies were carried out in conversion, although the response to vaccine responded after the primary this investigation owing to the unavail- primary vaccination in both cases was of vaccination with marked anti-rabies ability of sufficient naive wild dogs, the short duration. Using vaccines of similar neutralising antibody titres. The non- lack of suitably characterised challenge potency as used in this trial, booster doses responder only showed seroconversion virus strains, lack of facilities to safely of rabies will be necessary 1–3 months following the booster injection. In all dogs house rabid animals, and ethical consid- after the primary vaccination. We tenta- the titres following the primary vaccina- erations. Absence of neutralising anti- tively conclude that all the vaccines used tion declined to low concentrations body does not necessarily indicate the in this trial could be used in free-ranging within less than 2 months, a finding that is absence of protection; without controlled wild dogs, with the exception of inacti- similar to previous studies in domestic challenge studies it would be impossible vated distemper vaccine, which does not dogs vaccinated parenterally29. However, to accurately assess the significance of appear to be effective. other studies of parenterally and orally an undetected titre. The presence of neu- vaccinated dogs16,26 indicate that the tralising antibody is generally well corre- ACKNOWLEDGEMENTS primary antibody response is short in lated with protection against rabies in Virbac Laboratories, France is gratefully comparison with domestic dogs. As other species using SAG-2 and similar acknowledged for donating SAG-2 the neutralising antibodies following vaccines6,13. Similarly, it has been stated vaccine for this trial; Novartis South

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12 0038-2809 Tydskr.S.Afr.vet.Ver. (2002) 73(1): 8–12