COMPARISON OF ORAL AND INTRAMUSCULAR RECOMBINANT CANINE DISTEMPER VACCINATION IN AFRICAN WILD DOGS (LYCAON PICTUS) Author(s): Maren Connolly, D.V.M., Patrick Thomas, Ph.D., Rosie Woodroffe, D. Phil. and Bonnie L. Raphael, D.V.M., Dipl. A.C.Z.M. Source: Journal of Zoo and Wildlife Medicine, 44(4):882-888. Published By: American Association of Zoo Veterinarians DOI: http://dx.doi.org/10.1638/2012-0232R2.1 URL: http://www.bioone.org/doi/full/10.1638/2012-0232R2.1
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Journal of Zoo and Wildlife Medicine 44(4): 882–888, 2013 Copyright 2013 by American Association of Zoo Veterinarians
COMPARISON OF ORAL AND INTRAMUSCULAR RECOMBINANT CANINE DISTEMPER VACCINATION IN AFRICAN WILD DOGS (LYCAON PICTUS) Maren Connolly, D.V.M., Patrick Thomas, Ph.D., Rosie Woodroffe, D. Phil., and Bonnie L. Raphael, D.V.M., Dipl. A.C.Z.M.
Abstract: A series of three doses of recombinant canary-pox–vectored canine distemper virus vaccine was administered at 1-mo intervals, orally (n ¼ 8) or intramuscularly (n ¼ 13), to 21 previously unvaccinated juvenile African wild dogs (Lycaon pictus) at the Wildlife Conservation Society’s Bronx Zoo. Titers were measured by serum neutralization at each vaccination and at intervals over a period of 3.5–21.5 mo after the initial vaccination. All postvaccination titers were negative for orally vaccinated animals at all sampling time points. Of the animals that received intramuscular vaccinations, 100% had presumed protective titers by the end of the course of vaccination, but only 50% of those sampled at 6.5 mo postvaccination had positive titers. None of the three animals sampled at 21.5 mo postvaccination had positive titers. Key words: African wild dog, canine distemper virus, Lycaon pictus, vaccination.
INTRODUCTION The African wild dog (Lycaon pictus, AWD) is an endangered species, with fewer than 700 packs estimated to remain in the wild.42 The species’ decline reflects the expansion of human populations and the consequent fragmentation of habitat available to wildlife.12 AWDs fare poorly in human-dominated landscapes, partly because people perceive them as a threat to livestock and kill them, and partly because the domestic dogs that inevitably live alongside people carry diseases that can devastate AWD populations.4,16,39,40 Infectious diseases represent a particularly serious threat to very small AWD populations, including those being re-established through reintroductions.8,28 Outbreaks of canine distemper virus (CDV) have been associated with the loss of entire packs and devastated a captive colony in Tanzania.1,9,32 Given that CDV can infect domestic dogs as well as AWDs, it is often assumed that domestic dog vaccination will reduce the risks of transmission to wildlife. However, new evidence indicates that domestic dogs may not always be From Zoological Health, Wildlife Conservation Society, 2300 Southern Boulevard, Bronx, New York 10460, USA (Connolly, Raphael); Zoos and Aquarium Division, Wildlife Conservation Society, 2300 Southern Boulevard, Bronx, New York 10460, USA (Thomas); and Institute of Zoology, Regent’s Park, London, NW1 4RY, United Kingdom (Woodroffe). Present address (Connolly): Dallas Zoo and Children’s Aquarium at Fair Park, Dallas Zoo Management, 650 South R.L Thornton Freeway, Dallas, Texas 75203, USA. Correspondence should be directed to Dr. Connolly ([email protected]).
the reservoir of CDV in wildlife areas, and may not be a primary source of CDV infection for AWDs.6,25,26,41 Hence, CDV vaccination of domestic dogs might not protect AWDs. In the absence of alternative management approaches, and the threats of significant population impacts, range state wildlife authorities therefore recently recommended that efforts be made to ‘‘evaluate the conservation potential of vaccinating free ranging wild dogs against canid diseases.’’14 A number of challenges have impaired previous management attempts to vaccinate free-ranging AWDs against CDV. Responses to previous vaccination attempts of AWDs and other nondomestic carnivores with modified live canine distemper vaccines have varied, ranging from adequate titer response to poor seroconversion or even death associated with vaccination.7,10,17,18,20,22,30,33–35 Use of inactivated CDV vaccines in nondomestic carnivores, including AWDs, has resulted in poor to no detectable seroconversion.20,34 These problems are common to a variety of nondomestic carnivores, as well as domestic ferrets, and have resulted in the development of new recombinant vaccines.11,19,31 Both experimental and commercially available recombinant canine distemper vaccinations have been used safely in domestic dogs and other carnivore species, including AWDs.5,23,37,38 Some of those have been immunogenic when administered orally in other species.36,38 In particular, the commercially available recombinant canary-pox–vectored CDV vaccine (PurevaxTM Ferret, Merial Limited, Duluth, Georgia 30096, USA) appears to be both safe and potentially effective in a variety of threatened
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Table 1. Serum antibody titers against canine distemper virus (CDV) in African wild dogs (AWDs): oral vaccination.a Litter
Age at first vaccine (mo)
A A A A A B B B Mean Range % 32d
2 2 2 2 2 2 2 2
Neg 1 mo
First vacc
Second vacc
Third vacc
3.5 mo
,8 ,8 ,8 ,8 ,8 ,8 ,8 ,16 0 0–16 0
,8 ,16 ,8 ,16 ,8 ,16 ,8 ,16 0 0–16 0
,8 ,8 ,8 ,8 ,8
12
29 25 45 54 25 ,8b ,8 ,8 22.3 0–54 25.0
12 –c 0
0 0 0
5 mo
6.5 mo
,8 ,8 ,8 ,8 ,8 ,8 ,8 ,16 0 0–16 0
0 0 0
a Serum antibody titers against CDV for individual AWDs at each time point. Samples collected prior to vaccination are indicated at Neg 1 month. Samples collected at the time of each vaccination are indicated at # vacc. b Values reported as ,8 or ,16 were set as 0 for the purposes of the calculations as both indicate that no antibody was detected. c Not calculated. d Percent of pups with titers greater than or equal to 32 at each sampling point.
species.3,13 This vaccine has the potential to be effective via both the parenteral and oral routes, and has been used experimentally in AWDs without clinical side effects.23,36 The goal of this study was to evaluate novel protocols for vaccinating AWDs against CDV in a zoo setting by investigating humoral responses to the canary-pox–vectored vaccine administered either orally or parenterally.
MATERIALS AND METHODS Vaccination The study population was comprised of 21 previously unvaccinated AWD pups born at the Wildlife Conservation Society’s Bronx Zoo in three litters between 2005 and 2007. The parents of the litters had previously been vaccinated for CDV, although the vaccines used and frequency of vaccination varied. All pups were housed with the pack under similar conditions for the duration of the study. All study animals received a series of three vaccinations with a recombinant canary-pox–vectored canine distemper vaccine (PurevaxTM Ferret) administered either orally (PO) or intramuscularly (IM). An initial group of 15 animals (in two litters from different dams) were assigned to either a PO (n ¼ 8) or IM (n ¼ 7) vaccination group with the use of a random numbers table. Subsequently, a third litter of six pups all received IM vaccinations, for a total of 13 IM vaccinated animals. The initial vaccines were administered when the pups were 2 mo of age. Subsequent booster vaccines
were administered at 0.5–1.5-mo intervals (Tables 1 and 2). Initial vaccinations were performed with the animals under manual restraint; older animals were vaccinated while temporarily restrained in a chute. For PO vaccination, the animal was encouraged to bite a padded wooden block while a 6-in. metal ball-ended gavage tube and 5 ml syringe were used to spray 1 ml of vaccine onto the gingival mucosa. For IM vaccination, 1 ml of vaccine was administered into the gluteal muscles. Blood collection and analysis Blood was collected at the time of initial vaccination, during subsequent booster vaccinations, and intermittently thereafter (Table 1). Blood collection from PO-vaccinated animals ceased after 6.5 mo because of the lack of immune response noted up to that point. Collection intervals were partially determined by management needs. The number of animals sampled declined throughout the study period because of animals being sent to other zoos (n ¼ 3 PO, n ¼ 7 IM), death from unrelated causes (n ¼ 1 IM) and management concerns about pack hierarchy disruption during handling (n ¼ 2 IM). Blood was collected from the lateral saphenous vein. Blood samples were immediately placed in a standard serum separator tube. Serum was separated via centrifugation within 30 min of collection. Serum was then removed and stored at 808C until shipment to the laboratory. Titers were measured by serum neutralization at the Cornell University Animal Health Diagnostic Center (AHDC, Ithaca, New York, USA)
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Table 2. Serum antibody titers against canine distemper virus (CDV) in African wild dogs (AWDs): intramuscular vaccination.a
Litter
Age at first vaccine (mo)
A A A A B B B C C C C C C Mean Range % 32c
2 2 2 2 2 2 2 2 2 2 2 2 2
Neg 1 mo
,8b 24 16
13.3 0–24 0
First vacc
Second vacc
Third vacc
19 21 13 25 ,8 ,8 ,8 ,8 ,8 ,16 ,4 ,4 ,8 6.0 0–25 0
,8 192 24 24 96 48 16 128 128 64 48 ,16 48 62.8 0–192 61.5
,16 768 96 192 512 768 384 512 768 512 512 1,536 768 563.7 0–1,536 92.3
2.5 mo
3.5 mo
5 mo
768 512 192 128
6.5 mo
21.5 mo
128 96 ,16 24 96 96 128
192 192 96 384 384 384 272.0 96–384 100.0
,16 ,16 ,16
400.0 128–768 100.0
106.7 96–128 100.0
62.0 0–128 50.0
0 0 0
a Serum antibody titers against CDV for individual AWDs at each time point up to 21.5 mo. Samples collected prior to vaccination are indicated at Neg 1 month. Samples collected at the time of each vaccination are indicated at # vacc. b Values ,4, ,8, or ,16 were set as zero for the purposes of the calculations, as all indicate that no antibody was detected. c Percent of pups with titers greater than or equal to 32 at each sampling point.
and are reported as the inverse of the dilution. A value of 8 was considered to represent CDVspecific antibodies, whereas a value of 32 was interpreted as sufficient for protection based upon both recommendations made for domestic dogs at the Cornell University AHDC and values used in previous studies.2,17,21 Data analysis Separate analyses of antibody titers before and after completion of vaccine courses were conducted. Differences in log-transformed titers were analyzed with the use of generalized linear mixed models (GLMM), including random effects to account for nonindependence of data collected from the same animal or litter. GLMMs were fitted with the use of the lme procedure (in the statistical software package R).24,27 Individual identity was included as a random effect. For these analyses, undetectable titers (,8) were assigned zero values. To avoid problems associated with log-transforming zero values, 4 (equivalent to half the lowest nonzero value) was added to all values before taking the (natural) logs. To maximize consistency of the data, statistical comparisons were restricted to the two litters that received both treatments. However, data from the third litter, which received only intramuscular vaccination, are included in descriptive analyses.
RESULTS No clinical abnormalities were observed in any of the AWD pups, whether vaccinated PO or IM. Some pups showed evidence of maternal antibodies prior to vaccination. Of four pups sampled a month before vaccination (aged 1 mo), three had detectable serum antibody titers (8) although none were 32. None of these animals had detectable antibodies at the time of vaccination. However, nine pups from a different litter did have detectable serum antibody titers at the time of initial vaccination, and two of these had titers 32 (Tables 1 and 2). As the course of three CDV vaccinations was administered, the two treatment groups showed different temporal patterns in their titers. Pups given IM vaccination showed rising titers, whereas those given PO vaccination remained seronegative (Fig. 1). The interaction between treatment (PO vs. IM) and time since the start of vaccination was statistically significant at P , 0.001. This difference in temporal patterns continued after the vaccinations were completed. Animals vaccinated PO continued to be seronegative, whereas IM-vaccinated animals showed declining titers. The interaction between treatment (PO vs. IM) and time since the end of vaccination was statistically significant at P , 0.005. Of the 13 pups vaccinated by the IM route, titers 32 were recorded after a single dose of
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885
Figure 1. Changes in canine distemper virus (CDV) serum antibody titers in African wilds dogs (AWDs) given three successive doses of recombinant CDV vaccine, either intramuscularly (closed symbols) or orally (open symbols). Data are shown from the periods during (a) and after (b) administration of the vaccine courses. Circles, triangles, and diamonds indicate mean (and SE) values from litters A, B, and C, respectively; litter C received only intramuscular vaccination. Dashed lines and dots indicate titers at which domestic dogs are presumed to be protected (titer 32, log-transformed as 3.58), dashed lines indicate titers below which antibodies are no longer detectable (titer ,8, log-transformed as 2.48).
vaccine in 8 pups (62%), after two doses in 12 pups (92%), and after three doses in all 13 pups. All blood samples collected within 2 mo of completing the vaccine course revealed titers 32 (Fig. 1), but by 4 mo postvaccination only two of four animals sampled still had titers 32. None of three animals resampled 20 mo postvaccination had detectable antibodies (titers all ,8).
DISCUSSION Findings of this study suggest that the commercially available canary-pox–vectored recombinant CDV vaccine was safe, and potentially effective, for use in AWDs if delivered by the IM route. Vaccination by the PO route was safe but highly unlikely to be effective in providing protection against CDV. For the purposes of this study, a serum antibody titer of 32 was used to indicate potential protection against CDV. However, an assessment of actual protection against infection was not performed. The endangered status of AWDs renders a challenge study inadvisable. None of the pups vaccinated by the PO route displayed a clinically relevant humoral response, even though some had evidence of maternal antibodies before vaccination. Because of the degree of variation in parental vaccination history, as well as the lack of CDV titers obtained from the dams, no substantial conclusions can be drawn from this study in regards to maternal vaccination strategies and resulting maternal antibodies in the pups. The potential remains for maternal antibodies to interfere with initial vac-
cination. However, of those pups with detectable CDV titers at the time of initial vaccination, those that received IM vaccinations did display a rise in titers following booster injections. Those pups that received the PO vaccine did not display a corresponding increase in titers. The lack of a clinically relevant humoral response following PO administration contrasts with results obtained when administering the same vaccine orally to another canid species, the Channel Island fox (Urocyon littoralis), in which measurable antibody responses were present in over 80% of the study population.36 In addition, mucosal vaccination with other experimental recombinant canine distemper vaccines has been shown to be effective in both the Siberian polecat (Mustela eversmanni) and the domestic ferret (Mustela putorius).37,38 However, the Channel Island fox study population received a double dose of the vaccine (two 1-ml vials PO) whereas this study utilized the standard 1-ml dose vial previously demonstrated to be safe for use in AWDs.23 Future research might examine the possibility for increased efficacy of higher PO vaccine doses in AWDs. The reason for the poor titer response following PO vaccination in AWDs compared to other nondomestic carnivores remains otherwise unclear. In contrast with the negative titers obtained following PO vaccination, all pups receiving the series of three IM injections displayed a strong humoral immune response. However, the complete series of three injections was required to stimulate high titers in 100% of the pups (Table
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1). The humoral responses that were observed suggest that a minimum of two injections would be required to provide protection to the majority of pups. This requirement might be difficult or impractical to meet in a free-ranging situation. Moreover, even following three IM injections, titers began to fall rapidly. Comparable results were obtained in a previous study on the use of PurevaxTM Ferret in AWDs, in which a substantial portion of the animals no longer had protective titers 1 yr post IM vaccination series.23 This pattern differs from that observed in domestic dogs following vaccination with modified live products, in which studies have shown protective titers achieved following a single vaccination.2,29 In addition, in domestic dogs, titers to CDV have been shown to persist from 36 mo to as long as 5 yr following vaccination with a canary-pox–vectored CDV vaccine, and 9 yr following vaccination with modified live CDV vaccines, with minimal decrease in serum antibody titer noted during that time.15,29 Domestic dogs challenged with CDV 9 yr following vaccination resisted infection.29 In domestic dogs vaccinated against CDV, it has been suggested that the actual titer of antibodies is of lesser importance than simply the presence of detectable antibodies.29 Despite a low titer, domestic dogs will develop a rapid anamnestic humoral and cell-mediated immune response, protecting against infection and disease. Potentially, vaccinated AWDs might mount a protective immune response when exposed to CDV despite low titers. Unfortunately, because of the inadvisability of challenge studies in AWDs, the efficacy of the combined humoral and cell-mediated immune response following vaccination is difficult to assess. Future studies might examine the titer response following an additional booster vaccination administered after the initial series. Based on the findings in this study, PO vaccination with the PurevaxTM Ferret recombinant distemper vaccine appears not to be efficacious in AWD. Further studies might investigate the usefulness of alternate vaccines or doses. A series of three IM vaccinations resulted in a strong humoral immune response in all animals receiving the vaccine, although practical use in freeranging situations remains challenging. Acknowledgments: This study was funded in part by a One WCS grant. Thanks and appreciation are extended to all of the Bronx Zoo mammal department and Zoological Health staff that participated in this study.
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Journal of Zoo and Wildlife Medicine 44(4): 882–888, 2013 Copyright 2013 by American Association of Zoo Veterinarians
COMPARISON OF ORAL AND INTRAMUSCULAR RECOMBINANT CANINE DISTEMPER VACCINATION IN AFRICAN WILD DOGS (LYCAON PICTUS) Maren Connolly, D.V.M., Patrick Thomas, Ph.D., Rosie Woodroffe, D. Phil., and Bonnie L. Raphael, D.V.M., Dipl. A.C.Z.M.
Abstract: A series of three doses of recombinant canary-pox–vectored canine distemper virus vaccine was administered at 1-mo intervals, orally (n ¼ 8) or intramuscularly (n ¼ 13), to 21 previously unvaccinated juvenile African wild dogs (Lycaon pictus) at the Wildlife Conservation Society’s Bronx Zoo. Titers were measured by serum neutralization at each vaccination and at intervals over a period of 3.5–21.5 mo after the initial vaccination. All postvaccination titers were negative for orally vaccinated animals at all sampling time points. Of the animals that received intramuscular vaccinations, 100% had presumed protective titers by the end of the course of vaccination, but only 50% of those sampled at 6.5 mo postvaccination had positive titers. None of the three animals sampled at 21.5 mo postvaccination had positive titers. Key words: African wild dog, canine distemper virus, Lycaon pictus, vaccination.
INTRODUCTION The African wild dog (Lycaon pictus, AWD) is an endangered species, with fewer than 700 packs estimated to remain in the wild.42 The species’ decline reflects the expansion of human populations and the consequent fragmentation of habitat available to wildlife.12 AWDs fare poorly in human-dominated landscapes, partly because people perceive them as a threat to livestock and kill them, and partly because the domestic dogs that inevitably live alongside people carry diseases that can devastate AWD populations.4,16,39,40 Infectious diseases represent a particularly serious threat to very small AWD populations, including those being re-established through reintroductions.8,28 Outbreaks of canine distemper virus (CDV) have been associated with the loss of entire packs and devastated a captive colony in Tanzania.1,9,32 Given that CDV can infect domestic dogs as well as AWDs, it is often assumed that domestic dog vaccination will reduce the risks of transmission to wildlife. However, new evidence indicates that domestic dogs may not always be From Zoological Health, Wildlife Conservation Society, 2300 Southern Boulevard, Bronx, New York 10460, USA (Connolly, Raphael); Zoos and Aquarium Division, Wildlife Conservation Society, 2300 Southern Boulevard, Bronx, New York 10460, USA (Thomas); and Institute of Zoology, Regent’s Park, London, NW1 4RY, United Kingdom (Woodroffe). Present address (Connolly): Dallas Zoo and Children’s Aquarium at Fair Park, Dallas Zoo Management, 650 South R.L Thornton Freeway, Dallas, Texas 75203, USA. Correspondence should be directed to Dr. Connolly ([email protected]).
the reservoir of CDV in wildlife areas, and may not be a primary source of CDV infection for AWDs.6,25,26,41 Hence, CDV vaccination of domestic dogs might not protect AWDs. In the absence of alternative management approaches, and the threats of significant population impacts, range state wildlife authorities therefore recently recommended that efforts be made to ‘‘evaluate the conservation potential of vaccinating free ranging wild dogs against canid diseases.’’14 A number of challenges have impaired previous management attempts to vaccinate free-ranging AWDs against CDV. Responses to previous vaccination attempts of AWDs and other nondomestic carnivores with modified live canine distemper vaccines have varied, ranging from adequate titer response to poor seroconversion or even death associated with vaccination.7,10,17,18,20,22,30,33–35 Use of inactivated CDV vaccines in nondomestic carnivores, including AWDs, has resulted in poor to no detectable seroconversion.20,34 These problems are common to a variety of nondomestic carnivores, as well as domestic ferrets, and have resulted in the development of new recombinant vaccines.11,19,31 Both experimental and commercially available recombinant canine distemper vaccinations have been used safely in domestic dogs and other carnivore species, including AWDs.5,23,37,38 Some of those have been immunogenic when administered orally in other species.36,38 In particular, the commercially available recombinant canary-pox–vectored CDV vaccine (PurevaxTM Ferret, Merial Limited, Duluth, Georgia 30096, USA) appears to be both safe and potentially effective in a variety of threatened
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Table 1. Serum antibody titers against canine distemper virus (CDV) in African wild dogs (AWDs): oral vaccination.a Litter
Age at first vaccine (mo)
A A A A A B B B Mean Range % 32d
2 2 2 2 2 2 2 2
Neg 1 mo
First vacc
Second vacc
Third vacc
3.5 mo
,8 ,8 ,8 ,8 ,8 ,8 ,8 ,16 0 0–16 0
,8 ,16 ,8 ,16 ,8 ,16 ,8 ,16 0 0–16 0
,8 ,8 ,8 ,8 ,8
12
29 25 45 54 25 ,8b ,8 ,8 22.3 0–54 25.0
12 –c 0
0 0 0
5 mo
6.5 mo
,8 ,8 ,8 ,8 ,8 ,8 ,8 ,16 0 0–16 0
0 0 0
a Serum antibody titers against CDV for individual AWDs at each time point. Samples collected prior to vaccination are indicated at Neg 1 month. Samples collected at the time of each vaccination are indicated at # vacc. b Values reported as ,8 or ,16 were set as 0 for the purposes of the calculations as both indicate that no antibody was detected. c Not calculated. d Percent of pups with titers greater than or equal to 32 at each sampling point.
species.3,13 This vaccine has the potential to be effective via both the parenteral and oral routes, and has been used experimentally in AWDs without clinical side effects.23,36 The goal of this study was to evaluate novel protocols for vaccinating AWDs against CDV in a zoo setting by investigating humoral responses to the canary-pox–vectored vaccine administered either orally or parenterally.
MATERIALS AND METHODS Vaccination The study population was comprised of 21 previously unvaccinated AWD pups born at the Wildlife Conservation Society’s Bronx Zoo in three litters between 2005 and 2007. The parents of the litters had previously been vaccinated for CDV, although the vaccines used and frequency of vaccination varied. All pups were housed with the pack under similar conditions for the duration of the study. All study animals received a series of three vaccinations with a recombinant canary-pox–vectored canine distemper vaccine (PurevaxTM Ferret) administered either orally (PO) or intramuscularly (IM). An initial group of 15 animals (in two litters from different dams) were assigned to either a PO (n ¼ 8) or IM (n ¼ 7) vaccination group with the use of a random numbers table. Subsequently, a third litter of six pups all received IM vaccinations, for a total of 13 IM vaccinated animals. The initial vaccines were administered when the pups were 2 mo of age. Subsequent booster vaccines
were administered at 0.5–1.5-mo intervals (Tables 1 and 2). Initial vaccinations were performed with the animals under manual restraint; older animals were vaccinated while temporarily restrained in a chute. For PO vaccination, the animal was encouraged to bite a padded wooden block while a 6-in. metal ball-ended gavage tube and 5 ml syringe were used to spray 1 ml of vaccine onto the gingival mucosa. For IM vaccination, 1 ml of vaccine was administered into the gluteal muscles. Blood collection and analysis Blood was collected at the time of initial vaccination, during subsequent booster vaccinations, and intermittently thereafter (Table 1). Blood collection from PO-vaccinated animals ceased after 6.5 mo because of the lack of immune response noted up to that point. Collection intervals were partially determined by management needs. The number of animals sampled declined throughout the study period because of animals being sent to other zoos (n ¼ 3 PO, n ¼ 7 IM), death from unrelated causes (n ¼ 1 IM) and management concerns about pack hierarchy disruption during handling (n ¼ 2 IM). Blood was collected from the lateral saphenous vein. Blood samples were immediately placed in a standard serum separator tube. Serum was separated via centrifugation within 30 min of collection. Serum was then removed and stored at 808C until shipment to the laboratory. Titers were measured by serum neutralization at the Cornell University Animal Health Diagnostic Center (AHDC, Ithaca, New York, USA)
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Table 2. Serum antibody titers against canine distemper virus (CDV) in African wild dogs (AWDs): intramuscular vaccination.a
Litter
Age at first vaccine (mo)
A A A A B B B C C C C C C Mean Range % 32c
2 2 2 2 2 2 2 2 2 2 2 2 2
Neg 1 mo
,8b 24 16
13.3 0–24 0
First vacc
Second vacc
Third vacc
19 21 13 25 ,8 ,8 ,8 ,8 ,8 ,16 ,4 ,4 ,8 6.0 0–25 0
,8 192 24 24 96 48 16 128 128 64 48 ,16 48 62.8 0–192 61.5
,16 768 96 192 512 768 384 512 768 512 512 1,536 768 563.7 0–1,536 92.3
2.5 mo
3.5 mo
5 mo
768 512 192 128
6.5 mo
21.5 mo
128 96 ,16 24 96 96 128
192 192 96 384 384 384 272.0 96–384 100.0
,16 ,16 ,16
400.0 128–768 100.0
106.7 96–128 100.0
62.0 0–128 50.0
0 0 0
a Serum antibody titers against CDV for individual AWDs at each time point up to 21.5 mo. Samples collected prior to vaccination are indicated at Neg 1 month. Samples collected at the time of each vaccination are indicated at # vacc. b Values ,4, ,8, or ,16 were set as zero for the purposes of the calculations, as all indicate that no antibody was detected. c Percent of pups with titers greater than or equal to 32 at each sampling point.
and are reported as the inverse of the dilution. A value of 8 was considered to represent CDVspecific antibodies, whereas a value of 32 was interpreted as sufficient for protection based upon both recommendations made for domestic dogs at the Cornell University AHDC and values used in previous studies.2,17,21 Data analysis Separate analyses of antibody titers before and after completion of vaccine courses were conducted. Differences in log-transformed titers were analyzed with the use of generalized linear mixed models (GLMM), including random effects to account for nonindependence of data collected from the same animal or litter. GLMMs were fitted with the use of the lme procedure (in the statistical software package R).24,27 Individual identity was included as a random effect. For these analyses, undetectable titers (,8) were assigned zero values. To avoid problems associated with log-transforming zero values, 4 (equivalent to half the lowest nonzero value) was added to all values before taking the (natural) logs. To maximize consistency of the data, statistical comparisons were restricted to the two litters that received both treatments. However, data from the third litter, which received only intramuscular vaccination, are included in descriptive analyses.
RESULTS No clinical abnormalities were observed in any of the AWD pups, whether vaccinated PO or IM. Some pups showed evidence of maternal antibodies prior to vaccination. Of four pups sampled a month before vaccination (aged 1 mo), three had detectable serum antibody titers (8) although none were 32. None of these animals had detectable antibodies at the time of vaccination. However, nine pups from a different litter did have detectable serum antibody titers at the time of initial vaccination, and two of these had titers 32 (Tables 1 and 2). As the course of three CDV vaccinations was administered, the two treatment groups showed different temporal patterns in their titers. Pups given IM vaccination showed rising titers, whereas those given PO vaccination remained seronegative (Fig. 1). The interaction between treatment (PO vs. IM) and time since the start of vaccination was statistically significant at P , 0.001. This difference in temporal patterns continued after the vaccinations were completed. Animals vaccinated PO continued to be seronegative, whereas IM-vaccinated animals showed declining titers. The interaction between treatment (PO vs. IM) and time since the end of vaccination was statistically significant at P , 0.005. Of the 13 pups vaccinated by the IM route, titers 32 were recorded after a single dose of
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Figure 1. Changes in canine distemper virus (CDV) serum antibody titers in African wilds dogs (AWDs) given three successive doses of recombinant CDV vaccine, either intramuscularly (closed symbols) or orally (open symbols). Data are shown from the periods during (a) and after (b) administration of the vaccine courses. Circles, triangles, and diamonds indicate mean (and SE) values from litters A, B, and C, respectively; litter C received only intramuscular vaccination. Dashed lines and dots indicate titers at which domestic dogs are presumed to be protected (titer 32, log-transformed as 3.58), dashed lines indicate titers below which antibodies are no longer detectable (titer ,8, log-transformed as 2.48).
vaccine in 8 pups (62%), after two doses in 12 pups (92%), and after three doses in all 13 pups. All blood samples collected within 2 mo of completing the vaccine course revealed titers 32 (Fig. 1), but by 4 mo postvaccination only two of four animals sampled still had titers 32. None of three animals resampled 20 mo postvaccination had detectable antibodies (titers all ,8).
DISCUSSION Findings of this study suggest that the commercially available canary-pox–vectored recombinant CDV vaccine was safe, and potentially effective, for use in AWDs if delivered by the IM route. Vaccination by the PO route was safe but highly unlikely to be effective in providing protection against CDV. For the purposes of this study, a serum antibody titer of 32 was used to indicate potential protection against CDV. However, an assessment of actual protection against infection was not performed. The endangered status of AWDs renders a challenge study inadvisable. None of the pups vaccinated by the PO route displayed a clinically relevant humoral response, even though some had evidence of maternal antibodies before vaccination. Because of the degree of variation in parental vaccination history, as well as the lack of CDV titers obtained from the dams, no substantial conclusions can be drawn from this study in regards to maternal vaccination strategies and resulting maternal antibodies in the pups. The potential remains for maternal antibodies to interfere with initial vac-
cination. However, of those pups with detectable CDV titers at the time of initial vaccination, those that received IM vaccinations did display a rise in titers following booster injections. Those pups that received the PO vaccine did not display a corresponding increase in titers. The lack of a clinically relevant humoral response following PO administration contrasts with results obtained when administering the same vaccine orally to another canid species, the Channel Island fox (Urocyon littoralis), in which measurable antibody responses were present in over 80% of the study population.36 In addition, mucosal vaccination with other experimental recombinant canine distemper vaccines has been shown to be effective in both the Siberian polecat (Mustela eversmanni) and the domestic ferret (Mustela putorius).37,38 However, the Channel Island fox study population received a double dose of the vaccine (two 1-ml vials PO) whereas this study utilized the standard 1-ml dose vial previously demonstrated to be safe for use in AWDs.23 Future research might examine the possibility for increased efficacy of higher PO vaccine doses in AWDs. The reason for the poor titer response following PO vaccination in AWDs compared to other nondomestic carnivores remains otherwise unclear. In contrast with the negative titers obtained following PO vaccination, all pups receiving the series of three IM injections displayed a strong humoral immune response. However, the complete series of three injections was required to stimulate high titers in 100% of the pups (Table
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1). The humoral responses that were observed suggest that a minimum of two injections would be required to provide protection to the majority of pups. This requirement might be difficult or impractical to meet in a free-ranging situation. Moreover, even following three IM injections, titers began to fall rapidly. Comparable results were obtained in a previous study on the use of PurevaxTM Ferret in AWDs, in which a substantial portion of the animals no longer had protective titers 1 yr post IM vaccination series.23 This pattern differs from that observed in domestic dogs following vaccination with modified live products, in which studies have shown protective titers achieved following a single vaccination.2,29 In addition, in domestic dogs, titers to CDV have been shown to persist from 36 mo to as long as 5 yr following vaccination with a canary-pox–vectored CDV vaccine, and 9 yr following vaccination with modified live CDV vaccines, with minimal decrease in serum antibody titer noted during that time.15,29 Domestic dogs challenged with CDV 9 yr following vaccination resisted infection.29 In domestic dogs vaccinated against CDV, it has been suggested that the actual titer of antibodies is of lesser importance than simply the presence of detectable antibodies.29 Despite a low titer, domestic dogs will develop a rapid anamnestic humoral and cell-mediated immune response, protecting against infection and disease. Potentially, vaccinated AWDs might mount a protective immune response when exposed to CDV despite low titers. Unfortunately, because of the inadvisability of challenge studies in AWDs, the efficacy of the combined humoral and cell-mediated immune response following vaccination is difficult to assess. Future studies might examine the titer response following an additional booster vaccination administered after the initial series. Based on the findings in this study, PO vaccination with the PurevaxTM Ferret recombinant distemper vaccine appears not to be efficacious in AWD. Further studies might investigate the usefulness of alternate vaccines or doses. A series of three IM vaccinations resulted in a strong humoral immune response in all animals receiving the vaccine, although practical use in freeranging situations remains challenging. Acknowledgments: This study was funded in part by a One WCS grant. Thanks and appreciation are extended to all of the Bronx Zoo mammal department and Zoological Health staff that participated in this study.
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