Serosurvey of ex situ giant pandas (Ailuropoda melanoleuca) and red pandas (Ailurus fulgens) in China with implications for species conservation.

Conservation strategies for the giant panda (Ailuropoda melanoleuca) include the development of a self-sustaining ex situ population. This study examined the potential significance of infectious pathogens in giant pandas ex situ. Serologic antibody titers against canine distemper virus (CDV), canine parvovirus (CPV), canine adenovirus (CAV), canine coronavirus (CCV), canine herpesvirus, canine parainfluenza virus (CPIV), Toxoplasma gondii, Neospora caninum, and Leptospira interrogans were measured in 44 samples taken from 19 giant pandas between 1998 and 2003 at the Chengdu Research Base of Giant Panda Breeding in Sichuan, China. Seroassays also included samples obtained in 2003 from eight red pandas (Ailurus fulgens) housed at the same institution. All individuals had been vaccinated with a Chinese canine vaccine that included modified live CDV, CPV, CAV, CCV, and CPIV. Positive antibody titers were found only against CDV, CPV, and T. gondii. Sera were negative for antibodies against the other six pathogens. Results indicate that the quality of the vaccine may not be reliable and that it should not be considered protective or safe in giant pandas and red pandas. Positive antibody titers against T. gondii were found in seven of the 19 giant pandas. The clinical, subclinical, or epidemiologic significance of infection with these pathogens via natural exposure or from modified live vaccines in giant pandas is unknown. Research in this area is imperative to sustaining a viable population of giant pandas and other endangered species.     

圈养大熊猫麻疹病毒易感性流行病学调查

成都大熊猫繁育研究基地对圈养的7只8～21岁的大熊猫血清犬瘟热病毒(CDV)中和抗体滴度进行了测定.结果显示,尽管这些大熊猫自2003年起每年接受2次CDV弱毒疫苗接种,该弱毒疫苗是由未知家犬犬瘟热病毒株制作的,大熊猫血清犬瘟热病毒中和抗体滴度却在2×到256×的较大范围内(平价值=16)波动.通常单次的麻疹病毒弱毒疫苗注射足以导致宿主产生相应的稳定的免疫反应,在大熊猫上抗犬瘟热中和抗体变化幅度较大提示在宿主与疫苗的关系中存在某种程度的不足.     

Adjuvant effects of recombinant giant panda (Ailuropoda melanoleuca) IL-18 on the canine distemper disease vaccine in mice

Canine distemper virus (CDV) is a morbillivirus known to cause morbidity and mortality in a broad range of animals. Giant pandas (Ailuropoda melanoleuca), especially captive ones, are susceptible to natural infection with CDV. Interleukin-18 (IL-18) is a powerful adjuvant molecule that can enhance the development of antigen-specific immunity and vaccine efficacy. In this study, a giant panda IL-18 gene eukaryotic expression plasmid (pcAmIL-18) was constructed. Female BALB/c mice were muscularly inoculated with the plasmids pcAmIL-18, pcDNA3.1 and PBS, respectively. They were subsequently injected with an attenuated CDV vaccine for dogs, and the induced humoral and cellular responses were evaluated. The results showed that pcAmIL-18 remarkably improved the level of specific antibody, IFN-γ and IL-2 in mice sera, the T lymphocyte proliferation index and the percentage of CD4⁺ and CD8⁺ cells. These data indicated that pcAmIL-18 is a potential adjuvant that promotes specific immunity.     

大熊猫犬瘟热病毒LG株的分离鉴定

为获得大熊猫犬瘟热病毒株,采集死亡大熊猫的心脏、肺、肝病料,研磨并反复冻融后收集上清液接种Vero细胞,待出现细胞病变(CPE)后,收集病毒液。用逆转录-聚合酶链反应(RT-PCR)鉴定病毒分离株,测定病毒TCID_(50),动物回归试验测定其毒力。结果显示,盲传到第5代时,Vero细胞出现圆缩、聚集、脱落等病变;PCR扩增出287bp片段,与预期相符;测序结果显示,该毒株与已发表的CDV SD(14)11毒株(亚洲-Ⅰ型)的同源性为98%;毒株的TCID_(50)为10~(-5.2)/mL;幼犬感染分离毒株后出现体温升高、鼻头发干、拉稀、眼鼻分泌出水样分泌物等症状。本研究成功分离出大熊猫源犬瘟热病毒株。     

Serum antibody response to canine parvovirus, canine adenovirus-1, and canine distemper virus in dogs with known status of immunization: study of dogs in Sweden

Serum antibody titers to canine parvovirus (CPV), canine adenovirus-1 (CAV-1), and canine distemper virus (CDV) were measured in dogs with known immunization status. The dogs represented 3 groups: nonvaccinated dogs less than 12 months old; vaccinated dogs less than 12 months old; and adult dogs greater than 12 months old. For practical reasons, the population from which the specimens were obtained could be considered as free from natural infection with CAV-1 and CDV. In nonvaccinated dogs less than 12 months old, antibodies against all 3 viruses were measured at the time the dogs were given their first vaccination. Altogether, 50.7% of the dogs had titer greater than or equal to 1:10 to CPV, and 26.1 and 46.2% had titer greater than or equal to 1:8 to CAV-1 and CDV, respectively. The concentration of maternal antibody seemed to be of major importance for failure of immunization with use of inactivated CPV vaccine, but not with CAV-1 and CDV vaccination. In dogs less than 12 months old and vaccinated against CPV infection with inactivated virus, only 11.5% had titer greater than or equal to 1:80. In dogs vaccinated against infectious canine hepatitis and canine distemper, 63.2 and 78.3%, respectively, had titer greater than or equal to 1:16. In adult dogs greater than 2 months old and vaccinated against CPV infection, less than 50% had titer greater than or equal to 1:80, regardless of time after vaccination. There was no significant difference in titer between vaccinated and nonvaccinated dogs.(ABSTRACT TRUNCATED AT 250 WORDS)     

我国犬瘟热病毒的生态学调查研究

本研究应用电子显微镜技术检查了１７个毛皮动物和野生动物的６７６份材料,从犬,貂,貉,狐熊,小熊猫,大熊猫,狼,狮,虎,猞狮、金猫等１２种动物病料中,检出含有ＣＤＶ材料４８７份。应用间接ＥＬＩＳＡ、免疫荧光和中和试验等技术检测了８种动物１５８份血清,其中从犬,狐,小熊猫、虎、金猫,狼等６种动物的１０６份血清中检出了抗ＣＤＶ抗体。应用ＲＴ－ＰＣＲ和基因探针检查了４种动物的３７份材料,其中有２９份阳性。     

Three-year duration of immunity in dogs following vaccination against canine adenovirus type-1, canine parvovirus, and canine distemper virus

A challenge-of-immunity study was conducted to demonstrate immunity in dogs 3 years after their second vaccination with a new multivalent, modified-live vaccine containing canine adenovirus type 2 (CAV-2), canine parvovirus (CPV), and canine distemper virus (CDV). Twenty-three seronegative pups were vaccinated at 7 and 11 weeks of age. Eighteen seronegative pups, randomized into groups of six dogs, served as challenge controls. Dogs were kept in strict isolation for 3 years following the vaccination and then challenged sequentially with virulent canine adenovirus type 1 (CAV-1), CPV, and CDV. For each viral challenge, a separate group of six control dogs was also challenged. Clinical signs of CAV-1, CPV, and CDV infections were prevented in 100% of vaccinated dogs, demonstrating that the multivalent, modified-live test vaccine provided protection against virulent CAV-1, CPV, and CDV challenge in dogs 7 weeks of age or older for a minimum of 3 years following second vaccination.     

犬瘟热的诊断及其预防免疫的研究进展

本文对犬瘟热（CD）的诊断、预防免疫和免疫失败的影响因素及犬瘟热病毒（CDV）的宿主范围进行了综述。CDV不仅感染陆生食肉动物，而且也感染水生食肉动物，并且其宿主范围还在不断扩大。CDV感染主要采用病毒分离、特异性病毒抗原或特异性核酸检测等方法确诊。疫苗包括灭活的CDV疫苗、麻疹病毒（MV）异源苗及CDV弱毒活苗。疫苗接种犬的免疫反应主要取决于毒株特性及犬的应答能力，只有弱毒活苗能诱导产生持久而坚强的保护力。尽管多年来CDV弱毒活苗的使用控制了CD的发生，但最近免疫过的犬发生CD的病例并不少见。分析免疫失败的原因，主要是母源抗体干扰、疫苗质量差、其它病毒的免疫抑制以及CDV流行株可能发生了变异等因素的影响。     

Serologic response of domestic ferrets (Mustela putorius furo) to canine distemper and rabies virus vaccines

Nine unrelated 12-week-old naive domestic ferrets (Mustela putorius furo) were used to evaluate the serologic responses to commercial canine distemper virus (CDV) and rabies virus (RV) vaccines. Five of the ferrets (group 1) were inoculated 3 times at 2-week intervals with a multivalent modified-live virus vaccine of canine cell-line origin, containing CDV and an inactivated RV vaccine. Four of the ferrets (group 2) were inoculated once with the multivalent modified-live virus vaccine containing CDV and were not inoculated with the RV vaccine. Both group-1 and group-2 ferrets seroconverted to the CDV component of the vaccine. Group-1 ferrets also seroconverted after RV vaccination and maintained serum antibody titers to both CDV and RV for at least 7 months. Domestic ferret sera were found to have IgG epitopes similar to sera of domestic dogs and cats. Domestic ferret sera did not contain antibodies to feline coronavirus or FeLV antigens.     

Effect of vaccination with recombinant canine distemper virus vaccine immediately before exposure under shelter-like conditions

Vaccination with modified-live virus (MLV) canine distemper virus (CDV) vaccine has historically been recommended for animals in high-risk environments because of the rapid onset of immunity following vaccination. Recombinant CDV (rCDV) vaccine was deemed a suitable alternative to MLV-CDV vaccination in pet dogs, but insufficient data precluded its use where CDV was a serious threat to puppies, such as in shelters, kennels, and pet stores. In this study, dogs experimentally challenged hours after a single dose of rCDV or MLV vaccine became sick but recovered, whereas unvaccinated dogs became sick and died. Dogs vaccinated with a single dose of rCDV or MLV vaccine 1 week before being experimentally challenged remained healthy and showed no clinical signs. Dogs given one dose of rCDV vaccine hours before being placed in a CDV-contaminated environment did not become sick. These findings support the hypothesis that rCDV vaccine has a similar time-to-immunity as MLV-CDV vaccines and can likewise protect dogs in high-risk environments after one dose.     

Evaluation of a dot ELISA kit for measuring immunoglobulin M antibodies to canine parvovirus and distemper virus

A dot ELISA for the detection of immunoglobulin M (IgM) antibodies to canine distemper virus (CDC) and canine parvovirus (CPV) was assessed. The titres of IgM antibodies to CDV and CPV in 100 dogs were measured by the Immunocomb ELISA kit and compared with the results derived from the immunofluorescence assay (IFA). There was a strong correlation between the results of the dot ELISA technique and the IFA (P < 0.001). The dot ELISA kit was also used to assess the changes in the levels of immunoglobulin G (IgG) and IgM antibodies to CPV and CDV in 10 puppies vaccinated with a polyvalent vaccine. High levels of IgM antibodies to CPV were first detected seven days after they were vaccinated, and after nine days all the pups had high titres of IgG antibodies to CPV. High levels of IgM antibodies to CDV were detected after nine days and the highest average titres were recorded after 12 days. IgG antibodies to CDV were present from nine days after vaccination.     

Serosurvey of infectious disease agents of carnivores in captive red pandas (Ailurus fulgens) in China.

The future of the endangered red panda (Ailurusfulgens) depends in part on the development of protective measures against infectious diseases. The present study is a first step toward improved understanding of infectious diseases in the species' home regions. Serum samples obtained from 73 red pandas in 10 captive facilities in southwest, east, and northeast China from October to December 2004 were tested for antibodies against nine common infectious pathogens of carnivores. Antibody titers against canine distemper virus (CDV), canine parvovirus (CPV), and canine adenovirus (CAV) in the three facilities in which red pandas were vaccinated were highly variable. The CAV titer in one vaccinated red panda was high enough to suggest infection with the field virus following vaccination. Together with anecdotal reports of vaccine-associated morbidity and mortality, our results suggest that the Chinese vaccine is not suitable for this species. In the seven unvaccinated groups, CDV titers were low and occurred in 20-100% of the animals; antibody titers against CPV were found in seven of eight areas. Only one of 61 and two of 61 unvaccinated red pandas had CAV and canine coronavirus titers, respectively, and these titers were all low. Positive titers to Toxoplasma gondii were found in four locations (33-94% seropositive); the titers in 52% of seropositive individuals were of a magnitude consistent with active disease in other species (1:1,024 to > or = 1:4,096). One red panda in each of three locations was seropositive for Neospora caninum. Antibodies against canine herpesvirus and Brucella canis were not detected in any of the samples. Only one of the 73 red pandas had a weak positive influenza A titer. The results of this study emphasize the need for research on and protection against infectious diseases of red pandas and other endangered species in China.     

Comparative analyses of canine distemper viral isolates from clinical cases of canine distemper in vaccinated dogs

Sequence and phylogenetic analyses of three isolates of canine distemper virus (CDV) isolated from three dogs with a vaccination history were compared with the same analyses of vaccine virus isolated from a vaccine used for dogs. The three dogs showed clinical signs of a recent major type of CD in Japan, including oculonasal discharge and diarrhea, and pathological findings including non-suppurative encephalitis, pneumonia, mild gastroenteritis and lymphoid depletion. Inclusion bodies were in the stomach without inflammation and encephalitis was without clinical signs. One of the highest titers of CDV in different organs of the three dogs was commonly systemic lymphatic organs, including the spleen, lymph nodes and tonsils. New isolates of CDV joined to the clades of the Asia 1 group that is far from the vaccine group. These results surely indicate that wild strains of CDV from dogs with a vaccination history were not reversed vaccine virus, and that the dogs showed characteristics of recent CD in Japan.     

小熊猫源犬瘟热病毒株H、F基因的克隆及序列分析

为了解1株圈养小熊猫源犬瘟热病毒（CDV）GD-1的遗传变异情况,通过RT-PCR方法对该株CDV进行H、F基因的克隆、测序及序列分析。结果显示：该分离株的H基因序列与GenBank中丹麦报道的登录号为GU266280的犬源CDV毒株的核苷酸序列相似性最高,为96%;F基因序列与巴西报道的登录号为KY057355的犬源CDV的核苷酸序列相似性最高,为95.7%。下载CDV代表毒株序列进行遗传演化、氨基酸序列比对及分子特征分析。结果显示：H蛋白共有8个潜在的N-糖基化位点,分别位于19、149、309、391、422、456、587、603位点;H蛋白的SLAM受体结合位点氨基酸序列与欧亚野生型毒株一致,与疫苗株相比,530、549位氨基酸不同,与其他CDV参考毒株H蛋白相比还存在24、41等9处氨基酸位点发生明显变异,与标准强毒株A75/17的氨基酸相似性为95.2%,与Onderstepoort、Convac等5株疫苗株的氨基酸序列相似性为88.2%~89.3%;F蛋白共有6个N-糖基化位点,分别位于62、108、141、173、179、517位,与Onderstepoort等疫苗株氨基酸相似性为89.1%~89.7%;与其他参考毒株相比还存在115、130等11处氨基酸发生变异;构建基于H、F基因的遗传进化树,结果显示：该毒株位于Asia-4型的一个小的进化分支,这与目前我国流行毒株主要位于Asia-1型存在明显不同。本研究首次报道了小熊猫源的Asia-4基因型CDV野毒株,并对毒株的H及F基因进行了序列分析,对于了解我国CDV流行株的遗传变异情况、流行病学调查、疾病防控及疫苗研发等具有重要意义。     

Three-year rabies duration of immunity in dogs following vaccination with a core combination vaccine against canine distemper virus, canine adenovirus type-1, canine parvovirus, and rabies virus

Thirty-two seronegative pups were vaccinated at 8 weeks of age with modified-live canine distemper virus (CDV), canine adenovirus type-2 (CAV-2), and canine parvovirus (CPV) vaccine and at 12 weeks with a modified-live CDV, CAV-2, CPV, and killed rabies virus vaccine. An additional 31 seronegative pups served as age-matched, nonvaccinated controls. All test dogs were strictly isolated for 3 years after receiving the second vaccination and then were challenged with virulent rabies virus. Clinical signs of rabies were prevented in 28 (88%) of the 32 vaccinated dogs. In contrast, 97% (30 of 31) of the control dogs died of rabies infection. These study results indicated that no immunogenic interference occurred between the modified-live vaccine components and the killed rabies virus component. Furthermore, these results indicated that the rabies component in the test vaccine provided protection against virulent rabies challenge in dogs 12 weeks of age or older for a minimum of 3 years following vaccination.     

Duration of serologic response to five viral antigens in dogs

OBJECTIVE: To determine whether vaccinated dogs either remained seropositive or responded serologically to revaccination for 5 key viral antigens after extended periods since their last vaccination. DESIGN: Serologic survey. ANIMALS: 322 healthy client-owned dogs. PROCEDURE: Dogs were > or = 2 years old and vaccinated against canine distemper virus (CDV), canine adenovirus-1 (CAV-1), canine adenovirus-2 (CAV-2), canine parainfluenza virus (CPIV), and canine parvovirus (CPV). On day 0, dogs were revaccinated with a vaccine from the same vaccine line as they had historically received. Antibody titers were measured in sera collected at day 0 (prevaccination titer) and 5 to 7 days later (postvaccination titer). Dogs were considered to have responded serologically if they had a day-0 serum neutralization titer to CDV > or = 1:32; a serum neutralization titer to CAV-1, CAV-2, or CPIV > or = 1:16; a hemagglutination inhibition titer to CPV > or = 1:80; or a > or = 4-fold increase in antibody titer after revaccination. RESULTS: The percentage of dogs that had titers at or greater than the threshold values or responded to revaccination with a > or = 4-fold increase in titer was 98.1% for CDV, 98.4% for CAV-1, 99.0% for CAV-2, 100% for CPIV, and 98.1% for CPV. CONCLUSIONS AND CLINICAL RELEVANCE: In most dogs, vaccination induced a response that lasted up to and beyond 48 months for all 5 antigens. Although not equivalent to challenge-of-immunity studies as a demonstration of efficacy, results suggest that revaccination with the same vaccine provides adequate protection even when given less frequently than the traditional 1-year interval. The study provides valuable information for clinicians to help determine appropriate revaccination intervals.     

Evaluation of oral and subcutaneous delivery of an experimental canarypox recombinant canine distemper vaccine in the Siberian polecat (Mustela eversmanni).

We assessed the safety and efficacy of an experimental canarypox-vectored recombinant canine distemper virus (CDV) subunit vaccine in the Siberian polecat (Mustela eversmanni), a close relative of the black-footed ferret, (M. nigripes), an endangered species that is highly susceptible to the virus. Siberian polecats were randomized into six treatment groups. Recombinant canine distemper vaccine was administered s.c. at three dose levels (10(4.5), 10(5.0), and 10(5.5) plaque-forming units [PFU] per dose) and was administered orally by spraying the vaccine into the oropharnyx at two dose levels (10(5.5), 10(8.0) PFU per dose). The sixth group of control animals was not vaccinated. For both routes of administration, two 1-ml doses of reconstituted vaccine were delivered 4 wk apart, followed by live virus challenge 3 wk after the second vaccination. During the challenge, Synder Hill test strain CDV obtained from the National Veterinary Services Laboratory in Ames, Iowa, was administered i.p. Serial blood samples for CDV serology were collected immediately before vaccination and challenge, and 10, 15, and 20 days after challenge. Clinical signs and body weights were recorded up to 32 days after challenge. The survival rate in animals receiving vaccine at the highest oral dose (10(8.0) PFU per dose) was 83.3%. Survival rate was 50.0% in the high s.c. and 60.0% in the medium s.c. groups. All animals in the low-s.c. dose, low-oral dose, and control groups died after exposure. Vaccine dose overall (oral and s.c.) and dose in response to s.c. administration when considered alone were significant predictors of survival (P = 0.006 and P = 0.04, respectively). Among the polecats challenged with virulent virus, those that died became sick sooner than those that survived. Animals that died lost significantly more weight during the 10 days after challenge than did animals that survived (P = 0.02). Survival rates did not differ by sex, founder female status, or breeding pedigree in any of the treatment groups. Survival rates were higher in animals with increasing serum neutralization titers (P = 0.027). This study demonstrates the efficacy of oral delivery of a recombinant CDV vaccine in the Siberian polecat. Further studies are needed to evaluate the safety and efficacy of vectored recombinant vaccines in highly susceptible species and especially in those species in which vaccination with modified live CDV has led to disease.     

A comparison of the immune responses of dogs exposed to canine distemper virus (CDV) - Differences between vaccinated and wild-type virus exposed dogs

Canine distemper virus (CDV)-specific immune response was measured in different dog populations. Three groups of vaccinated or wild-type virus exposed dogs were tested: dogs with a known vaccination history, dogs without a known vaccination history (shelter dogs), and dogs with potential exposure to wild-type CDV. The use of a T-cell proliferation assay demonstrated a detectable CDV-specific T-cell response from both spleen and blood lymphocytes of dogs. Qualitatively, antibody assays [enzyme-linked immunosorbent assay (ELISA) and neutralization assay] predicted the presence of a T-cell response well, although quantitatively neither antibody assays nor the T-cell assay correlated well with each other. An interesting finding from our study was that half of the dogs in shelters were not vaccinated (potentially posing a public veterinary health problem) and that antibody levels in dogs living in an environment with endemic CDV were lower than in vaccinated animals.     

Prevalence of positive antibody test results for canine parvovirus (CPV) and canine distemper virus (CDV) and response to modified live vaccination against CPV and CDV in dogs entering animal shelters

Canine parvovirus (CPV) and canine distemper virus (CDV) infections are relatively common in animal shelters and are important population management issues since the immune status of incoming dogs is usually unknown. This study aimed to determine the prevalence of positive antibody test results for CPV and CDV in incoming dogs aged ≥ 4 months and to measure antibody response over 2 weeks following vaccination with a modified live vaccine (MLV). Dogs aged 4-24 months entering an adoption-guarantee shelter (Shelter 1, n=51) and aged ≥ 4 months entering a limited admission shelter (Shelter 2; n=51) were enrolled. Dogs from Shelter 1 had been vaccinated with MLV at a municipal shelter 5 days before enrollment, whereas dogs from Shelter 2 had no known history of vaccination at enrollment. Sera were obtained on day 1, immediately prior to CPV/CDV MLV, and tested using an in-clinic ELISA kit to detect CPV/CDV antibodies. Dogs negative for CPV and/or CDV were retested at day 6-8 and those dogs still negative at day 6-8 were retested at day 13-15. Prior to CPV/CDV MLV on day 1, more dogs tested positive for CPV (Shelter 1 - 68.6%; Shelter 2 - 84.3%) than for CDV (Shelter 1 - 37.3%; Shelter 2 - 41.2%). On day 1, prior to MLV, all spayed/neutered animals tested CPV antibody-positive (n=17/102) and CPV antibody-positive dogs were older than serologically negative dogs (Shelter 1, P=0.0029; Shelter 2, P=0.0042). By day 13-15, almost all dogs were CPV antibody-positive (Shelter 1 - 97.9%; Shelter 2 - 100.0%) and CDV antibody-positive (Shelter 1 - 93.8%; Shelter 2 - 97.8%). MLV induces protective antibody titers against CPV/CDV in almost all dogs after 13-15 days.