Development of a case definition for clinical feline herpesvirus infection in cheetahs (Acinonyx jubatus) housed in zoos.

The identification of feline herpesvirus (FHV) infected cheetahs (Acinonyx jubatus) and characterization of shedding episodes is difficult due to nonspecific clinical signs and limitations of diagnostic tests. The goals of this study were to develop a case definition for clinical FHV and describe the distribution of signs. Medical records from six different zoologic institutions were reviewed to identify cheetahs with diagnostic test results confirming FHV. Published literature, expert opinion, and results of a multiple correspondence analysis (MCA) were used to develop a clinical case definition based on 69 episodes in FHV laboratory confirmed (LC) cheetahs. Four groups of signs were identified in the MCA: general ocular signs, serious ocular lesions, respiratory disease, and cutaneous lesions. Ocular disease occurred with respiratory signs alone, with skin lesions alone, and with both respiratory signs and skin lesions. Groups that did not occur together were respiratory signs and skin lesions. The resulting case definition included 1) LC cheetahs; and 2) clinically compatible (CC) cheetahs that exhibited a minimum of 7 day's duration of the clinical sign groupings identified in the MCA or the presence of corneal ulcers or keratitis that occurred alone or in concert with other ocular signs and skin lesions. Exclusion criteria were applied. Application of the case definition to the study population identified an additional 78 clinical episodes, which represented 58 CC cheetahs. In total, 28.8% (93/322) of the population was identified as LC or CC. The distribution of identified clinical signs was similar across LC and CC cheetahs. Corneal ulcers and/or keratitis, and skin lesions were more frequently reported in severe episodes; in mild episodes, there were significantly more cheetahs with ocular-only or respiratory-only disease. Our results provide a better understanding of the clinical presentation of FHV, while presenting a standardized case definition that can both contribute to earlier diagnoses and be used for population-level studies.

Herpesvirus surveillance and discovery in zoo-housed ruminants.

Gammaherpesvirus infections are ubiquitous in captive and free-ranging ruminants and are associated with a variety of clinical diseases ranging from subclinical or mild inflammatory syndromes to fatal diseases such as malignant catarrhal fever. Gammaherpesvirus infections have been fully characterized in only a few ruminant species, and the overall diversity, host range, and biologic effects of most are not known. This study investigated the presence and host distribution of gammaherpesviruses in ruminant species at two facilities, the San Diego Zoo and San Diego Zoo Safari Park. We tested antemortem (blood, nasal or oropharyngeal swabs) or postmortem (internal organs) samples from 715 healthy or diseased ruminants representing 96 species and subspecies, using a consensus-based herpesvirus PCR for a segment of the DNA polymerase (DPOL) gene. Among the 715 animals tested, 161 (22.5%) were PCR and sequencing positive for herpesvirus, while only 11 (6.83%) of the PCR positive animals showed clinical signs of malignant catarrhal fever. Forty-four DPOL genotypes were identified of which only 10 have been reported in GenBank. The data describe viral diversity within species and individuals, identify host ranges of potential new viruses, and address the proclivity and consequences of interspecies transmission during management practices in zoological parks. The discovery of new viruses with wide host ranges and presence of co-infection within individual animals also suggest that the evolutionary processes influencing Gammaherpesvirus diversity are more complex than previously recognized.

ASSESSMENT OF DISEASE RISK ASSOCIATED WITH POTENTIAL REMOVAL OF ANTHROPOGENIC BARRIERS TO MOJAVE DESERT TORTOISE (GOPHERUS AGASSIZII) POPULATION CONNECTIVITY.

The Mojave Desert tortoise (Gopherus agassizii), federally listed as threatened, has suffered habitat loss and fragmentation due to human activities. Upper respiratory tract disease (URTD), a documented health threat to desert tortoises, has been detected at the Large-Scale Translocation Study Site (LSTS) in southwestern Nevada, US, a fenced recipient site for translocated animals. Our study aimed to 1) estimate prevalence of URTD and Mycoplasma infection at LSTS and three nearby unfenced sites; 2) assess whether Mycoplasma infection status was associated with developing clinical signs of URTD; and 3) determine whether such an association differed between LSTS and unfenced areas. We sampled 421 tortoises in 2016 to describe the current status of these populations. We evaluated three clinical signs of URTD (nasal discharge, ocular discharge, nasal erosions) and determined individual infection status for Mycoplasma agassizii and Mycoplasma testudineum by quantitative PCR and enzyme-linked immunosorbent assay. In 2016, LSTS had the highest prevalence of M. agassizii (25.0%; 33/132), M. testudineum (3.0%; 4/132), and URTD clinical signs (18.9%; 25/132). Controlling for other factors, clinical sign(s) were positively associated with M. agassizii infection (odds ratio [OR]=7.7, P=0.001), and this effect was similar among study sites (P>0.99). There was no association with M. testudineum status (P=0.360). Of the 196 tortoises in a longitudinal comparison of 2011-14 with 2016, an estimated 3.2% converted from M. agassizii-negative to positive during the study period, and incidence was greater at LSTS (P=0.002). Conversion to positive M. agassizii status was associated with increased incidence of clinical signs in subsequent years (OR=11.1, P=0.018). While M. agassizii and URTD are present outside the LSTS, there is a possibility that incidence of Mycoplasma infection and URTD would increase outside LSTS if these populations were to reconnect. Population-level significance of this risk appears low, and any risk must be evaluated against the potential long-term benefits to population viability through increased connectivity.

Genetic characterization and epidemiology of Helicobacters in non-domestic animals.

BACKGROUND: Novel helicobacter infections and associated disease are being recognized with increasing frequency in animals and people. Yet, the pervasiveness of infection in distantly related animal taxa, genetic diversity of helicobacters, and their transmissability are not known. AIM: To better understand the ecology of helicobacters, we did a PCR survey and epidemiologic analysis of 154 captive or wild vertebrate taxa originating from 6 continents. MATERIALS AND METHODS: One hundred twenty nine helicobacter 16s rRNA gene segments were amplified by PCR and sequenced from ninety-three mammalian, reptilian, avian, or amphibian host species. Prevalence estimates were generated, and univariate logistic regression analyses were used to explore relationships between infection status and the health and characteristics of the 220 individual animals. RESULTS: One hundred and nineteen novel helicobacter DNA sequences were found. No significant relationship between infection and host health was found; however, multi-infection or infections with particular genotypes were associated with mild clinical signs. Phylogenetic and genetic comparisons of helicobacters suggested prolonged co-adaptation and niche-associated divergence as well as periodic inter-species transmission. CONCLUSION: The genus Helicobacter should accordingly be viewed as a collection of hundreds of organisms that have colonized most tetrapod taxa and have the potential to expand into new hosts as contact among animals and between animals and people increases.

Investigation of factors predicting disease among zoo birds exposed to avian mycobacteriosis.

OBJECTIVE: To characterize infection patterns and identify factors associated with avian mycobacteriosis among zoo birds that were housed with infected enclosure mates. DESIGN: Matched case-control study. ANIMALS: 79 birds with avian mycobacteriosis (cases) and 316 nondiseased birds (controls) of similar age and taxonomic group that were present in the bird collection of the Zoological Society of San Diego from 1991 through 2005. PROCEDURES: Inventory and necropsy records from all eligible, exposed birds (n = 2,413) were examined to determine disease incidence and prevalence in the exposed cohort. Cases were matched in a 1:4 ratio to randomly selected controls of similar age and taxonomic grouping. Risk factors for mycobacteriosis (demographic, temporal, enclosure, and exposure characteristics as well as translocation history) were evaluated with univariate and multivariable conditional logistic regression analyses. RESULTS: Disease prevalence and incidence were estimated at 3.5% and 8 cases/1,000 bird-years at risk, respectively. In the multivariable model, cases were more likely to have been imported into the collection, exposed to mycobacteriosis at a young age, exposed to the same bird species, and exposed in small enclosures than were controls. Odds for disease increased with an increasing amount of time spent with other disease-positive birds. CONCLUSIONS AND CLINICAL RELEVANCE: The low incidence of mycobacteriosis and the risk factors identified suggested that mycobacteria may not be easily transmitted through direct contact with infected enclosure mates. Identification of risk factors for avian mycobacteriosis will help guide future management of this disease in zoo bird populations.

Association between Mycobacterium avium subsp. paratuberculosis infection among offspring and their dams in nondomestic ruminant species housed in a zoo.

The objective of the present study was to determine whether offspring of dams infected with Mycobacterium avium subsp. paratuberculosis (Map) have an increased risk for Map infection. Antemortem and postmortem disease surveillance data were used to identify positive and test-negative ruminants born at the Zoological Society of San Diego (ZSSD) between 1991 and 2007 and to estimate cumulative lifetime incidence. A matched case-control study, nested within the population, was conducted and conditional logistic regression analyses were used to quantify the association between infection status of offspring and their dams. Cases (infected ruminants, n = 47) were matched to controls (test-negative ruminants, n = 152) by species, birth date, birth enclosure, and follow-up time to control for confounding factors. The overall cumulative lifetime incidence of infection was estimated at 2.2%, but it decreased over time and varied by species. There was a significant association between infection status of offspring and their dams (odds ratio [OR] = 6.8, P < 0.01), which is consistent with studies in domestic livestock species. The association was stronger for animals whose dam was diagnosed within 2 years of their birth (OR = 9.0, P < 0.01) than for animals whose dam was diagnosed more than 2 years after their birth (OR = 6.0, P < 0.01) compared to animals with test-negative dams. For positive animals born to a positive dam, 85.3% of the Map infections were attributable to having a positive dam. For the entire population of ZSSD ruminants, 28.8% [corrected] of the cases were attributable to having a positive dam. Findings will help guide future management of Map infection in zoo ruminant populations.

Epidemiologic analysis of factors associated with local disappearances of native ranid frogs in Arizona.

We examined factors that may independently or synergistically contribute to amphibian population declines. We used epidemiologic case-control methodology to sample and analyze a large database developed and maintained by the Arizona Game and Fish Department that describes historical and currently known ranid frog localities in Arizona, U.S.A. Sites with historical documentation of target ranid species (n= 324) were evaluated to identify locations where frogs had disappeared during the study period (case sites) and locations where frog populations persisted (control sites). Between 1986 and 2003, 117 (36%) of the 324 sites became case sites, of which 105 were used in the analyses. An equal number of control sites were sampled to control for the effects of time. Risk factors, or predictor variables, were defined from environmental data summarized during site surveys and geographic information system data layers. We evaluated risk factors with univariate and multifactorial logistic-regression analyses to derive odds ratios (OR). Odds for local population disappearance were significantly related to 4 factors in the multifactorial model. Disappearance of frog populations increased with increasing elevation (OR = 2.7 for every 500 m, p < 0.01). Sites where disappearances occurred were 4.3 times more likely to have other nearby sites that also experienced disappearances (OR = 4.3, p < 0.01), whereas the odds of disappearance were 6.7 times less (OR = 0.15, p < 0.01) when there was a source population nearby. Sites with disappearances were 2.6 times more likely to have introduced crayfish than were control sites (OR = 2.6, p= 0.04). The identification of factors associated with frog disappearances increases understanding of declines occurring in natural populations and aids in conservation efforts to reestablish and protect native ranids by identifying and prioritizing implicated threats.

Investigation of characteristics and factors associated with avian mycobacteriosis in zoo birds.

The objective of the current study was to identify factors associated with avian mycobacteriosis in zoo birds. Inventory data, population health records, and necropsy data from eligible birds in the Zoological Society of San Diego's (ZSSD) collection from 1991-2005 (n = 13,976) were used to describe disease incidence, prevalence, and postmortem findings. A matched case-control study was then conducted to identify factors describing demographic, temporal, and enclosure characteristics, along with move and exposure histories. Cases (disease-positive birds; n = 167) were matched in a 17 ratio with controls (disease-negative birds; n = 1169) of similar age and taxonomic grouping. Potential risk factors were evaluated using univariate and multivariable conditional logistic regression. Disease prevalence and incidence were estimated for the study period at 1.2% and 3 cases/(1,000 bird-years at risk), respectively. Lesion characteristics and order prevalence are described. In the multivariable model, case birds were more likely to have been previously housed with a bird with mycobacterial disease involving the intestinal tract (odds ratio [OR] = 5.6, P < 0.01) or involving only nonintestinal sites (OR = 2.0, P < 0.01). Cases were more likely to have been imported into the collection than hatched at the ZSSD (OR = 4.2, P < 0.01). Cases were moved among ZSSD enclosures more than controls (OR = 1.1 for each additional move, P < 0.01). Findings will help guide future management of this disease for zoo bird populations.

Early-life exposures and Johne's disease risk in zoo ruminants.

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic, progressive bacterial enteritis of ruminants that can cause serious losses in both livestock and exotic species. Infection risk in exotic ruminants is associated with maternal infection status, but the effect of other herdmates on risk of infection has not been reported, to our knowledge. We conducted a retrospective cohort study to evaluate the association between MAP infection status and early-life contact with infected herdmates. The study population included 3,234 individuals representing 128 species at San Diego Zoo Global facilities between 1991 and 2010. Animal movement, health, and pathology records were used to trace enclosure-sharing contacts between members of the study population and any MAP-infected animal. Contact-days were counted by age of the reference animal and the number of unique infected individuals contacted. Herdmate infection status was stratified by stage of infection (180 d prior to diagnosis), age, and whether relevant lesions were found at autopsy. Having an infected herdmate was a strong risk factor for infection (OR = 4.4; 95% CI: 1.9-10.3), and each method of defining herdmate infection status showed significant differences in infection risk. The best predictor was number of contact-days within the first week of life, with a 2-fold increase in risk associated with each doubling in the number of contact-days (OR = 2.1; 95% CI: 1.1-4.0). We conclude that early contact with infected animals is an important predictor of MAP infection risk, although the effect size is smaller than that previously described for maternal infection status.

Whole-genome analysis of mycobacteria from birds at the San Diego Zoo.

METHODS: Mycobacteria isolated from more than 100 birds diagnosed with avian mycobacteriosis at the San Diego Zoo and its Safari Park were cultured postmortem and had their whole genomes sequenced. Computational workflows were developed and applied to identify the mycobacterial species in each DNA sample, to find single-nucleotide polymorphisms (SNPs) between samples of the same species, to further differentiate SNPs between as many as three different genotypes within a single sample, and to identify which samples are closely clustered genomically. RESULTS: Nine species of mycobacteria were found in 123 samples from 105 birds. The most common species were Mycobacterium avium and Mycobacterium genavense, which were in 49 and 48 birds, respectively. Most birds contained only a single mycobacterial species, but two birds contained a mixture of two species. The M. avium samples represent diverse strains of M. avium avium and M. avium hominissuis, with many pairs of samples differing by hundreds or thousands of SNPs across their common genome. By contrast, the M. genavense samples are much closer genomically; samples from 46 of 48 birds differ from each other by less than 110 SNPs. Some birds contained two, three, or even four genotypes of the same bacterial species. Such infections were found in 4 of 49 birds (8%) with M. avium and in 11 of 48 birds (23%) with M. genavense. Most were mixed infections, in which the bird was infected by multiple mycobacterial strains, but three infections with two genotypes differing by ≤ 10 SNPs were likely the result of within-host evolution. The samples from 31 birds with M. avium can be grouped into nine clusters within which any sample is ≤ 12 SNPs from at least one other sample in the cluster. Similarly, the samples from 40 birds with M. genavense can be grouped into ten such clusters. Information about these genomic clusters is being used in an ongoing, companion study of mycobacterial transmission to help inform management of bird collections.

Epidemiology of clinical feline herpesvirus infection in zoo-housed cheetahs (Acinonyx jubatus).

OBJECTIVE To determine the incidence of and risk factors for clinical feline herpesvirus (FHV) infection in zoo-housed cheetahs and determine whether dam infection was associated with offspring infection. DESIGN Retrospective cohort study. ANIMALS 144 cheetah cubs born in 6 zoos from 1988 through 2007. PROCEDURES Data were extracted from the health records of cheetahs and their dams to identify incident cases of clinical FHV infection and estimate incidence from birth to 18 months of age. Univariate and multivariable Cox proportional hazards models, controlling for correlations among cheetahs with the same dam, were used to identify risk factors for incident FHV infection. RESULTS Cumulative incidence of FHV infection in cheetah cubs was 35% (50/144). No significant association between dam and offspring infection was identified in any model. Factors identified as significant through multivariable analysis varied by age group. For cheetahs up to 3 months of age, the most important predictor of FHV infection was having a dam that had received a preparturition FHV vaccine regimen that included a modified-live virus vaccine versus a dam that had received no preparturition vaccine. Other risk factors included being from a small litter, being born to a primiparous dam, and male sex. CONCLUSIONS AND CLINICAL RELEVANCE This study provided the first population-level characterization of the incidence of and risk factors for FHV infection in cheetahs, and findings confirmed the importance of this disease. Recognition that clinical FHV infection in the dam was not a significant predictor of disease in cubs and identification of other significant factors have implications for disease management.

Survey of Plasmodium spp. in free-ranging neotropical primates from the Brazilian Amazon region impacted by anthropogenic actions.

This study investigated Plasmodium spp. infection in free-ranging neotropical primates from Brazilian Amazon regions under the impact of major anthropogenic actions. Blood samples from 19 new world primates were collected and analyzed with microscopic and molecular procedures. The prevalence of Plasmodium infection was 21.0% (4/19) and PCR positive samples were identified as P. brasilianum. Considering the social-economic changes that the Amazon is facing, the prevalence of P. brasilianum infection highlights the necessity to closely monitor the movement of both human and non-human primate populations, in order to mitigate pathogen exposure and the introduction of new agents into previously naïve areas.