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.

Isolation of a Bohle-like iridovirus from boreal toads housed within a cosmopolitan aquarium collection.

A captive 'survival assurance' population of 56 endangered boreal toads Anaxyrus boreas boreas, housed within a cosmopolitan collection of amphibians originating from Southeast Asia and other locations, experienced high mortality (91%) in April to July 2010. Histological examination demonstrated lesions consistent with ranaviral disease, including multicentric necrosis of skin, kidney, liver, spleen, and hematopoietic tissue, vasculitis, and myriad basophilic intracytoplasmic inclusion bodies. Initial confirmation of ranavirus infection was made by Taqman real-time PCR analysis of a portion of the major capsid protein (MCP) gene and detection of iridovirus-like particles by transmission electron microscopy. Preliminary DNA sequence analysis of the MCP, DNA polymerase, and neurofilament protein (NFP) genes demonstrated highest identity with Bohle iridovirus (BIV). A virus, tentatively designated zoo ranavirus (ZRV), was subsequently isolated, and viral protein profiles, restriction fragment length polymorphism analysis, and next generation DNA sequencing were performed. Comparison of a concatenated set of 4 ZRV genes, for which BIV sequence data are available, with sequence data from representative ranaviruses confirmed that ZRV was most similar to BIV. This is the first report of a BIV-like agent outside of Australia. However, it is not clear whether ZRV is a novel North American variant of BIV or whether it was acquired by exposure to amphibians co-inhabiting the same facility and originating from different geographic locations. Lastly, several surviving toads remained PCR-positive 10 wk after the conclusion of the outbreak. This finding has implications for the management of amphibians destined for use in reintroduction programs, as their release may inadvertently lead to viral dissemination.

Molecular methods to detect Mycoplasma spp. And Testudinid herpesvirus 2 in desert tortoises (Gopherus agassizii) and implications for disease management.

Abstract Mycoplasmas are an important cause of upper respiratory tract disease (URTD) in desert tortoises (Gopherus agassizii) and have been a main focus in attempts to mitigate disease-based population declines. Infection risk can vary with an animal's population of origin, making screening tests popular tools for determining infection status in individuals and populations. To provide additional methods for investigating URTD we developed quantitative PCR (qPCR) assays specific for agents causing clinical signs of URTD: Mycoplasma agassizii, Mycoplasma testudineum, and Testudinid herpesvirus 2 (TeHV2) and tested necropsied desert tortoises housed at the Desert Tortoise Conservation Center in Las Vegas, Nevada, USA, as well as wild desert tortoises (n=3), during 2010. Findings were compared with M. agassizii enzyme-linked immunosorbent assay (ELISA) data. Based on qPCR, the prevalence of M. agassizii was 75% (33/44) and the prevalence of TeHV2 was 48% (20/42) in the evaluated population. Both agents were also present in the wild tortoises. Mycoplasma testudineum was not detected. The M. agassizii ELISA and qPCR results did not always agree. More tortoises were positive for M. agassizii by nasal mucosa testing than by nasal flush. Our findings suggest that mycoplasmas are not the only agents of concern and that a single M. agassizii ELISA or nasal flush qPCR alone failed to identify all potentially infected animals in a population. Caution should be exercised in using these tests for disposition decisions.

Treatment of chytridiomycosis with reduced-dose itraconazole.

Effective treatment methods to eliminate infection with Batrachochytrium dendrobatidis (Bd) are required for development of sustainable captive survival assurance populations of amphibians and to reduce the risk of introducing Bd to new locations as part of amphibian trade or reintroduction programs. Treatment with itraconazole baths at 100 mg l-1 is commonly used in captive amphibians, but side effects are observed in some amphibian species and life stages. Naturally occurring outbreaks of chytridiomycosis in Wyoming toads Anaxyrus baxteri and White's tree frogs Litoria caerulea were treated with lower-dose itraconazole baths (e.g. 50 mg l-1 for White's tree frogs) and followed post-treatment with serial Taqman PCR testing to confirm elimination of Bd infection. Post-treatment PCR tests were consistently negative for the presence of Bd and treatment was deemed successful. Although this was not a controlled clinical trial, results suggest that lower doses of itraconazole may be effective for treatment of chytridiomycosis with resulting cost savings to amphibian conservation programs and a potential for a reduction in dose-related side effects from itraconazole treatment. Prospective clinical trials of alternative itraconazole treatment protocols are encouraged.

Serologic and molecular evidence for Testudinid herpesvirus 2 infection in wild Agassiz's desert tortoises, Gopherus agassizii.

Following field observations of wild Agassiz's desert tortoises (Gopherus agassizii) with oral lesions similar to those seen in captive tortoises with herpesvirus infection, we measured the prevalence of antibodies to Testudinid herpesvirus (TeHV) 3 in wild populations of desert tortoises in California. The survey revealed 30.9% antibody prevalence. In 2009 and 2010, two wild adult male desert tortoises, with gross lesions consistent with trauma and puncture wounds, respectively, were necropsied. Tortoise 1 was from the central Mojave Desert and tortoise 2 was from the northeastern Mojave Desert. We extracted DNA from the tongue of tortoise 1 and from the tongue and nasal mucosa of tortoise 2. Sequencing of polymerase chain reaction products of the herpesviral DNA-dependent DNA polymerase gene and the UL39 gene respectively showed 100% nucleotide identity with TeHV2, which was previously detected in an ill captive desert tortoise in California. Although several cases of herpesvirus infection have been described in captive desert tortoises, our findings represent the first conclusive molecular evidence of TeHV2 infection in wild desert tortoises. The serologic findings support cross-reactivity between TeHV2 and TeHV3. Further studies to determine the ecology, prevalence, and clinical significance of this virus in tortoise populations are needed.

Molecular epidemiology of mycobacteriosis in wildlife and pet animals.

The ecology of mycobacteria is shifting in accordance with environmental change and new patterns of interaction between wildlife, humans, and nondomestic animals. Infection of vertebrate hosts throughout the world is greater now than ever and includes a growing prevalence in free ranging and captive wild animals. Molecular epidemiologic studies using standardized methods with high discriminatory power are useful for tracking individual cases and outbreaks, identifying reservoirs, and describing patterns of transmission and are used with increasing frequency to characterize disease wildlife. This review describes current features of mycobacteriosis in wildlife species based on traditional descriptive studies and recent molecular applications.

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.

Molecular characterization of a novel gammaretrovirus in killer whales (Orcinus orca).

There are currently no published data documenting the presence of retroviruses in cetaceans, though the occurrences of cancers and immunodeficiency states suggest the potential. We examined tissues from adult killer whales and detected a novel gammaretrovirus by degenerate PCR. Reverse transcription-PCR also demonstrated tissue and serum expression of retroviral mRNA. The full-length sequence of the provirus was obtained by PCR, and a TaqMan-based copy number assay did not demonstrate evidence of productive infection. PCR on blood samples from 11 healthy captive killer whales and tissues from 3 free-ranging animals detected the proviral DNA in all tissues examined from all animals. A survey of multiple cetacean species by PCR for gag, pol, and env sequences showed homologs of this virus in the DNA of eight species of delphinids, pygmy and dwarf sperm whales, and harbor porpoises, but not in beluga or fin whales. Analysis of the bottlenose dolphin genome revealed two full-length proviral sequences with 97.4% and 96.9% nucleotide identity to the killer whale gammaretrovirus. The results of single-cell PCR on killer whale sperm and Southern blotting are also consistent with the conclusion that the provirus is endogenous. We suggest that this gammaretrovirus entered the delphinoid ancestor's genome before the divergence of modern dolphins or that an exogenous variant existed following divergence that was ultimately endogenized. However, the transcriptional activity demonstrated in tissues and the nearly intact viral genome suggest a more recent integration into the killer whale genome, favoring the latter hypothesis. The proposed name for this retrovirus is killer whale endogenous retrovirus.

New hosts for equine herpesvirus 9.

Equine herpesvirus 9 was detected in a polar bear with progressive encephalitis; the source was traced to 2 members of a potential equid reservoir species, Grevy's zebras. The virus was also found in an aborted Persian onager. Thus, the natural host range is extended to 6 species in 3 mammalian orders.

Hepatic hemorrhage, hemocoelom, and sudden death due to Haemoproteus infection in passerine birds: eleven cases.

Haemoproteus spp. are ancient apicomplexan hemoparasites that have undergone extensive coevolution with their natural hosts and are typically species specific, with inapparent or minimal pathogenicity. A promiscuous genotype of Haemoproteus capable of undergoing host switching on a familial level was identified. This protozoan caused severe disease with high mortality in 6 species of exotic passerine birds housed in California at the San Diego Zoo's Wild Animal Park: Surinam crested oropendola (Psarocolius decumanus decumanus), Guianan turquoise tanager (Tangara mexicana mexicana), blue-necked tanager (Tangara cyanicollis caeruleocephala, Guianan red-capped cardinal (Paroaria gularis gularis), magnificent bird of paradise (Diphyllodes magnificus hunsteini), and superb bird of paradise (Lophorina superba). The birds had few or no clinical signs. Necropsy findings consisted of hemocoelom and irregularly scattered areas of hemorrhage and hepatocellular necrosis. Affected areas of liver contained solitary protozoal megaloschizonts in varied states of degeneration and peripheral nonsuppurative inflammation. No other parasite life stages were found in parenchymal organs or blood smears. Polymerase chain reaction using consensus primers for an avian malarial mitochondrial cytochrome B gene segment was positive in all cases. Sequencing and BLAST analysis identified the protozoan as a Haemoproteus sp. related to Haemoproteus spp. found in asymptomatic passerine birds native to North America. In situ hybridization was performed in 3 animals with a mitochondrial cytochrome B probe and was positive only in megaloschizonts. These findings suggest the recognition of a genotype of Haemoproteus that exhibits high levels of host infidelity and causes severe disease in captive birds exotic to North America.

Evolution of mammals and their gut microbes.

Mammals are metagenomic in that they are composed of not only their own gene complements but also those of all of their associated microbes. To understand the coevolution of the mammals and their indigenous microbial communities, we conducted a network-based analysis of bacterial 16S ribosomal RNA gene sequences from the fecal microbiota of humans and 59 other mammalian species living in two zoos and in the wild. The results indicate that host diet and phylogeny both influence bacterial diversity, which increases from carnivory to omnivory to herbivory; that bacterial communities codiversified with their hosts; and that the gut microbiota of humans living a modern life-style is typical of omnivorous primates.

Molecular epidemiology of Mycobacterium avium subsp. avium and Mycobacterium intracellulare in captive birds.

Mycobacterium avium subsp. avium and Mycobacterium intracellulare are primary causes of mycobacteriosis in captive birds throughout the world, but little is known about how they are transmitted. To define the local epidemiology of infection, we strain-typed 70 M. avium subsp. avium and 15 M. intracellulare culture isolates obtained over a 4-year period from captive birds. Typing was performed using randomly amplified polymorphic DNA (RAPD) PCR, amplified fragment length polymorphic (AFLP) fragment analyses, and for a subset of isolates, DNA sequencing of a segment of the 16S-23S rRNA internal transcribed spacer region. Six strain clusters comprising 43 M. avium subsp. avium, isolates were identified; 42 isolates had unique typing patterns, including all M. intracellulare isolates. Phylo-geographical analyses using RAPD and AFLP fingerprints and animal confinement histories showed no correlation between housing of infected birds and mycobacterial strain-type, except for two animals. The diversity of M. avium subsp. avium and M. intracellulare isolates and minimal evidence for bird-to-bird transmission suggest that environmental reservoirs may be important sources of infection in captivity.

Molecular assays for detection of falcon adenovirus.

Falcon adenovirus is a newly recognized member of the family Aviadenoviridae and includes 2 closely related strains that are pathogenic to several species of falcons. Peregrine falcons appear to be one of the primary reservoirs, but recent outbreaks suggest that other carrier species probably exist. To allow screening of captive birds for virus shedding and investigations of disease outbreaks, conventional and real-time, quantitative polymerase chain reaction (PCR) assays and an in situ hybridization technique were developed. The diagnostic protocols were used on tissue and fecal samples from 7 species or subspecies of falcons infected with adenovirus as well as adenoviruses from other birds and mammals. The assays were specific for falcon adenovirus and detected both strains of virus in fecal samples from living animals or frozen and formalin-fixed, paraffinized tissues. Together with established serologic tests for falcon adenovirus, these molecular assays are valuable tools for management and conservation of falcons in captivity and the wild.

Falcon adenovirus infection in breeding Taita falcons (Falco fasciinucha).

Four female and 3 male Taita falcons (Falco fasciinucha) out of a breeding colony of 14 Taita falcons (7 pairs) died during the breeding season after showing lethargy and anorexia for 1 to 2 days. All animals were submitted for necropsy. Gross lesions in the female falcons were characterized by anemia secondary to marked hemorrhage into the ovary and oviduct, serofibrinous effusion into the cardioabdominal cavity and serosal petechiae. In addition, marked necrotizing splenitis and pulmonary hemorrhage were present. Histologically, the female falcons had mild necrotizing hepatitis with numerous intranuclear inclusion bodies and necrotizing splenitis with rare inclusion bodies. There were no gross lesions in the male falcons, and the histological lesions were characterized by urate deposition and rare intranuclear inclusion bodies in the renal tubular epithelial cells. Adenoviral particles were found by electron microscopy in the cloacal contents of the female Taita falcons but not in the male falcons. DNA in situ hybridization revealed widespread aviadenoviral nucleic acid within the nuclei of hepatocytes, renal tubular epithelial cells, and adrenal cells in the female falcons but no aviadenoviral nucleic acid in 1 male falcon and only a low quantity of adenoviral nucleic acid in the liver and kidney of another male Taita falcon. PCR amplified aviadenoviral DNA in the liver and intestine of all Taita falcons. The amplicons were sequenced, and the virus was identified as falcon adenovirus. The deaths of the female and male birds were attributed to the aviadenovirus infection.

Molecular characterization of isosporoid coccidia (Isospora and Atoxoplasma spp.) in passerine birds.

Prevalence and disease caused by isosporoid coccidia in passerine birds are well recognized, but confusion about the life cycles of the parasites has led to taxonomic inconsistencies. In this study, we characterized segments of the chromosomal small and large-subunit ribosomal RNA (rRNA) genes of coccidial parasites from 23 species of passerine birds, as well as heat shock protein 70, apicoplast rRNA, and chromosomal 5.8s rRNA genes from a subgroup of these animals, and we correlated genetic data with morphologic findings for different parasite developmental stages, host phylogeny, and overall taxonomic relations within the phylum Apicomplexa. Our findings indicate that isosporoid coccidia of passerine birds are monophyletic but exhibit substantial diversity, with most avian species having one or several unique parasite lineages that underwent synchronous speciation with their hosts, interrupted by sporadic episodes of lateral transmission across species and families. Molecular analyses support a homoxenous life cycle, with sexual forms occurring chiefly in the intestines and asexual merozoites present systemically. Rarely, extraintestinal sexual stages can occur. The passerine coccidia are genetically most closely related to species of Eimeria rather than Isospora. We suggest that these parasites, whether identified from blood merozoite stages or fecal oocysts, be provisionally grouped as a homogeneous clade of individual species in a single taxon and formally named when reliable criteria allowing reclassification of related genera in the suborder Eimeriina are clarified.

Characterization of a new species of adenovirus in falcons.

In 1996, a disease outbreak occurred at a captive breeding facility in Idaho, causing anorexia, dehydration, and diarrhea or sudden death in 72 of 110 Northern aplomado falcons (Falco femoralis septentrionalis) from 9 to 35 days of age and in 6 of 102 peregrine falcons (Falco peregrinus) from 14 to 25 days of age. Sixty-two Northern aplomado and six peregrine falcons died. Epidemiologic analyses indicated a point source epizootic, horizontal transmission, and increased relative risk associated with cross-species brooding of eggs. Primary lesions in affected birds were inclusion body hepatitis, splenomegaly, and enteritis. The etiology in all mortalities was determined by molecular analyses to be a new species of adenovirus distantly related to the group I avian viruses, serotypes 1 and 4, Aviadenovirus. In situ hybridization and PCR demonstrated that the virus was epitheliotropic and lymphotropic and that infection was systemic in the majority of animals. Adeno-associated virus was also detected by PCR in most affected falcons, but no other infectious agents or predisposing factors were found in any birds. Subsequent to the 1996 epizootic, a similar disease caused by the same adenovirus was found over a 5-year period in orange-breasted falcons (Falco deiroleucus), teita falcons (Falco fasciinucha), a merlin (Falco columbarius), a Vanuatu peregrine falcon (Falco peregrinus nesiotes), and gyrfalcon x peregrine falcon hybrids (Falco rusticolus/peregrinus) that died in Wyoming, Oklahoma, Minnesota, and California. These findings indicate that this newly recognized adenovirus is widespread in western and midwestern North America and can be a primary pathogen in different falcon species.

Isolation and epidemiology of falcon adenovirus.

An adenovirus was detected by electron microscopy in tissues from falcons that died during an outbreak of inclusion body hepatitis and enteritis that affected neonatal Northern aplomado (Falco femoralis septentrionalis) and peregrine (Falco peregrinus anatum) falcons. Molecular characterization has identified the falcon virus as a new member of the aviadenovirus group (M. Schrenzel, J. L. Oaks, D. Rotstein, G. Maalouf, E. Snook, C. Sandfort, and B. Rideout, J. Clin. Microbiol. 43:3402-3413, 2005). In this study, the virus was successfully isolated and propagated in peregrine falcon embryo fibroblasts, in which it caused visible and reproducible cytopathology. Testing for serum neutralizing antibodies found that infection with this virus was limited almost exclusively to falcons. Serology also found that wild and captive peregrine falcons had high seropositivity rates of 80% and 100%, respectively, although clinical disease was rarely reported in this species. These data implicate peregrine falcons as the natural host and primary reservoir for the virus. Other species of North American falcons, including aplomado falcons, had lower seropositivity rates of 43 to 57%. Falcon species of tropical and/or island origin were uniformly seronegative, although deaths among adults of these species have been described, suggesting they are highly susceptible. Chickens and quail were uniformly seronegative and not susceptible to infection, indicating that fowl were not the source of infection. Based on the information from this study, the primary control of falcon adenovirus infections should be based on segregation of carrier and susceptible falcon species.

Systemic disease in Vaal rhebok (Pelea capreolus) caused by mycoplasmas in the mycoides cluster.

In the winter of 2002, an outbreak of mycoplasma infection in Vaal rhebok (Pelea capreolus) originating from South Africa occurred 15 weeks after their arrival in San Diego, Calif. Three rhebok developed inappetence, weight loss, lethargy, signs related to pulmonary or arthral dysfunction, and sepsis. All three rhebok died or were euthanized. Primary postmortem findings were erosive tracheitis, pleuropneumonia, regional cellulitis, and necrotizing lymphadenitis. Mycoplasmas were detected in numerous tissues by electron microscopy, immunohistochemistry, and PCR. The three deceased rhebok were coinfected with ovine herpesvirus-2, and two animals additionally had a novel gammaherpesvirus. However, no lesions indicative of herpesvirus were seen microscopically in any animal. The rheboks' mycoplasmas were characterized at the level of the 16S rRNA gene, the 16S-23S intergenic spacer region, and the fructose biphosphate aldolase gene. Denaturing gradient gel electrophoresis was carried out to address the possibility of infection with multiple strains. Two of the deceased rhebok were infected with a single strain of Mycoplasma capricolum subsp. capricolum, and the third animal had a single, unique strain most closely related to Mycoplasma mycoides subsp. mycoides large-colony. A PCR survey of DNA samples from 46 other ruminant species demonstrated the presence of several species of mycoplasmas in the mycoides cluster, including a strain of M. capricolum subsp. capricolum identical to that found in two of the rhebok. These findings demonstrate the pervasiveness of mycoplasmas in the mycoides cluster in small ruminants and the potential for interspecies transmission and disease when different animal taxa come in contact.

Oral papillomas and papilliform lesions in rhesus macaques (Macaca mulatta).

Oral papillomas in two male rhesus macaques that were diagnosed morphologically as filiform and squamous types are described. Two additional macaques had oral papilliform lesions consistent histologically with papillary hyperplasia. Immunohistochemistry, along with electron microscopy and PCR assays, failed to demonstrate evidence of papillomavirus in any of the tumors; however, such results are often lacking when suspect oral lesions in humans and other species are assessed. Other potential causes of the papillary masses include chronic irritation and perhaps a genetic susceptibility. Benign tumors of the oral epithelium in macaques have not been reported previously; they appear to be rare and of variable clinical significance.