Otarine herpesvirus-1: a novel gammaherpesvirus associated with urogenital carcinoma in California sea lions (Zalophus californianus)

The incidence of neoplasia in California sea lions (CSLs) is considered to be unusually high. Electron microscopic examination of some of these urogenital tumours revealed the presence of virions with typical herpes-like structure. While current attempts to cultivate this virus have not been successful, molecular studies employing DNA extracted from tumour tissues allowed both the classification of the agent and its identification in tumours and archived tissue samples. Two genome fragments generated using degenerate primers in PCR demonstrated highest identities with other mammalian gammaherpesviruses. Phylogenetic analysis showed that this novel virus, tentatively designated Otarine herpesvirus-1 (OtHV-1), grouped with members of the gammaherpesvirus subfamily and was distinct from PHV-2, a previously described pinniped gammaherpesvirus. An OtHV-1 specific PCR was established and used to investigate the presence of this virus in CSL tissues. PCR of DNA isolated from animals with these tumours, demonstrated that this virus was present in 100% (16/16) of tumours. Furthermore, DNA extracted from archived brain and muscle tissues was also positive in 29% (4/14) and 50% (7/14) of cases examined. This preliminary study provides evidence to support the hypothesis that the presence of this novel gammaherpesvirus is a factor in the development of urogenital carcinoma in CSLs.     

Common metastatic carcinoma of California sea lions (Zalophus californianus): evidence of genital origin and association with novel gammaherpesvirus

Tissues from 10 adult California sea lions (Zalophus californianus, seven females and three males) that had metastatic carcinoma in sublumbar area lymph nodes were examined histologically. A distinctive epithelial proliferative lesion interpreted as intraepithelial neoplasia was found in genital tracts of all ten animals; in vagina (5/7), cervix (7/7), uterus (3/7), penis (3/3) and prepuce (3/3). Intraepithelial neoplasia closely resembled metastatic carcinomas and was directly contiguous with invasive carcinoma in one animal. Rare eosinophilic intranuclear inclusion bodies were found in penile and preputial intraepithelial neoplasia (one animal), cervical intraepithelial neoplasia (one animal), invasive cervical carcinoma (one animal) and metastatic carcinoma (two animals). Electron microscopic examination of tissues from two sea lions (one with intraepithelial neoplasia and one with metastatic carcinoma) demonstrated viral particles consistent with a herpesvirus. An immunohistochemical stain for the latent membrane protein of Epstein-Barr virus was positive in intraepithelial neoplasia in one sea lion. Herpesvirus DNA sequences were detected by consensus primer polymerase chain reaction (PCR) in metastatic carcinomas from all four sea lions from which unfixed tumor samples were available. Results of sequencing were consistent with a novel gammaherpesvirus in the genus Rhadinovirus. DNA extracted from the four metastatic carcinomas also was tested for papillomavirus by Southern blot and PCR with consensus papillomavirus primers; all samples were negative by both methods. These findings support the genital origin of the sea lion carcinoma and implicate a novel gammaherpesvirus as a possible cause.     

Aerobic bacterial flora of the vagina and prepuce of California sea lions (Zalophus californianus) and investigation of associations with urogenital carcinoma

To investigate the association between genital bacterial infection and urogenital carcinoma in California sea lions (Zalophus californianus), vaginal and preputial swabs for bacterial isolation were taken from 148 free-ranging and 51 stranded California sea lions including 16 animals with urogenital carcinoma. Cytological examination of vaginal or preputial smears showed a majority (65.5%, 57/87) of animals examined had mild or no inflammation. Aerobic bacteria were isolated from 116 (78.4%) wild sea lions and 100% of stranded animals. A total of 403 isolates were identified representing 51 unique bacterial species. The median number of isolates per animal increased with age in the wild group, but there was no difference in the number of isolates per animal between wild and stranded adults. The most common bacteria isolated from the wild sea lions were Psychrobacter phenylpyruvicus (39 isolates), non-hemolytic Streptococcus (35 isolates), Corynebacterium spp. (30 isolates), and Escherichia coli (20 isolates). More bacterial species were isolated from stranded animals than wild animals (33 versus 26) and there was significantly less growth of P. phenylpyruvicus, Corynebacterium spp., and Moraxella-like spp. in the stranded animals. Beta-hemolytic Streptococcus was the only bacterium significantly associated with urogenital carcinomas in California sea lions, but only in females.     

Fecal shedding of Helicobacter spp. by co-housed Australian sea lions (Neophoca cinerea) and Australian fur seals (Arctocephalus pusillus doriferus)

With the emergence of Helicobacter species as agents of gastrointestinal disease within a broad range of animal hosts, there is growing awareness of the need to identify such species and the potential role(s) they play within the intestine. Of interest in this study are captive seals and sea lions, where close proximity to one another may enhance the transmission of pathogens, in particular Helicobacter. The feces of several captive Australian sea lions and Australian fur seals were assessed for the occurrence of Helicobacter over 31 days. The presence of Helicobacter, detected by the polymerase chain reaction (PCR) varied over time and at times could not be detected. Helicobacter species were detected in five of the six animals examined of which two species were identified. This is the first report of Helicobacter species in captive seals and demonstrates the diversity and potential role(s) they may play in the gut of these animals.     

Influence of age,sex, capture technique,and restraint on hematologic measurements and serum chemistries of wild California sea otters

Hematologic and/or serum chemical analyses were done on a total of 27 non-tranquilized adult and juvenile wild sea otters, 66 tranquilized adult and juvenile wild sea otters, and 26 wild sea otter pups. The median and inner 90 percentile range were determined for the adult, juvenile, and pup groups and for the following subgroups: adult male versus adult female, juvenile male versus juvenile female, pup male versus pup female, captured with dip net versus captured with Wilson trap, and tranquilized adults and juveniles versus non-tranquilized adults and juveniles. When values for adults were compared to values for juveniles and pups, hematocrits, red blood cell counts, and hemoglobin levels were significantly lower in pups. This is consistent with documented findings in other species. White cell counts were also somewhat lower in younger animals. Among the subgroups, significantly higher hemoglobin levels, white cell counts and neutrophil counts were found in adult females than in adult males. This is in direct contrast to what is seen in other mammalian species and warrants further documentation. Method of capture and tranquilization did not appear to influence either hematologic or serum biochemical determinations from a clinical perspective.     

Seroprevalence of Toxoplasma gondii in mainland and sub-Antarctic New Zealand sea lion (Phocarctos hookeri) populations

AIMS: To investigate the seroprevalence of antibodies to Toxoplasma gondii in New Zealand sea lions (Phocarctos hookeri), as a potential contributor to reproductive failure.

METHODS: Archived sera were sourced from New Zealand sea lions from two recolonising mainland populations in the Otago Peninsula (n=15) and Stewart Island (n=12), as well as a declining population at Enderby Island (n=28) in the New Zealand sub-Antarctic. Sera were tested for antibodies to T. gondii using a commercially available ELISA (with samples considered positive if the sample to positive ratio was?>30%), and latex agglutination test (LAT; with titres ≥1:32 considered positive). Western blot analysis was used to validate the results of a subset of 14 samples.

RESULTS: Five samples from sea lions in mainland locations were confirmed positive for antibodies to T. gondii. Two adult females exhibited high LAT antibody titres (min 1:2048, max 1:4096) on both occasions when sampled 1 and 2 years apart, respectively. No animals from Enderby Island were seropositive.

CONCLUSIONS: Toxoplasma gondii infection is unlikely to be a major contributor to poor reproductive success in New Zealand sea lions. However, continued surveillance is pertinent to assess subclinical and clinical impacts of the parasite on these threatened populations. The commercial tests evaluated here, with further species-specific threshold refinement could provide a fast, inexpensive and reliable indicator of T. gondii exposure in New Zealand sea lions.     

Detection of bovine herpesvirus-1 in peripheral blood mononuclear cells eight months postinfection.

Peripheral blood mononuclear cells (PBMCs) from 5 calves (3 controls and 2 vaccinates) used in a bovine herpesvirus 1 (BHV-1) vaccine study with a BHV-1 Cooper strain challenge were collected 6 months after challenge. The PBMCs from the control animals were positive by immunofluorescence for the BHV-1 glycoprotein D (gD) while the vaccinates were negative. The PBMC samples from 4 of the 5 animals were examined for BHV-1 DNA by polymerase chain reaction (PCR) and for gD immunofluorescence at 8 months after challenge. The BHV-1 DNA and viral antigen were detected in PBMC samples at 8 months postinfection, but no virus was isolated.     

Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction.

Equine herpesvirus-1 (EHV-1) infection is common in young horses throughout the world, resulting in respiratory disease, epidemic abortion, sporadic myelitis, or latent infections. To improve on conventional diagnostic tests for EHV-1, a real-time polymerase chain reaction (PCR) technique was developed, using primers and probes specific for the EHV-1 gB gene. Amplification efficiencies of 100% +/- 5% were obtained for DNA isolated from a plasmid, infected peripheral blood mononuclear cells (PBMCs), and nasal secretions from infected ponies. The dynamic range of the assay was 8 log10 dilutions, and the lower limit of detection was 6 DNA copies. Fifteen ponies, seronegative for EHV-1, were experimentally infected with EHV-1, and nasal samples were used to quantify shedding of virus by both virus isolation and real-time PCR analysis. Virus isolation identified nasal shedding of EHV-1 in 12/15 ponies on a total of 25 days; real-time PCR detected viral shedding in 15/15 ponies on 75 days. Viremia was quantified using PBMC DNA, subsequent to challenge infection in 3 additional ponies. Viremia was identified in 1/3 ponies on a single day by virus isolation; real-time PCR detected viremia in 3/3 ponies on 17 days. When real-time PCR was used to analyze PBMC DNA from 11 latently infected ponies (documented by nested PCR), EHV-1 was not detected. We conclude that real-time PCR is a sensitive and quantitative test for EHV-1 nasal shedding and viremia and provides a valuable tool for EHV-1 surveillance, diagnosis of clinical disease, and investigation of vaccine efficacy.     

Risk factors associated with development of poxvirus lesions in hospitalized California sea lions

OBJECTIVE: To identify risk factors that may predispose California sea lions (Zalophus californianus) to development of cutaneous poxvirus nodules during hospitalization in a rehabilitation center. DESIGN: Retrospective case-control study. ANIMALS: 90 California sea lions admitted to a rehabilitation center. PROCEDURE: Hospital records of 275 stranded California sea lions admitted to the rehabilitation center between January 1 and December 31, 2002, were reviewed. All California sea lions (n = 18) that developed > or = 1 cutaneous poxvirus nodule during hospitalization were classified as cases. Seventy-two California sea lions that did not develop poxvirus lesions during hospitalization were randomly selected (control group). The frequencies of various exposure factors prior to admission, at admission, and during hospitalization for cases and control sea lions were compared by use of logistic regression. RESULTS: California sea lions that had previously been admitted to the rehabilitation center were 43 times as likely to develop poxvirus lesions as sea lions admitted for the first time; those with high band neutrophil counts (> 0.69 X 10(3) bands/microL) at admission were 20 times less likely to develop poxvirus lesions than sea lions with counts within reference limits. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that sea lions with a history of prior hospitalization or band neutrophil counts within reference limits at admission were more likely to develop poxvirus lesions during hospitalization. Sea lions with histories of hospitalization should be kept in quarantine and infection control measures implemented to help prevent disease transmission to attending personnel and other hospitalized animals.     

Enzootic reticuloendotheliosis in the endangered Attwater's and greater prairie chickens

Reticuloendotheliosis (RE) in captive greater prairie chickens (GPC, Tympanuchus cupido pinnatus) and Attwater's prairie chickens (APC, Tympanuchus cupido attwateri) was first reported in 1998. RE is caused by avian reticuloendotheliosis virus (REV), an oncogenic and immunosuppressive retrovirus infecting multiple species of wild and domestic birds. During August 2004 through May 2006 a captive population of prairie chickens was affected simultaneously with a neoplastic condition and also avian pox, the latter being detected in 7.4% (2 of 27) of all birds submitted for histopathology. A survey for REV was conducted in order to examine its possible role in mortality observed primarily in juvenile and adult specimens of prairie chickens. The investigative procedures included postmortem examinations, histopathology, molecular detection, and virus isolation. In total, 57 Attwater's prairie chickens and two greater prairie chickens were included in the study. REV infection was diagnosed using virus isolation or polymerase chain reaction (PCR) or both in 59.5% (28 of 47) of blood samples and/or tumors from suspect birds. Lymphosarcomas were detected in the tissues of 37% (10 of 27) of the birds submitted for histopathology. Such lymphosarcomas suggestive of RE represented the most frequent morphologic diagnosis on histopathology among 27 separate submissions of naturally dead prairie chickens. Overall, REV was detected or RE diagnosed in 34 of 59 prairie chickens (57.62%). The average death age of all birds diagnosed with lymphosarcomas on histopathology was 2.2 yr, ranging from <1 to 4 yr. Although deaths associated with neoplasia occurred in males and females in equal proportions based on submissions, overall more males were diagnosed as REV infected or RE affected (16 males vs. 7 females, and 11 birds of undetermined gender). Reticuloendotheliosis virus was confirmed as a significant cause of mortality in captive prairie chickens.     

Using quantitative polymerase chain reaction to correlate Chlamydia pecorum infectious load with ocular, urinary and reproductive tract disease in the koala (Phascolarctos cinereus)

Complex interactions between Chlamydia pecorum infection, the immune response and disease exist in the koala. We used quantitative polymerase chain reaction to investigate the relationship between C. pecorum infectious load and ocular and urogenital tract disease. Chlamydia pecorum shedding was generally higher in animals with chronic, active disease than in animals with inactive disease. The absence of ocular disease was generally associated with low levels of shedding, but relatively high levels of shedding in the urogenital tract were detected in some koalas without clinical disease signs. These results suggest a complex disease pathogenesis and clinical course in C. pecorum-infected koalas.     

Pathology of domoic acid toxicity in California sea lions (Zalophus californianus)

Over 100 free-ranging adult California sea lions (Zalophus californianus) and one Northern fur seal (Callorhinus ursinus), predominantly adult females, were intoxicated by domoic acid (DA) during three harmful algal blooms between 1998 and 2000 in central and northern California coastal waters. The vector prey item was Northern anchovy (Engraulis mordax) and the primary DA-producing algal diatom was Psuedonitzschia australis. Postmortem examination revealed gross and histologic findings that were distinctive and aided in diagnosis. A total of 109 sea lions were examined, dying between 1 day and 10 months after admission to a marine mammal rehabilitation center. Persistent seizures with obtundation were the main clinical findings. Frequent gross findings in animals dying acutely consisted of piriform lobe malacia, myocardial pallor, bronchopneumonia, and complications related to pregnancy. Gross findings in animals dying months after intoxication included bilateral hippocampal atrophy. Histologic observations implicated limbic system seizure injury consistent with excitotoxin exposure. Peracutely, there was microvesicular hydropic degeneration within the neuropil of the hippocampus, amygdala, pyriform lobe, and other limbic structures. Acutely, there was ischemic neuronal necrosis, particularly apparent in the granular cells of the dentate gyrus and the pyramidal cells within the hippocampus cornu ammonis (CA) sectors CA4, CA3, and CA1. Dentate granular cell necrosis has not been reported in human or experimental animal DA toxicity and may be unique to sea lions. Chronically, there was gliosis, mild nonsuppurative inflammation, and loss of laminar organization in affected areas.     

Clinical reference values for serum protein electrophoresis for the llama (Lama glama)

Serum protein electrophoresis was performed on 71 clinically healthy juvenile and adult llamas (6 juvenile males, 7 juvenile females, 25 adult males, 13 adult females, and 20 pregnant females) to determine normal serum protein concentrations. Values were reported for each of the 5 groups because the groups were not homogeneous in all 8 peaks. Although the values reported here may serve as reference values for adults, they represent only a guideline for the juveniles because of the limited number of animals in each of these groups.     

Clinical relevance of novel Otarine herpesvirus-3 in California sea lions (Zalophus californianus): lymphoma,esophageal ulcers,and strandings

Herpesviruses have been recognized in marine mammals, but their clinical relevance is not always easy to assess. A novel otarine herpesvirus-3 (OtHV3) was detected in a geriatric California sea lion (Zalophus californianus), and using a newly developed quantitative PCR assay paired with histology, OtHV3 was associated with esophageal ulcers and B cell lymphoblastic lymphoma in this animal. The prevalence and quantities of OtHV3 were then determined among buffy coats from 87 stranded and managed collection sea lions. Stranded sea lions had a higher prevalence of OtHV3 compared to managed collection sea lions (34.9% versus 12.5%; p = 0.04), and among the stranded sea lions, yearlings were most likely to be positive. Future epidemiological studies comparing the presence and viral loads of OtHV3 among a larger population of California sea lions with and without lymphoid neoplasia or esophageal ulcers would help elucidate the relevance of OtHV3-associated pathologies to these groups.     

Detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from Yorkshire, Hampshire, and Landrace boars.

Because transmission of porcine reproductive and respiratory syndrome virus (PRRSV) can occur through boar semen, it is important to identify persistently infected boars. However, even for boars given the same PRRSV strain and dose, variability in the duration of viral shedding in semen has been observed, suggesting that host factors are involved in PRRSV persistence. To determine whether there are host genetic factors, particularly litter and breed differences related to the persistence of PRRSV, 3 litters from 3 purebred swine breeds were used for this study. It was also determined whether PRRSV could be detected for a longer period of time in serum, semen, or peripheral blood mononuclear cells (PBMC) and if PRRSV could still be detected in tissues after these antemortem specimens were PRRSV negative for a minimum of 2-3 weeks. Three Hampshire, 3 Yorkshire, and 2 Landrace PRRSV-naive boars were obtained and inoculated intranasally with a wild-type PRRSV isolate (SD-23983). All boars within each breed were from the same litter, and litters were within 9 days of age. Serum and PBMC were collected twice weekly from each boar and analyzed for the presence of PRRSV by virus isolation and the polymerase chain reaction (PCR). Serum was also used to obtain virus neutralization titers and enzyme-linked immunosorbent assay S/P values. Semen was collected twice weekly from 7 of 8 boars and analyzed by PCR. After all specimens were PRRSV negative for a minimum of 2-3 weeks, each boar was euthanized, and 21 tissues plus saliva, serum, feces, and urine were collected. All postmortem specimens were evaluated by virus isolation. Specimens that were PRRSV negative by virus isolation were then evaluated by PCR. The mean number of days (+/-SD) for the duration of PRRSV shedding in semen was 51+/-26.9 days, 7.5+/-4.9 days, and 28.3+/-17.5 days for Landrace, Yorkshire, and Hampshire boars, respectively. Because of small sample sizes and large SDs, the differences in duration of PRRSV shedding in semen between breeds were not considered significant. However, the trend suggested that Yorkshire boars were more resistant to PRRSV shedding in semen than were Landrace boars, requiring further investigation using a larger numbers of boars. PRRSV was detected for a longer period in semen than in serum or PBMC in 4 of 7 boars. Viremia could be detected for a longer period in serum than in PBMC in 6 of 8 boars. After a minimum of 2-3 weeks of PRRSV-negative serum, semen, and PBMC, PRRSV could still be detected in the tonsil of 3 of 8 boars by virus isolation, indicating that boars still harbor PRRSV within the tonsil even though antemortem specimens are PRRSV negative.     

Experimental intravaginal infection of goats with caprine herpesvirus 1

Three adult goats, seronegative to caprine herpesvirus 1 (CpHV.1), were intravaginally inoculated with BA.1 strain of CpHV.1. The animals were kept under observation for 1 month and daily both clinical examinations and white blood cell count were performed. Ocular, nasal, rectal and vaginal swabs and heparinized blood samples were collected every day to attempt virus isolation on cell cultures and detect viral DNA by polymerase chain reaction (PCR) assay. The virus was isolated and detected by PCR only from the vaginal swabs for 5-7 days post-infection. The animals showed transient fever and leukopenia and typical necrotic lesions on the vulva and vagina.     

Pathogenesis of experimental vesicular stomatitis virus (New Jersey serotype) infection in the deer mouse (Peromyscus maniculatus)

The pathogenesis of vesicular stomatitis virus (VSV) infection has not been investigated previously in native New World rodents that may have a role in the epidemiology of the disease. In the present study, 45 juvenile and 80 adult deer mice (Peromyscus maniculatus) were inoculated intranasally with VSV New Jersey serotype (VSV-NJ) and examined sequentially over a 7-day period. Virus was detected by means of immunohistochemistry and in situ hybridization in all tissues containing histologic lesions. Viral antigen and mRNA were observed initially in olfactory epithelium neurons, followed by olfactory bulbs and more caudal olfactory pathways in the brain. Virus also was detected throughout the ventricular system in the brain and central canal of the spinal cord. These results support both viral retrograde transneuronal transport and viral spread within the ventricular system. Other tissues containing viral antigen included airway epithelium and macrophages in the lungs, cardiac myocytes, and macrophages in cervical lymph nodes. In a second experiment, 15 adult, 20 juvenile, and 16 nestling deer mice were inoculated intradermally with VSV-NJ. Adults were refractory to infection by this route; however, nestlings and juveniles developed disseminated central nervous system infections. Viral antigen also was detected in cardiac myocytes and lymph node macrophages in these animals. Viremia was detected by virus isolation in 35/72 (49%) intranasally inoculated juvenile and adult mice and in 17/36 (47%) intradermally inoculated nestlings and juveniles from day 1 to day 3 postinoculation. The documentation of viremia in these animals suggests that they may have a role in the epidemiology of vector-borne vesicular stomatitis.     

Prevalence of Infectious Hypodermal and Hematopoietic NecrosisVirus (IHHNV) in Wild Adult Blue Shrimp Penaeus stylirostris from the Northern Gulf of California,Mexico

Abstract

Conventional histopathology and molecular methods, including dot blot hybridization with a specific DNA probe and polymerase chain reaction (PCR), were used to assess the prevalence and degree of severity of infectious hypodermal and hematopoietic necrosis virus (IHHNV) infection in wild adult blue shrimp Penaeus stylirostris captured in the Northern Gulf of California. Through histopathological analysis, a presumptive diagnosis of IHHNV was initially determined by demonstrating the presence of Cowdry type A inclusion bodies. Prevalence rate ranged from 80% to 100% in females and was 60% in males. Using the dot blot technique, the results showed that the presence of IHHNV varied in females from 86% to 89% and in males from 56% to 57%. Results were further validated using PCR to assess the prevalence of IHHNV in eggs and sperm. The PCR results showed that IHHNV prevalence in unfertilized eggs was 100% and in sperm was 60%. We concluded that wild adults of P. stylirostris are infected with IHHNV but that they do not show clinical signs of disease. Our data suggest that in the 10th year of the IHHNV epizootic in the Gulf of California, a host–pathogen relationship might have reached a putative equilibrium.     

High prevalence of mycoplasmas in the genital tract of asymptomatic stallions in Austria

Mycoplasma equigenitalium and M. subdolum have been implicated in genital disorders and infertility of horses. The reported cytopathic effects of M. equigenitalium observed in vitro underscore its potential pathogenic role in reproductive dysfunction in mares. This study was initiated to determine the prevalence of mycoplasmas in the genital tract of stallions in relationship to age, clinical signs, geographic location and semen quality. For this purpose the mycoplasma flora of the genital tract of 116 stallions of the Noric breed was determined by isolation and colony immunoblotting and by polymerase chain reaction (PCR) assays. Of 438 swabs from the genital tract, pre-ejaculatory fluid and semen samples, 352 (80%) samples were positive by PCR and 125 (29%) were positive by culture. Mycoplasmas were isolated predominantly from the fossa glandis and urethra and less frequently from the penis shaft and from semen. M. equigenitalium (89 isolates) and M. subdolum (70 isolates) were the predominant species identified. M. equirhinis and M. felis were detected in 27 and 8 samples, respectively. Comparison of these isolations with clinical signs, semen quality, and age of the stallions revealed no significant correlation. However, geographical location of the stallion significantly correlated with mycoplasma detection. These results suggest that mycoplasmas are present as commensals in the genital tract of stallions. Thus, clinically healthy stallions may present a permanent reservoir for infection of mares via venereal transmission.