A novel test for determination of wild felid-domestic cat hybridization.

In October 2018, Colorado Parks and Wildlife seized an animal believed to be an illegally possessed bobcat. The owner claimed the animal was a bobcat/domestic cat hybrid, exempted from license requirements. Burden of proof lay with CPW to determine the lineage of the animal. Commercial microsatellite arrays and DNA barcoding have not been developed for identification of bobcat/domestic cat hybrids, and limited time and resources prevented development of such tests for this application. Instead, we targeted endogenous feline leukemia virus (enFeLV) to quickly and inexpensively demonstrate the absence of domestic cat DNA in the contested animal. Using this assay, we were able to confirm that the contested animal lacked enFeLV, and therefore was not a domestic cat hybrid.

Temporal association of large granular lymphocytosis, neutropenia, proviral load, and FasL mRNA in cats with acute feline immunodeficiency virus infection.

During acute feline immunodeficiency virus-C(PGammar) (FIV-C-PG) infection, we observed that cats develop large granular lymphocyte (LGL) lymphocytosis concurrent with a marked neutropenia that is temporally associated with the rise and fall of FIV-C-PG proviral loads. LGLs, generally considered to be analogous to natural killer (NK) cells, can also be highly cytolytic CD8/CD57 T cells. Neutropenia has been reported during acute human immunodeficiency virus (HIV-1) infection, but there is a paucity of information describing the pathogenesis of this condition. During HIV-1 infection, LGLs have been shown to be both CD16(+) NK cells and CD8(+)/CD57(+) T cells, but an association with neutropenia has not been described. However, neutropenia with concurrent LGL lymphocytosis has been demonstrated in both LGL leukemia and common variable immunodeficiency syndrome in people, and in both syndromes, an increase in soluble Fas ligand (FasL) has been associated with neutrophil apoptosis leading to neutropenia. Flow cytometric analysis demonstrated increases in CD56 and CD8 peripheral blood cell surface expression during acute FIV-C-PG infection. Expression of FasL mRNA was increased at the same time points as these peripheral hematologic abnormalities, and also decreased as FIV-C-PG proviral load reached set point. We describe an interesting temporal association between innate immune responses and viral load during acute FIV-C-PG infection, which has similarities to HIV-1 infection and other immune dyscrasias of people, and which may contribute to the neutropenia and LGL lymphocytosis during FIV-C-PG infection.

Frequent transmission of immunodeficiency viruses among bobcats and pumas.

With the exception of human immunodeficiency virus (HIV), which emerged in humans after cross-species transmissions of simian immunodeficiency viruses from nonhuman primates, immunodeficiency viruses of the family Lentiviridae represent species-specific viruses that rarely cross species barriers to infect new hosts. Among the Felidae, numerous immunodeficiency-like lentiviruses have been documented, but only a few cross-species transmissions have been recorded, and these have not been perpetuated in the recipient species. Lentivirus seroprevalence was determined for 79 bobcats (Lynx rufus) and 31 pumas (Puma concolor) from well-defined populations in Southern California. Partial genomic sequences were subsequently obtained from 18 and 12 seropositive bobcats and pumas, respectively. Genotypes were analyzed for phylogenic relatedness and genotypic composition among the study set and archived feline lentivirus sequences. This investigation of feline immunodeficiency virus infection in bobcats and pumas of Southern California provides evidence that cross-species infection has occurred frequently among these animals. The data suggest that transmission has occurred in multiple locations and are most consistent with the spread of the virus from bobcats to pumas. Although the ultimate causes remain unknown, these transmission events may occur as a result of puma predation on bobcats, a situation similar to that which fostered transmission of HIV to humans, and likely represent the emergence of a lentivirus with relaxed barriers to cross-species transmission. This unusual observation provides a valuable opportunity to evaluate the ecological, behavioral, and molecular conditions that favor repeated transmissions and persistence of lentivirus between species.

T-lymphocyte profiles in FIV-infected wild lions and pumas reveal CD4 depletion.

Feline immunodeficiency virus (FIV) is a lentivirus related to human immunodeficiency virus (HIV) that causes feline AIDS in the domestic cat (Felis catus). Serological surveys indicate that at least 25 other species of cat possess antibodies that cross-react with domestic cat FIV. Most infected nondomestic cat species are without major symptoms of disease. Long-term studies of FIV genome variation and pathogenesis reveal patterns consistent with coadaptation of virus and host in free-ranging FIV-Ple-infected African lions (Panthera leo) and FIV-Pco-infected pumas (Puma concolor) populations. This report examined correlates of immunodeficiency in wild and captive lions and pumas by quantifying CD5(+), CD4(+), and CD8(+) T-cell subsets. Free-ranging FIV-Ple-infected lions had immunofluorescence flow cytometry (IFC) profiles marked by a dramatic decline in CD4(+) subsets, a reduction of the CD4(+)/CD8(+) ratio, reduction of CD8(+)beta(high) cells, and expansion of the CD8(+)beta(low) subset relative to uninfected lions. An overall significant depletion in CD5(+) T-cells in seropositive lions was linked with a compensatory increase in total CD5(-) lymphocytes. The IFC profiles were altered significantly in 50% of the seropositive individuals examined. The FIV-Pco-infected pumas had a more generalized response of lymphopenia expressed as a significant decline in total lymphocytes, CD5(+) T-cells, and CD5(-) lymphocytes as well as a significant reduction in CD4(+) T-cells. Like lions, seropositive pumas had a significant decline in CD8(+)beta(high) cells but differed by not having compensatory expansion of CD8(+)beta(low) cells relative to controls. Results from FIV-infected lions and pumas parallel human and Asian monkey CD4(+) diminution in HIV and SIV infection, respectively, and suggest there may be unrecognized immunological consequences of FIV infection in these two species of large cats.

Hip dysplasia in rabbits: association with nest box flooring.

PURPOSE: To study etiologic aspects of hip dysplasia in a colony of Dutch-belted rabbits. METHODS: Rabbits used in the study were part of a reproductive toxicologic study. Incidence of hip dysplasia among 296 Dutch-Belted rabbit kits raised on waxed cardboard, smooth Plexiglas, or Plexiglas covered with textured adhesive strips was recorded. All animals were examined at 2 to 4 weeks of age for inability to adduct one or more limbs, then were classified as normal or dysplastic. A subset of 16 juvenile male rabbits (4 normal, 12 affected) raised on Plexiglas flooring were given a physical examination at 12 weeks of age followed by complete necropsy. In four animals (one normal, three affected), pelvic radiography and neurologic examination were performed. RESULTS: Seven percent of the rabbits kits reared on waxed cardboard flooring and 22% of those reared on smooth Plexiglas flooring developed hip dysplasia. Animals reared on Plexiglas floor with traction strips did not have evidence of hip dysplasia. Among the animals selected for detailed analysis, body weight was similar between rabbits with or without splay leg. Affected animals had splaying of one or both hind limbs, various degrees of flattening and reduction of the size of the femoral head, subluxation of the hip, valgus deformity, and patellar luxation. Histologically, there was marked thickening of the hip joint capsule with fibrocartilage formation, mild trabecular bone loss, and bony sclerosis of the proximal portion of the femur and adductor muscle hypoplasia. CONCLUSIONS: Provision of non-slippery flooring during the postnatal period is critical in preventing development of hip dysplasia in rabbits. Hip dysplasia resulted in significant musculoskeletal changes, but not abnormal neurologic development.

Dot immunobinding assay for detection of bovine herpesvirus 4 antibodies in rabbits.

A dot immunobinding assay (DIA) was used for the detection of antibody against bovine herpesvirus 4 (BHV-4) in experimentally infected specific-pathogen-free male and female rabbits. A semipurified virus preparation was used as the antigen, and protein A/G-horseradish-peroxidase conjugate and diaminobenzidine tetrahydrochloride were used as the detection system. Results of the DIA procedure on serum samples of experimentally infected male and female rabbits were compared with those of a complement-dependent virus neutralization (VN) test. None of the tested sera (0/60 samples) from either male or female rabbits were positive by the complement-dependent VN test. Results of the DIA procedure for the same tested sera were positive in 35 of 60 samples (58%) from BHV-4 infected rabbits, indicating higher sensitivity of DIA procedure as compared with the complement-dependent VN test.

Interstitial cell tumour and germ cell tumour with carcinoma in situ in rabbit testes.

Simultaneous occurrence of a well-demarcated interstitial cell tumour and an intratubular seminoma-like tumour, which was beginning to invade peritubular areas, in the contralateral testes of a 3-year-old Dutch-Belted rabbit is described. Morphological hallmarks of carcinoma in situ, which have not been reported previously for the rabbit, were observed in association with the seminoma. These observations indicate that carcinoma in situ, preceding a seminoma-like tumour, occurs in the rabbit and that the rabbit may serve as a practically useful animal model for studying testicular germ cell neoplasia.

Growth of lion and puma lentiviruses in domestic cat cells and comparisons with FIV.

Feline immunodeficiency virus (FIV-Fca) is a lentivirus that causes gradual immunological deterioration in domestic cats. Lentiviruses related to FIV have been detected in several nondomestic feline species; the biologic significance of these viruses remains to be defined. To examine the in vitro cell tropism of these nondomestic cat lentiviruses, prototypical puma and lion lentiviruses (FIV-Pco and FIV-Ple) were cultured in a variety of feline cell cultures. A domestic cat T lymphoma cell line, 3201, best supported the replication of both FIV-Pco and FIV-Ple. Moreover, FIV-Ple was lytic for these cells. RT-PCR amplification of a conserved pol gene region demonstrated species-specific primer homology. Sequence and phylogenetic analyses of this amplification product confirmed the identity of the replicating viruses and classified two previously uncharacterized viruses within predictable lion and puma clades. Sequence analysis of a conserved pol region demonstrated homology with previously characterized FIV-Ple and FIV-Pco. Western blot analysis using domestic cat anti-FIV-Fca sera showed that both FIV-Pco and FIV-Ple were antigenically related, to differing degrees, to three serotypes of FIV-Fca. These studies demonstrate that though nondomestic cat lentiviruses differ significantly from FIV-Fca and that a viral-specific protocol may be necessary for sensitive viral detection, these viruses can replicate in cells of domestic cats. suggesting the potential for cross-species transmission.

Infectivity of lion and puma lentiviruses for domestic cats.

Infection of domestic cats with feline immunodeficiency virus (FIV) causes progressive immunological deterioration similar to that caused by human immunodeficiency virus (HIV). Lentiviruses related to but phylogenetically distinct from FIV have been detected in several non-domestic feline species. Serological cross-reactivity of these viruses raises the question as to whether inter-species transmission may occur. To address this issue, we asked whether lion lentivirus (FIV-Ple) or two strains of puma lentivirus (FIV-Pco) could replicate or cause disease in domestic cats. We found that domestic cats inoculated with FIV-Ple developed persistent cell-associated viraemia, transient cell-free viraemia and antiviral antibody. Clinical disease was not detected throughout a 6 month observation period. Two of four cats inoculated with FIV-Pco developed cell-associated viraemia, seroconverted and exhibited transient lymphadenopathy. No changes in white blood cell parameters or other haematological abnormalities were detected in any of the infected cats. Virus-specific RNA was detected in co-cultivated lymphocytes of all infected cats by RT-PCR. These findings reveal that non-domestic cat lentiviruses are infectious for domestic cats and can establish persistent infection in the absence of disease.

Characterization of dermatologic changes in geriatric rhesus macaques.

Nonhuman primates are frequently used for aging studies. We observed a high prevalence of skin disease among a group of geriatric rhesus monkeys (mean age = 25 years; n = 9) used in aging behavioral studies. Gross and histopathologic changes in the skin of these geriatric rhesus were compared with skin from control adult monkeys (mean age = 10; n = 4) and sun-exposed monkeys (mean age = 11; n = 4) to characterize age-related skin changes. Biopsy specimens were taken from four specified skin locations (lateral to bridge of nose, ventral midline, dorsal midline, perineal area) and from additional areas where skin lesions were present. Samples were routinely processed and evaluated by light microscopy. Blood samples were collected and tested for estrogen, thyroid-stimulating hormone, triiodothyronine thyroxine, and cortisol levels. The axilla was swabbed and samples were obtained for bacterial culturing. All nine of the geriatric monkeys had notable dermal lesions, while one of the control monkeys and one of the sun-exposed monkeys had abnormal findings. Major gross findings included increased areas of erythematous skin, wrinkling, focal skin scaling, thinning of hair, foot calluses, and exudative lesions. Histologic skin changes included subacute dermatitis, acanthotic dermatitis, and a lesion resembling an early solar lentigo in the sun-exposed animal. These changes were not associated with hormonal abnormalities or bacterial pathogens. Histologic changes are compatible with nonspecific skin changes observed in elderly humans. This study establishes a baseline of dermatologic changes of the aging rhesus macaque.

Infection with Borna disease virus: molecular and immunobiological characterization of the agent.

Borna disease virus (BDV), which seems to be distinct from all other known viruses, exhibits a unique mechanism of pathogenesis. This review highlights several aspects of the biology of infection with this virus and summarizes the preliminary characterization of the agent. Studies on BDV may help to illuminate several important areas of neurobiology, including the mechanisms regulating the replication of a new type of RNA virus in the nuclei of neural cells, the neuroinvasiveness and neurotropism of such viruses, their T cell-mediated immunopathology, tolerance in newborn animals to persistent viral infection of the central nervous system, and behavioral diseases and eating disorders induced by such agents.

The role of Branhamella catarrhalis in the "bloody-nose syndrome" of cynomolgus macaques.

During a 15-month period, 25 cynomolgus macaques (Macaca fascicularis) at the Johns Hopkins University were observed to have nasal discharge. Fifteen (60%) of these animals had positive nasal cultures for Branhamella catarrhalis. Clinical signs associated with infection by this bacterium were sneezing, epistaxis, and mucohemorrhagic nasal discharge. Treatment with antibiotics resulted in prompt resolution of clinical signs. Post-therapeutic nasal cultures were negative for B. catarrhalis. Two groups of clinically normal, culture-negative, cynomolgus macaques were inoculated with natural isolates of B. catarrhalis which had been passaged in culture for various amounts of time. Five of the eight animals inoculated became culture-positive and had mild nasal discharge. Presence of blood on nasal swabs was indicative of infection with B. catarrhalis. Three of the inoculated animals had post-swabbing epistaxis. This report documents the role of B. catarrhalis as an upper respiratory pathogen in the cynomolgus monkey which causes mild self-limiting disease reminiscent of the so-called "Bloody-Nose Syndrome." In addition to the obvious clinical significance of this finding to primate clinicians, development of an animal model for human disease caused by this organism may be possible.

A borna virus cDNA encoding a protein recognized by antibodies in humans with behavioral diseases.

Borna disease virus (BDV) causes a rare neurological disease in horses and sheep. The virus has not been classified because neither an infectious particle nor a specific nucleic acid had been identified. To identify the genome of BDV, a subtractive complementary DNA expression library was constructed with polyadenylate-selected RNA from a BDV-infected MDCK cell line. A clone (B8) was isolated that specifically hybridized to RNA isolated from BDV-infected brain tissue and BDV-infected cell lines. This clone hybridized to four BDV-specific positive strand RNAs (10.5, 3.6, 2.1, and 0.85 kilobases) and one negative strand RNA (10.5 kilobases) in BDV-infected rat brain. Nucleotide sequence analysis of the clone suggested that it represented a full-length messenger RNA which contained several open reading frames. In vitro transcription and translation of the clone resulted in the synthesis of the 14- and 24-kilodalton BDV-specific proteins. The 24-kilodalton protein, when translated in vitro from the clone, was recognized by antibodies in the sera of patients (three of seven) with behavioral disorders. This BDV-specific clone will provide the means to isolate the other BDV-specific nucleic acids and to identify the virus responsible for Borna disease. In addition, the significance of BDV or a BDV-related virus as a human pathogen can now be more directly examined.