Patricia A. Pesavento

Assessment of Melamine and Cyanuric Acid Toxicity in Cats

The major pet food recall associated with acute renal failure in dogs and cats focused initially on melamine as the suspect toxicant. In the course of the investigation, cyanuric acid was identified in addition to melamine in the offending food. The purpose of this study was to characterize the toxicity potential of melamine, cyanuric acid, and a combination of melamine and cyanuric acid in cats. In this pilot study, melamine was added to the diet of 2 cats at 0.5% and 1%, respectively. Cyanuric acid was added to the diet of 1 cat at increasing doses of 0.2%, 0.5%, and 1% over the course of 10 days. Melamine and cyanuric acid were administered together at 0%, 0.2%, 0.5%, and 1% to 1 cat per dose group. No effect on renal function was observed in cats fed with melamine or cyanuric acid alone. Cats dosed with a combination were euthanized at 48 hours after dosing because of acute renal failure. Urine and touch impressions of kidneys from all cats dosed with the combination revealed the presence of fan-shaped, birefringent crystals. Histopathologic findings were limited to the kidneys and included crystals primarily within tubules of the distal nephron, severe renal interstitial edema, and hemorrhage at the corticomedullary junction. The kidneys contained estimated melamine concentrations of 496 to 734 mg/kg wet weight and estimated cyanuric acid concentrations of 487 to 690 mg/kg wet weight. The results demonstrate that the combination of melamine and cyanuric acid is responsible for acute renal failure in cats.

Viruses in diarrhoeic dogs include novel kobuviruses and sapoviruses

The close interactions of dogs with humans and surrounding wildlife provide frequent opportunities for cross-species virus transmissions. In order to initiate an unbiased characterization of the eukaryotic viruses in the gut of dogs, this study used deep sequencing of partially purified viral capsid-protected nucleic acids from the faeces of 18 diarrhoeic dogs. Known canine parvoviruses, coronaviruses and rotaviruses were identified, and the genomes of the first reported canine kobuvirus and sapovirus were characterized. Canine kobuvirus, the first sequenced canine picornavirus and the closest genetic relative of the diarrhoea-causing human Aichi virus, was detected at high frequency in the faeces of both healthy and diarrhoeic dogs. Canine sapovirus constituted a novel genogroup within the genus Sapovirus, a group of viruses also associated with human and animal diarrhoea. These results highlight the high frequency of new virus detection possible even in extensively studied animal species using metagenomics approaches, and provide viral genomes for further disease-association studies.

Divergent Astrovirus Associated with Neurologic Disease in Cattle

Using viral metagenomics of brain tissue from a young adult crossbreed steer with acute onset of neurologic disease, we sequenced the complete genome of a novel astrovirus (BoAstV-NeuroS1) that was phylogenetically related to an ovine astrovirus. In a retrospective analysis of 32 cases of bovine encephalitides of unknown etiology, 3 other infected animals were detected by using PCR and in situ hybridization for viral RNA. Viral RNA was restricted to the nervous system and detected in the cytoplasm of affected neurons within the spinal cord, brainstem, and cerebellum. Microscopically, the lesions were of widespread neuronal necrosis, microgliosis, and perivascular cuffing preferentially distributed in gray matter and most severe in the cerebellum and brainstem, with increasing intensity caudally down the spinal cord. These results suggest that infection with BoAstV-NeuroS1 is a potential cause of neurologic disease in cattle.

A Clonal Outbreak of Acute Fatal Hemorrhagic Pneumonia in Intensively Housed (Shelter) Dogs Caused by Streptococcus equi subsp. zooepidemicus

An outbreak of acute, fatal, hemorrhagic pneumonia was observed in more than 1,000 mixed breed dogs in a single animal shelter. The Department of Anatomic Pathology at the University of California at Davis School of Veterinary Medicine performed necropsies on dogs that were found moribund in acute respiratory distress or found dead with evidence of nasal bleeding. All dogs had hemothorax and an acute, fibrinosuppurative pneumonia. Large numbers of gram-positive cocci were observed within the lungs of all dogs and within septic thromboemboli of remote organs in about 50% of cases. Bacterial cultures from the dogs and their environment revealed widespread beta-hemolytic Streptococus equi subspecies zooepidemicus (Lancefield Group C). Extensive diagnostic testing failed to reveal the consistent presence of copathogens in individual cases. The clinical, epidemiologic, molecular biologic, and pathologic data indicate that a single clone of S. zooepidemicus was the cause of an acutely fatal respiratory infection in these dogs.

Pathologic, Immunohistochemical, and Electron Microscopic Findings in Naturally Occurring Virulent Systemic Feline Calicivirus Infection in Cats:

Infection with feline calicivirus (FCV) is a common cause of upper respiratory and oral disease in cats. FCV infection is rarely fatal, however, virulent, systemic strains of FCV (VS-FCV) that cause alopecia, cutaneous ulcers, subcutaneous edema, and high mortality in affected cats have recently been described. Seven cats with natural VS-FCV infection all had subcutaneous edema and ulceration of the oral cavity, with variable ulceration of the pinnae, pawpads, nares, and skin. Other lesions that were present in some affected cats included bronchointerstitial pneumonia, and pancreatic, hepatic, and splenic necrosis. Viral antigen was present within endothelial and epithelial cells in affected tissues as determined by immunohistochemical staining with a monoclonal antibody to FCV. Mature intranuclear and intracytoplasmic virions in necrotic epithelial cells were identified by transmission electron microscopy. VS-FCV infection causes epithelial cell cytolysis and systemic vascular compromise in susceptible cats, leading to cutaneous ulceration, severe edema, and high mortality.

Significance of Coronavirus Mutants in Feces and Diseased Tissues of Cats Suffering from Feline Infectious Peritonitis

The internal FECV→FIPV mutation theory and three of its correlates were tested in four sibs/half-sib kittens, a healthy contact cat, and in four unrelated cats that died of FIP at geographically disparate regions. Coronavirus from feces and extraintestinal FIP lesions from the same cat were always >99% related in accessory and structural gene sequences. SNPs and deletions causing a truncation of the 3c gene product were found in almost all isolates from the diseased tissues of the eight cats suffering from FIP, whereas most, but not all fecal isolates from these same cats had intact 3c genes. Other accessory and structural genes appeared normal in both fecal and lesional viruses. Deliterious mutations in the 3c gene were unique to each cat, indicating that they did not originate in one cat and were subsequently passed horizontally to the others. Compartmentalization of the parental and mutant forms was not absolute; virus of lesional type was sometimes found in feces of affected cats and virus identical to fecal type was occasionally identified in diseased tissues. Although 3c gene mutants in this study were not horizontally transmitted, the parental fecal virus was readily transmitted by contact from a cat that died of FIP to its housemate. There was a high rate of mutability in all structural and accessory genes both within and between cats, leading to minor genetic variants. More than one variant could be identified in both diseased tissues and feces of the same cat. Laboratory cats inoculated with a mixture of two closely related variants from the same FIP cat developed disease from one or the other variant, but not both. Significant genetic drift existed between isolates from geographically distinct regions of the Western US.

Circovirus in Tissues of Dogs with Vasculitis and Hemorrhage

We characterized the complete genome of a novel dog circovirus (DogCV) from the liver of a dog with severe hemorrhagic gastroenteritis, vasculitis, and granulomatous lymphadenitis. DogCV was detected by PCR in fecal samples from 19/168 (11.3%) dogs with diarrhea and 14/204 (6.9%) healthy dogs and in blood from 19/409 (3.3%) of dogs with thrombocytopenia and neutropenia, fever of unknown origin, or past tick bite. Co-infection with other canine pathogens was detected for 13/19 (68%) DogCV-positive dogs with diarrhea. DogCV capsid proteins from different dogs varied by up to 8%. In situ hybridization and transmission electron microscopy detected DogCV in the lymph nodes and spleens of 4 dogs with vascular compromise and histiocytic inflammation. The detection of a circovirus in tissues of dogs expands the known tropism of these viruses to a second mammalian host. Our results indicate that circovirus, alone or in co-infection with other pathogens, might contribute to illness and death in dogs.

Comparative pathogenesis of epidemic and enzootic Chikungunya viruses in a pregnant Rhesus macaque model

Since 2004, an East African genotype of Chikungunya virus (CHIKV) has emerged, causing significant epidemics of an arthralgic syndrome. In addition, this virus has been associated for the first time with neonatal transmission and neurological complications. In the current study, pregnant Rhesus macaques were inoculated with an enzootic or epidemic strain of CHIKV to compare pathogenesis and transplacental transmission potential. Viremias were similar for both strains and peaked at 2-3 days post-inoculation (dpi). Viral RNA was detected at necropsy at 21 dpi in maternal lymphoid, joint-associated, and spinal cord tissues. The absence of detectable viral RNA and the lack of germinal center development in fetuses indicated that transplacental transmission did not occur. Neutralizing antibodies were detected in all dams and fetuses. Our study establishes a non-human primate model for evaluating vaccines and antiviral therapies and indicates that Rhesus macaques could serve as a competent enzootic reservoir.

Faecal virome of cats in an animal shelter

We describe the metagenomics-derived feline enteric virome in the faeces of 25 cats from a single shelter in California. More than 90 % of the recognizable viral reads were related to mammalian viruses and the rest to bacterial viruses. Eight viral families were detected: Astroviridae, Coronaviridae, Parvoviridae, Circoviridae, Herpesviridae, Anelloviridae, Caliciviridae and Picobirnaviridae. Six previously known viruses were also identified: feline coronavirus type 1, felid herpes 1, feline calicivirus, feline norovirus, feline panleukopenia virus and picobirnavirus. Novel species of astroviruses and bocaviruses, and the first genome of a cyclovirus in a feline were characterized. The RNA-dependent RNA polymerase region from four highly divergent partial viral genomes in the order Picornavirales were sequenced. The detection of such a diverse collection of viruses shed within a single shelter suggested that such animals experience robust viral exposures. This study increases our understanding of the viral diversity in cats, facilitating future evaluation of their pathogenic and zoonotic potentials.

Virulent systemic feline calicivirus infection: local cytokine modulation and contribution of viral mutants.

Virulent systemic feline calicivirus (VS-FCV) is a novel, emerging pathogen with mortality up to 67% even in previously healthy adult cats; VS-FCV has resulted in at least six epidemics since 1998. Affected cats have systemic vascular compromise and hemorrhagic-fever like signs in part due to viral invasion of epithelium and endothelium, coupled with host cytokine responses. Affected skin tissues had, on average, 3.8 elevated cytokines compared with control tissue, with prominent upregulation in IL-10, TNF-α, and MIP-1α. Sequencing of most of the genomes of two VS-FCV strains documented patterns of virus relatedness and implicated changes in the capsid gene in the emerging phenotype, possibly through initiation of immune mechanisms manifest in the cytokine changes. Understanding the features contributing to the emergence of this disease is critical for management and prevention of this and similar outbreaks attributable to RNA viruses in animals and humans.