Serological survey for antibodies to Leptospira in dogs and raccoons in Washington State.

A high number of reported canine leptospirosis cases occurred in Washington State from 2004 to 2006. This prompted a serosurvey of healthy dogs from around the state to determine the distribution of exposure risk and to provide insight into serovar epidemiology in the region. In addition, a convenience sample of sera from injured raccoons was also tested, and clinical serological data from the Washington Animal Disease Diagnostic Laboratory were examined. The proportion of dogs with an antibody titre (>or=1:100) to any serovar was 27/158 (17.1%, 95% CI 11.6-23.9), and that proportion among raccoons was 22/115 (19.1%, 95% CI 12.4-27.5) suggesting that the potential for exposure in Washington state is not uncommon. The most frequently detected serovars in healthy dogs were Autumnalis, Icterohemorrhagiae and Canicola, in clinical canine samples Autumnalis, Bratislava and Pomona were more frequent and in sick or injured raccoons Autumnalis, and Pomona were most frequently detected. Clinical canine serology demonstrated a late summer-fall seasonality that was consistent with other reports. An outbreak of canine leptospirosis occurred during 2004-2006 and was located primarily in western Washington counties, as were three reported human cases in 2005. Canine leptospirosis surveillance is an important tool for detecting human risk of exposure and may provide insights into which serovars are currently of clinical importance.

Clinicopathologic features of a systemic coronavirus-associated disease resembling feline infectious peritonitis in the domestic ferret (Mustela putorius).

From 2002 to 2007, 23 ferrets from Europe and the United States were diagnosed with systemic pyogranulomatous inflammation resembling feline infectious peritonitis (FIP). The average age at the time of diagnosis was 11 months. The disease was progressive in all cases, and average duration of clinical illness was 67 days. Common clinical findings were anorexia, weight loss, diarrhea, and large, palpable intra-abdominal masses; less frequent findings included hind limb paresis, central nervous system signs, vomiting, and dyspnea. Frequent hematologic findings were mild anemia, thrombocytopenia, and hypergammaglobulinemia. Grossly, whitish nodules were found in numerous tissues, most frequently the mesenteric adipose tissue and lymph nodes, visceral peritoneum, liver, kidneys, spleen, and lungs. One ferret had a serous abdominal effusion. Microscopically, pyogranulomatous inflammation involved especially the visceral peritoneum, mesenteric adipose tissue, liver, lungs, kidneys, lymph nodes, spleen, pancreas, adrenal glands, and/or blood vessels. Immunohistochemically, all cases were positive for coronavirus antigen using monoclonal antibody FIPV3-70. Electron microscopic examination of inflammatory lesions identified particles with coronavirus morphology in the cytoplasm of macrophages. Partial sequencing of the coronavirus spike gene obtained from frozen tissue indicates that the virus is related to ferret enteric coronavirus.

Myofasciitis in the domestic ferret.

Since late 2003, an inflammatory disease of muscle and fascia has been diagnosed in several ferrets at Northwest ZooPath, and this report describes the condition in 17 ferrets. It is a disease of young ferrets, characterized by rapid onset of clinical signs, high fever, neutrophilic leukocytosis, treatment failure, and death (or euthanasia). Gross lesions include atrophy of skeletal muscle; red and white mottling and dilatation of the esophagus; and splenomegaly. Histologically, moderate to severe suppurative to pyogranulomatous inflammation is in the skeletal muscle and the fascia at multiple sites, including esophagus, heart, limbs, body wall, head, and lumbar regions. Myeloid hyperplasia of spleen and/or bone marrow also is a prominent feature. Ultrastructural lesions include mitochondrial swelling, intracellular edema, disruption of myofibrils and Z bands. Bacterial and viral cultures, electron microscopy, immunohistochemistry, and polymerase chain reaction were negative for a variety of infectious agents. The clinical presentation and distribution of lesions suggests that polymyositis in domestic ferrets is likely a distinct entity. The etiopathogenesis if this condition is not known.

Canine coronavirus-associated puppy mortality without evidence of concurrent canine parvovirus infection.

This report presents 2 cases in which puppy fatalities were associated with canine coronavirus (CCV), but no evidence of concurrent canine parvovirus (CPV-2) disease was observed. Case 1 involved a 7-week-old, male short-haired Chihuahua, which had become lethargic 24 hours after purchase from a pet store. Within 72 hours, the puppy began to vomit, had diarrhea, and was admitted to the veterinary clinic, where it was placed on IV fluids. The parvovirus Cite test was negative. The puppy died within 12 hours of admission and was submitted for diagnostic workup. Gross pathology revealed an enteritis suggestive of CPV-2. Histopathology on intestines showed scattered dilated crypts with necrotic cellular debris and neutrophils. There was moderate depletion and necrosis of lymphoid follicles. Electron microscopy (EM) on intestinal contents was positive for coronavirus and negative for parvovirus. Immunohistochemistry (IHC) on gut sections was positive for CCV and negative for CPV-2. Case 2 was an 8-week-old, male Shih Tzu, which was admitted to the veterinary clinic exhibiting symptoms of severe gastroenteritis with abdominal pain. The referring veterinarian euthanized the puppy, and the entire body was submitted for diagnostic evaluation. Necropsy revealed a severe ileo-cecal intussusception and segmental necrotic enteritis of the small intestine. Electron microscopy of the intestinal contents was positive for coronavirus and negative for parvovirus. Immunohistochemistry on sections of affected gut were positive for CCV and negative for CPV-2. These cases emphasize the importance of pursuing a diagnosis of CCV in young puppies when CPV-2 disease has been ruled out by IHC.

Immunohistochemical analysis of two strains of lion (Panthera leo)-adapted canine distemper virus in ferrets (Mustela putorius furo).

Canine distemper virus (CDV) caused epizootics in lions (Panthera leo) in Tanzania's Serengeti National Park in 1994 and in captive lions and other Panthera spp. in the USA in 1991-1992. In this study, immunohistochemistry was used to compare viral distribution in tissues collected from ferrets (Mustela putorius furo) inoculated with one of the two lion-derived CDV isolates, either from Serengeti (A94-11/13) or from California (A92-27/20). The California isolate resulted in severe morbidity in all nine ferrets, whereas the Serengeti isolate resulted in severe morbidity in five of the nine ferrets. A slightly higher proportion of infected cells was found in many tissues in the Serengeti isolate-inoculated ferrets. These findings indicate that the pathogenicity of the California isolate is not directly related to the number of infected cells.

Detection and molecular characterization of cultivable caliciviruses from clinically normal mink and enteric caliciviruses associated with diarrhea in mink.

Enteric caliciviruses are emerging pathogens responsible for diarrhea or gastroenteritis in their respective hosts. In this report, mink enteric caliciviruses (MEC) were detected in feces from diarrheic mink by both immune electron microscopy (IEM) and RT-PCR using a broadly reactive primer pair (p289/290) targeting the highly conserved RNA polymerase regions of the enteric caliciviruses, Norwalk-like viruses (NLVs) and Sapporo-like viruses (SLVs). The MEC possess classical caliciviral morphology with typical cup-shaped depressions on the viral surface. Sequence analyses based on nucleotide and predicted amino acid (aa) sequences of the RT-PCR products indicated that MEC is most closely related genetically to SLVs of humans and animals. The MEC shared the highest aa identities (64-71%) in the RNA polymerase region with both human SLVs and the porcine enteric calicivirus (PEC) Cowden strain SLV, indicating that MEC may belong to an individual genogroup or subgroup in the SLV genus. The MEC shared only limited aa identities in the RNA polymerase region with vesiviruses (40-51%) and NLVs (29-33%). The RNA polymerase regions of the cultivable, non-enteric mink caliciviruses (MCV) were also amplified by RT-PCR using the primer pair Pol1/Pol3 based on sequences of vesiviruses, and the primer pair p289/290. Sequence analysis indicated that these MCV shared higher aa identities in the RNA polymerase region with vesiviruses (58-81%) than with SLVs (43-51%) including the MEC, lagoviruses (35-37%) and NLVs (27-35%), suggesting that they are most closely related genetically to vesiviruses. The MEC associated with diarrhea in mink are morphologically similar to but are genetically distinct from the cultivable MCV and likely represent a new member of the SLV genus.

Pathogenesis of two strains of lion (Panthera leo) morbillivirus in ferrets (Mustela putorius furo).

Canine distemper virus (CDV) was previously considered to have a host range restricted to the canid family. In 1994, the virus was associated with sporadic outbreaks of distemper in captive felids. However, after severe mortality occurred in the Serengeti lions (Panthera leo), attention became focused on the pathogenesis of the virus and a concerted effort was made to identify the virus as CDV or a closely related feline morbillivirus. The present study was designed to explore the susceptibility of ferrets to challenge with two morbilliviruses isolated from lions and the protective effects of a modified-live mink distemper vaccine. Because mortality in ferrets infected with pathogenic CDV approaches 100%, the ferret was selected as a test animal. Two strains of lion morbillivirus were used as a challenge, A92-27/20 (California lion isolate) and A94-11/13 (Serengeti lion isolate). The two strains of lion morbillivirus were antigenically related to CDV (Rockborn strain), and ferrets were susceptible to both of the viruses when inoculated intraperitoneally. The inoculated ferrets were anorectic at 5-6 days postinoculation (PI), exhibited oculonasal discharge at 9-12 days PI, and became moribund at 12-22 days PI. Severe bilateral conjunctivitis was the typical clinical sign. Inclusion bodies characteristic of morbillivirus (eosinophilic, intranuclear, and intracytoplasmic) were distributed in many epithelial cells, including those of the skin, conjunctiva, gallbladder, liver, pancreas, stomach, trachea, lung, urinary bladder, and kidney. Virus was reisolated from selected lung tissues collected at necropsy and identified by CDV-specific immunofluorescence. Ferrets vaccinated with the mink distemper vaccine (Onderstepoort strain) were protected from challenge with the two lion strains, adding further support to the premise that the viruses are closely related to CDV.

Serologic and molecular characterization of an abortigenic strain of equine arteritis virus isolated from infective frozen semen and an aborted equine fetus.

A virus isolated from an aborted equine fetus was determined to be antigenically distinct from several other strains of equine arteritis virus (EAV) by use of a neutralization assay with a large panel of neutralizing monoclonal antibodies. The virus was readily neutralized by polyclonal equine anti-EAV serum. Comparative nucleotide and amino acid sequence analyses indicated that the virus (WA97) isolated from the aborted fetus was virtually identical to a virus (S1971) isolated from imported semen used to inseminate another mare on the farm. Phylogenetic analysis indicated that the WA97/S1971 virus was more related to European than to North American strains of EAV. These sensitive molecular procedures may be useful for epidemiologic investigations of EAV infections. Screening and certification of stallions and frozen equine semen would prevent dissemination of pathogenic strains of EAV.

Bluetongue virus detection: a safer reverse-transcriptase polymerase chain reaction for prediction of viremia in sheep.

A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the use of the highly toxic methyl mercury hydroxide denaturant.

Occurrence of puma lentivirus infection in cougars from Washington.

Puma lentivirus (PLV) antibodies were detected in 13 (25%) of 52 serum samples obtained from cougars (Felis concolor) collected by hunters. The serum samples were collected from November 1993 through January 1994 from four specific regions throughout the state of Washington (USA), and included the Olympic Mountains, the Cascade Mountains, the Blue Mountains, and the Selkirk Mountains. More (38%) seropositive cougar samples originated from the Cascade Mountains than from any other site. The overall seroprevalence for PLV infection in Washington cougars was higher than previously reported for cougars sampled in Oregon and Idaho (USA), but lower than in cougars sampled in Arizona, Colorado, and California (USA).

Laboratory diagnostic tests for retroviral infections in dairy and beef cattle.

Detection of bovine retroviruses stretches our diagnostic creativity to its limits. The nucleic acid-based, PCR-amplified assays are finding increased clinical use as the veterinary and livestock industry seek earlier detection of infection for eventual corrective management decisions. We are evolving from a point of disease diagnosis by tumor identification through conventional histopathology, to molecular diagnostics for early identification of retroviral nucleic acid (provirus). The clinical use of antibody-based assays lies in the simplicity of testing large numbers of animals, the relative sensitivity of the assays, and the low cost of testing. Although the pathogenicity of bovine leukemia virus (BLV) for cattle has been well documented, the disease potential for bovine immunodeficiency-like virus (BIV) for cattle is still being determined. Nevertheless, pressure to test for retroviral infections of livestock and, when feasible, removal of these infected animals from the herd will be increased.

Serological evidence of morbillivirus infection in polar bears (Ursus maritimus) from Alaska and Russia.

One-hundred-and-ninety-one samples of blood serum collected from 186 polar bears (Ursus maritimus) between 1987 and 1992 were analysed for morbillivirus antibodies. The samples were collected in the Bering, Chukchi and East Siberian seas. Sixty-eight samples (35.6 per cent) had morbillivirus antibody titres > 5; the percentage of positive samples ranged from 26.2 to 46.2 per cent from year to year. The proportions of adults, sub-adults and cubs which were seropositive were 43.9, 35.7 and 37.9 per cent respectively. Some seropositive dams had seronegative young and some that were seronegative had seropositive young. One litter of two cubs, in which the dam was seronegative, had one seropositive and one seronegative cub. Seropositive bears occurred in all the areas from which the samples were collected but there was a significantly greater incidence in the bears sampled in Russia. The high prevalence of seropositive bears over the period suggests that the bear morbillivirus is endemic in these regions of the Arctic, but its source is unknown.

Evolution of canine parvovirus involved loss and gain of feline host range.

Canine parvovirus (CPV) type-2 emerged as a new virus infecting dogs in 1978, and it was probably derived as a variant of feline panleukopenia virus or of a closely related virus infecting another carnivore. CPV type-2 was subsequently replaced in nature by antigenically variant viruses (CPV type-2a and CPV type-2b) which now coexist in dog populations worldwide. We show that CPV type-2 isolates did not replicate in cats, but that both CPV type-2a and CPV type-2b isolates replicated efficiently. About 10% of the viruses isolated from cats with natural parvovirus disease were antigenically indistinguishable from CPV type-2a or type-2b. The capsid protein gene sequence of a 1990 feline parvovirus isolate ("FPV-24") was essentially identical to the sequence of CPV type-2b viruses from dogs. The loss and reacquisition of the feline host range in CPV was most likely due in each case to small numbers of changes in a region of the virus capsid where three protein monomers interact.

Diagnosis of naturally occurring bovine viral diarrhea virus infections in ruminants using monoclonal antibody-based immunohistochemistry.

Formalin-fixed paraffin-embedded tissues from 50 spontaneous cases (39 bovine, nine ovine, two caprine) of bovine viral diarrhea virus (BVDV) infection diagnosed by virus isolation were retrospectively examined for BVDV antigen by immunohistochemistry using anti-BVDV gp-43 monoclonal antibody (Mab 15C5). The cases were separated into enteric disease syndrome, respiratory disease syndrome, and abortion/weak calf syndrome based upon clinical disease. The purposes of the study were to 1) compare routine virus isolation with immunohistochemistry in determining BVDV infection and 2) define tissue and cellular distribution of BVDV in various clinical manifestations of infection. In bovids, there was 100% concordance of virus isolation and immunohistochemistry using Mab 15C5 in cases of enteric disease (mucosal disease, acute and chronic diarrhea, neonatal diarrhea), respiratory disease, and abortion. When laboratory tests were restricted to gastrointestinal tissue and/or feces, virus isolation detected BVDV in only 65% of cattle, whereas immunohistochemistry detected BVDV antigen in 100% of cattle. Immunohistochemical detection of pestivirus was poor in cases of ovine abortion, ovine hairy shaker syndrome, and caprine abortion. The tissue distribution of BVDV antigen was widespread in individual cattle with all clinical forms of BVDV infection. Viral antigen accumulation was spatially correlated with tissue lesions (in the absence of other pathogens) only in the gastrointestinal tract, lymphoid tissue, lung, placenta, and eye. This study demonstrates the utility of immunohistochemistry using Mab 15C5 to diagnose BVDV infections in cattle with a broad spectrum of clinical disease.