Development of an enzyme immunoassay for detection of antibodies against Coccidioides in dogs and other mammalian species.

Coccidioides is a soil-dwelling fungus that causes coccidioidomycosis, a disease also known as Valley fever, which affects humans and a variety of animal species. Recent findings of Coccidioides in new, unexpected areas of the United States have demonstrated the need for a better understanding of its geographic distribution. Large serological studies on animals could provide important information on the geographic distribution of this pathogen. To facilitate such studies, we used protein A/G, a recombinant protein that binds IgG antibodies from a variety of mammalian species, to develop an enzyme immunoassay (EIA) that detects IgG antibodies against Coccidioides in a highly sensitive and high-throughput manner. We showed the potential of this assay to be adapted to multiple animal species by testing a collection of serum and/or plasma samples from dogs, mice, and humans with or without confirmed coccidioidomycosis. We then evaluated the performance of the assay in dogs, using sera from dogs residing in a highly endemic area, and found seropositivity rates significantly higher than those in dogs of non-endemic areas. We further evaluated the specificity of the assay in dogs infected with other fungal pathogens known to cross-react with Coccidioides. Finally, we used the assay to perform a cross-sectional serosurvey investigating dogs from Washington, a state in which infection with Coccidioides has recently been documented. In summary, we have developed a Coccidioides EIA for the detection of antibodies in canines that is more sensitive and has higher throughput than currently available methods, and by testing this assay in mice and humans, we have shown a proof of principle of its adaptability for other animal species.

Development and performance evaluation of calf diarrhea pathogen nucleic acid purification and detection workflow.

Calf diarrhea (scours) is a primary cause of illness and death in young calves. Significant economic losses associated with this disease include morbidity, mortality, and direct cost of treatment. Multiple pathogens are responsible for infectious diarrhea, including, but not limited to, Bovine coronavirus (BCV), bovine Rotavirus A (BRV), and Cryptosporidium spp. Identification and isolation of carrier calves are essential for disease management. Texas Veterinary Medical Diagnostic Laboratory current methods for calf diarrhea pathogen identification include electron microscopy (EM) for BCV and BRV and a direct fluorescent antibody test (DFAT) for organism detection of Cryptosporidium spp. A workflow was developed consisting of an optimized fecal nucleic acid purification and multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) for single tube concurrent detection of BCV, BRV, and Cryptosporidium spp., and an internal control to monitor nucleic acid purification efficacy and PCR reagent functionality. In "spike-in" experiments using serial dilutions of each pathogen, the analytical sensitivity was determined to be <10 TCID(50)/ml for BCV and BRV, and <20 oocysts for Cryptosporidium spp. Analytical specificity was confirmed using Canine and Feline coronavirus, Giardia spp., and noninfected bovine purified nucleic acid. Diagnostic sensitivity was ≥98% for all pathogens when compared with respective traditional methods. The results demonstrate that the newly developed assay can purify and subsequently detect BCV, BRV, and Cryptosporidium spp. concurrently in a single PCR, enabling simplified and streamlined calf diarrhea pathogen identification.

Natural cases of 2009 pandemic H1N1 Influenza A virus in pet ferrets.

Respiratory swab samples were collected from 5 pet ferrets (Mustela putorius furo) exhibiting influenza-like illness. The ferrets represented 3 households in 2 states. In each case, the owners reported influenza-like illness in themselves or family members prior to the onset of a similar illness in the ferrets. Real-time reverse transcription polymerase chain reaction assays designed for the detection of the 2009 H1N1 Influenza A virus were conducted in the state animal health laboratories. The assays included detection of the matrix gene of Influenza A virus and neuraminidase gene specific for 2009 H1N1 virus. Samples were positive for both screening assays. The samples were confirmed positive by the National Veterinary Services Laboratories. The history of illness in family members prior to illness in the ferrets suggests that Influenza A virus was transmitted from humans to the ferrets.

Characterization of Cervidpoxvirus isolates from Oregon, California, and eastern Canada.

The present report describes the analysis of 4 Deerpox virus isolates from California, Oregon, and Ontario, Canada. All 4 isolates were associated with cutaneous crusting lesions. Examination of selected samples by electron microscopy demonstrated that the viruses were morphologically similar to orthopoxviruses. Phylogenetic analysis of the A21 gene, which is found in all poxviruses, indicated that the 4 isolates form a lineage distinct from other members except for those belonging to the genus Cervidpoxvirus of the subfamily Chordopoxvirinae. Members of the Cervidpoxvirus lineage encode a set of genes not found in other poxviruses. These include homologs of genes encoding interleukin 1 receptor antagonists (IL-1Ra) and C-type lectin-like receptors (CTLR). In the current investigation, genes encoding homologs of IL-1Ra and CTLR were amplified from all the isolates and were found to be closely related to orthologs found in the Cervidpoxvirus genus, which further supports the inclusion of these isolates in the Cervidpoxvirus genus.

Persistent infection with bovine viral diarrhea virus in an alpaca.

CASE DESCRIPTION: A 2.5-month-old female alpaca that had been born prematurely was examined because of moderate mucopurulent nasal discharge and high rectal temperature. CLINICAL FINDINGS: In addition to pyrexia and clinical signs of disease of the upper portion of the respiratory tract, the cria had inappetence and was in an unthrifty condition. Hematologic abnormalities included low WBC count, low hemoglobin concentration, and low PCV. Samples of blood were submitted for bovine viral diarrhea virus (BVDV) isolation and serologic evaluation. Other adults and newborn crias in the herd were similarly examined. Bovine viral diarrhea virus was detected in the cria, and a diagnosis of persistent infection with BVDV was made at 5.5 months of age. Persistent BVDV infection was suspected in another cria born into the herd but was not identified in any of the adult alpacas. TREATMENT AND OUTCOME: Despite several treatments with antimicrobials, no permanent improvement of the cria's condition was achieved. Because of the poor prognosis, the owners requested euthanasia of the cria; BVDV was isolated from specimens of multiple organs collected at necropsy. CLINICAL RELEVANCE: To date, BVDV infection in New World camelids has not been regarded as a major disease entity. Findings in the cria of this report illustrate that some strains of BVDV readily infect alpacas. Clinical description of the disease plus clinicopathologic findings suggest that persistent BVDV infection may be greatly overlooked as a cause of chronic anemia and failure to thrive in alpacas.

West Nile virus infection in two alpacas.

A male alpaca acutely developed signs of anorexia and fever. Within 2 days, neurologic signs (head tremors and asymmetric ataxia) developed. West Nile virus (WNV) infection was considered a primary differential diagnosis on the basis of 6 previous cases on nearby alpaca farms on which animals had similar clinical signs. Four days after the male alpaca became ill, a female alpaca from the same farm developed similar neurologic signs. In addition to anti-inflammatory and supportive treatments, both alpacas received a transfusion of llama plasma with antibodies against WNV Seven days after the onset of clinical signs, the female alpaca had made a full recovery; however, the more severely affected male died. West Nile virus infection was confirmed post mortem by use of reverse transcriptase-polymerase chain reaction assay and immunohistochemical staining.

Humoral response to West Nile virus vaccination in alpacas and llamas.

OBJECTIVE: To determine humoral responses to an equine West Nile virus (WNV) vaccine in healthy alpacas and llamas and compare responses in alpacas and llamas with responses in horses. DESIGN: Clinical trial. ANIMALS: 28 alpacas, 56 llamas, and 16 horses. PROCEDURE: Horses received 2 vaccinations at 4-week intervals, and alpacas and llamas received 3 vaccinations at 3-week intervals. Fifty-five llamas received a fourth vaccination 3 weeks after the third. Blood samples were collected immediately prior to each vaccination, 3 weeks after the last vaccination for alpacas and llamas, and 4 weeks after the last vaccination for horses and tested for virus-neutralizing antibodies. Samples from 29 randomly selected vaccinated llamas were used. RESULTS: None of the animals developed any local or systemic adverse reactions. Four of 28 (14%) alpacas, 4 of 29 (14%) llamas, and 7 of 16 (44%) horses were seropositive 3 (llamas and alpacas) or 4 (horses) weeks after administration of the first vaccination; 27 of 28 (96%) alpacas, 26 of 29 (90%) llamas, and 15 of 16 (94%) horses were seropositive after administration of the second vaccination; and all 28 alpacas and 28 of 29 (97%) llamas were seropositive 3 weeks after administration of the third vaccination. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that vaccination with the equine WNV vaccine is safe in alpacas and llamas. Administration of 3 vaccinations generally resulted in virus-neutralizing antibody titers similar to those observed following 2 vaccinations in horses; however, because it is not known what antibody titer would be protective against clinical WNV disease in alpacas or llamas, we cannot conclude that the vaccine was efficacious.

Potential pathogens in feces from unweaned llamas and alpacas with diarrhea.

OBJECTIVE: To identify potential pathogens in feces from llama and alpaca crias with diarrhea. DESIGN: Prospective observational study. ANIMALS: 45 unweaned crias with diarrhea. PROCEDURE: Fecal samples were evaluated for Eimeria spp, Giardia spp, Cryptosporidium spp, enteric viruses, and Salmonella spp. A questionnaire yielded information concerning herd management and presence of other affected camelids. RESULTS: 28 crias were < or = 31 days old, 11 were 32 to 62 days old, and 6 were 63 to 210 days old. Potential pathogens were isolated from feces from 32 of the 45 crias. A total of 39 potential pathogens were obtained, including coronavirus (n = 19 crias; 42%), Giardia spp (8; 18%), Eimeria spp (6; 13%), Cryptosporidium spp (4; 9%), rotavirus (1; 2%), and nematode ova (1; 2%). Salmonella spp were not isolated. Most crias from which potential pathogens were isolated were identified during outbreaks of diarrhea involving other camelids, although only coronavirus was isolated from crias identified during outbreaks involving adult camelids. Coronavirus was detected throughout the year, whereas protozoa were most commonly isolated during the fall and winter. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that a variety of potential pathogens may be isolated from young crias with diarrhea. Many crias shed coronavirus, which may also have been affecting older camelids. Protozoa were isolated most often during wetter months, suggesting that crias born during these months may have greater exposure to protozoal pathogens.