Daniel S. Bradway

Temporal Patterns of Diagnostic Results in Serial Samples from Cattle with Advanced Paratuberculosis Infections

As part of investigating diagnostic strategies for Mycobacterium avium subsp. paratuberculosis (Map), serial results from polymerase chain reaction (PCR) on extraintestinal tissues (blood, milk, and liver) were compared with those from more conventional detection methods including serum enzyme-linked immunosorbent assay (ELISA), fecal culture, and fecal PCR. Three cows previously identified as being subclinically infected with Map were selected for the study. Blood, milk, and feces were collected daily and liver biopsies were obtained weekly for a 30-day period. Unexpectedly, a substantial daily variation in serum ELISA sample to positive (S/P) ratios was observed in all 3 cows. In contrast, fecal culture results were consistently positive. However, whereas fecal culture colony counts were consistently high for 2 cows throughout the study, colony counts from the third cow varied from day to day. Diagnostic sensitivity of PCR for fecal, blood, milk, and liver samples in these advanced subclinically infected cows was 87%, 40%, 96%, and 93%, respectively.

Disseminated Bovine viral diarrhea virus in a persistently infected alpaca (Vicugna pacos) cria.

Bovine viral diarrhea virus (BVDV) is an emerging infectious pathogen of concern to the alpaca industry. A 4-month-old, intact, male alpaca cria was diagnosed as persistently infected with BVDV on the basis of repeated positive antemortem polymerase chain reaction (PCR) and virus isolation (VI) assays and negative serologic titers to BVDV. Immunohistochemistry, real-time reverse transcription PCR, and VI performed on tissues collected at necropsy demonstrated disseminated BVDV-1b infection. Virus was detected in multiple tissues, including parotid salivary gland, testes, prostate, kidneys, skin, and gastrointestinal tract. Demonstration of BVDV in previously unreported tissues suggests additional potential routes of BVDV transmission in alpacas.

Sensitivity and Specificity of the Complement Fixation Test for Detection of Cattle Persistently Infected with Anaplasma Marginale

The complement fixation (CF) test commonly is used to identify cattle infected with Anaplasma marginale prior to interstate or international movement. Estimates of the accuracy of the CF test in detecting animals persistently infected with A. marginale vary widely. In this study, the sensitivity and specificity of the CF test for detection of carrier animals was determined using serum from 232 cattle previously defined as A. marginale positive or negative by nested polymerase chain reaction methods and hybridization. Considering results from 2 independent laboratories and interpreting a 1:5 suspect reaction as positive, the best estimate of CF test sensitivity was 20%, with a specificity of 98%. Using a 1:10 cutoff, sensitivity decreased to 14% and specificity increased to 99%. Results of this study indicate that the CF test is ineffective for identifying cattle persistently infected with A. marginale and thus is inadequate for anaplasmosis regulatory and surveillance programs.

Evidence for persistent Bovine viral diarrhea virus infection in a captive mountain goat (Oreamnos americanus).

Bovine viral diarrhea (BVD) viruses are pestiviruses that have been isolated from domestic and wild ruminants. There is serologic evidence of pestiviral infection in more than 40 species of free-range and captive mammals. Vertical transmission can produce persistently infected animals that are immunotolerant to the infecting strain of Bovine viral diarrhea virus (BVDV) and shed virus throughout their lives. Seven species (white-tailed deer, mouse deer, eland, domestic cattle, alpaca, sheep, and pigs) have been definitively identified as persistently infected with BVDV. This study provides serological, molecular, immunohistochemical, and histological evidence for BVDV infection in 2 captive mountain goats from a zoological park in Idaho. The study was triggered by isolation of BVDV from tissues and immunohistochemical identification of viral antigen within lesions of a 7-month-old male mountain goat (goat 1). Blood was collected from other mountain goats and white-tailed and mule deer on the premises for BVDV serum neutralization, viral isolation, and reverse transcription polymerase chain reaction. One 3-month-old mountain goat (goat 2) was antibody negative and BVDV positive in serum samples collected 3 months apart. This goat subsequently died, and though still antibody negative, BVDV was isolated from tissues and identified by immunohistochemistry within lesions. Sequencing and phylogenetic analysis identified the isolates as BVDV-2. These findings provide evidence of persistent infection in a mountain goat, underscoring the need for pestivirus control strategies for wild ruminants in zoological collections.

Gastric Cryptosporidiosis in Freshwater Angelfish (Pterophyllum Scalare)

A freshwater angelfish (Pterophyllum scalare) hatchery experienced variable levels of emaciation, poor growth rates, swollen coelomic cavities, anorexia, listlessness, and increased mortality within their fish. Multiple chemotherapeutic trials had been attempted without success. In affected fish, large numbers of protozoa were identified both histologically and ultrastructurally associated with the gastric mucosa. The youngest cohort of parasitized fish was the most severely affected and demonstrated the greatest morbidity and mortality. The protozoa were morphologically most consistent with Cryptosporidium. All of the protozoan life stages were identified ultrastructurally and protozoal genomic DNA was isolated from parasitized tissue viscera and sequenced. Histological, ultrastructural, genetic, and phylogenetic analyses confirmed this protozoal organism to be a novel species of Cryptosporidium.

Phaeohyphomycosis of the Carapace in an Aldabra Tortoise (Geochelone gigantea)

Abstract An adult male Aldabra tortoise (Geochelone gigantea) presented with a deep flaking area of the carapace, and histologic examination of biopsies from this area revealed phaeohyphomycosis of the superficial keratinized layers. The disease progressed rapidly and spread to numerous sites on the carapace. After several weeks of regular debridement, deep bone involvement was evident and was confirmed through histologic examination. Fungal culture was attempted but was unsuccessful at isolating the infectious agent. Polymerase chain reaction analysis of extracted DNA from the fixed tissue block identified the fungus as Exophiala oligosperma. Initial treatment included weekly debridement and oral and topical antifungal agents. A nuclear scintigraphy bone scan was performed to determine the extent and status of the infection. Multiple foci of uptake of the radiopharmaceutical marker were present within the carapace, indicating active lesions. The tortoise was maintained on oral antifungal treatment, and lesions resolved over several months. A repeat bone scan performed 1 yr after initial presentation showed reduction in marker uptake, indicating a response to treatment in the deeper lesions. Phaeohyphomycosis should be considered as a differential diagnosis for cases of shell lesions in chelonians.

An outbreak of Sarcocystis calchasi encephalitis in multiple psittacine species within an enclosed zoological aviary

A total of 5 psittacine birds in an enclosed zoological exhibit, including 2 princess parrots and 3 cockatoos of 2 different species, developed severe central nervous system clinical signs over a 2–3-month period and died or were euthanized. Histologically, all birds had a lymphoplasmacytic and histiocytic encephalitis with intralesional protozoa consistent with a Sarcocystis species in addition to intramuscular tissue sarcocysts. By immunohistochemical staining, merozoites in brain and tissue cysts in muscle did not react with polyclonal antisera against Sarcocystis falcatula, Sarcocystis neurona, Toxoplasma gondii, and Neospora caninum, or with a monoclonal antibody to S. neurona. Transmission electron microscopy on sarcocyst tissue cyst walls from 2 birds was morphologically consistent with Sarcocystis calchasi. Polymerase chain reaction (PCR) amplification and sequencing of partial 18S ribosomal RNA from muscle tissue cysts and brain schizonts from 3 birds was consistent with a clade containing S. calchasi and Sarcocystis columbae but could not distinguish these closely related Sarcocystis species. However, PCR amplification and sequencing of the internal transcribed spacer 1 RNA segment in the brain from 2 birds and muscle from 2 birds specifically identified the isolates as S. calchasi. The current report documents that multiple psittacine species are susceptible intermediate hosts of S. calchasi, and that infection can cause encephalitis resulting in significant morbidity and mortality in psittacine aviaries.

A novel papillomavirus isolated from proliferative skin lesions of a wild American beaver (Castor canadensis)

Cutaneous papillomatosis was diagnosed in an adult American beaver (Castor canadensis). Gross lesions included numerous exophytic, roughly circular, lightly pigmented lesions on hairless areas of fore and hind feet and the nose. The most significant histopathologic findings were multifocal papilliform hyperplasia of the superficial stratified squamous epithelium, with multifocal koilocytes, and multiple cells with large, darkly basophilic intranuclear inclusion bodies. A virus with properties consistent with papillomavirus (PV) was recovered by virus isolation of skin lesions, utilizing rabbit and feline kidney cell lines. The presence of the virus was confirmed by PV-specific polymerase chain reaction. The partial sequences of E1 and L1 genes did not closely match those of any PVs in GenBank, suggesting that this might be a new type of PV. Partial E1 and L1 nucleotide sequences of the beaver papillomavirus (hereafter, ARbeaver-PV1) were used to create a phylogenetic tree employing the complete E1 and L1 open reading frame nucleotide sequences of 68 PVs. The phylogenetic tree placed the ARbeaver-PV1 in a clade that included the Mupapillomavirus (HPV1 and HPV63) and Kappapapillomavirus (OcPV1 and SfPV1) genera. The present article confirms the papillomaviral etiology of cutaneous exophytic lesions in the beaver.

Proventricular Nematodiasis in Wrinkled Hornbills (Aceros corrugatus)

Abstract Three immature Sunda wrinkled hornbills (Aceros corrugatus) were diagnosed postmortem with proventricular spirurid nematodiasis. Concurrent severe disseminated larval granulomatosis in other visceral organs was considered contributory to mortality in each case. Clinical signs of nematodiasis were vague but generally consisted of weight loss, anorexia, and lethargy. Frequent antemortem fecal examinations were negative for spirurid eggs. In these present cases, based on routine histopathology, both prophylactic and empirically based therapeutic anthelmintic treatments had no evident benefit in the elimination of the proventricular nematodes. Spirurid nematodiasis may be an important cause of mortality in young hornbills.

Evaluation of a commercial bovine viral diarrhea virus vaccine in nonpregnant female alpacas (Vicugna pacos)

Bovine viral diarrhea virus (BVDV) is an emerging pathogen in alpacas and many questions still persist regarding disease mechanisms and control strategies. The purpose of this study was to evaluate a commercial BVDV vaccine for safety and efficacy in alpacas. Five nonpregnant alpacas were vaccinated with a modified-live BVDV vaccine and challenged 25 days post-immunization by nasal and ocular inoculation with a BVDV Type 1b strain isolated from a confirmed BVDV persistently infected alpaca. Two nonpregnant alpacas served as non-vaccinated controls and were similarly challenged. Results indicated that BVDV virus could not be detected from the vaccinated alpacas but was detected in the unvaccinated alpacas. Results suggest that administration of modified-live BVDV vaccine protected the alpacas in this study from experimental challenge and no adverse effects from the vaccine were observed.