Seroprevalence of Toxoplasma gondii in American Black Bears (Ursus americanus) in Nevada, USA, using an Enzyme-linked Immunosorbent Assay.

Archived serum samples taken between 1997 and 2017 from 170 American black bears (Ursus americanus) in the Lake Tahoe area between California and Nevada, US, were tested for Toxoplasma antibodies to assess the seroprevalence of this agent. Samples were screened using a commercial porcine Toxoplasma (enzyme-linked immunosorbent assay [ELISA]) modified with Protein A/G peroxidase and compared to a traditional fluorescent antibody test. Results were analyzed to determine if there were differences in seroprevalence based on the test used, sex of bears, or habitat usage (urban-suburban vs. wildland). No significant differences in seroprevalence were attributable to any of these parameters. The overall seropositivity for bears was 36% (62/170), with urban-suburban bears scoring lower (31%; 37/119) than rural-wildland bears (40%; 18/45). Our results strongly support the use of a Protein A/G-modified ELISA for determining Toxoplasma exposure in black bears. We found somewhat lower levels of Toxoplasma antibodies in black bears from this region than in several reports from populations in the eastern US.

Identification of gammaherpesvirus infection in free-ranging black bears (Ursus americanus).

Herpesvirus infection was investigated in black bears (Ursus americanus) with neurological signs and brain lesions of nonsuppurative encephalitis of unknown cause. Visible cytopathic effects (CPE) could only be observed on days 3-5 post-infection in HrT-18G cell line inoculated with bear tissue extracts. The observed CPE in HrT-18G cells included syncytia, intranuclear inclusions, and cell detachments seen in herpesvirus infection in vitro. Herpesvirus-like particles were observed in viral culture supernatant under the electron microscope, however, capsids ranging from 60 nm to 100 nm in size were often observed in viral cultures within the first two passages of propagation. Herpesvirus infection in the bear tissues and tissue cultures were detected by PCR using degenerate primers specific to the DNA polymerase gene (DPOL) and glycoprotein B gene (gB). DNA sequencing of the amplicon revealed that the detected herpesvirus has 94-95% identity to Ursid gammaherpesvirus 1 (UrHV-1) DNA sequences of DPOL. Phylogenetic analysis of DPOL sequences indicates that black bear herpesviruses and UrHV-1 are closely related and have small distances to members of Rhadinovirus. Interestingly, black bear herpesvirus infections were also found in bears without neurological signs. The DPOL DNA sequence of black bear herpesviruses detected in neurological bears were similar to the those detected in the non-neurological bears. However, the gB DNA sequence detected from the neurological bear is different from non-neurological bear and has only 64.5%-70% identity to each other. It is possible that at least two different types of gammaherpesviruses are present in the U. americanus population or several gammaherpesviruses exist in ursine species.

Pigmented tumors in fallow deer ( Dama dama): 11 cases.

Pigmented tumors have been reported infrequently in captive deer. We document herein the clinical progression and gross and histopathologic features of pigmented tumors diagnosed as melanoma and pigmented schwannoma in 11 white fallow deer ( Dama dama). Affected animals were part of a captive herd maintained at a drive-through park in southern Oregon and were 5-17 y of age during the study period (2004-2013). Primary lesion locations included periocular, perineal, and neck tissues, with cutaneous and internal metastases later identified at autopsy in some cases of malignant melanoma. Diagnoses included 7 malignant melanomas, 2 benign melanomas, and 2 pigmented schwannomas. Diagnosis of melanoma was based on typical histomorphologic features, and final diagnosis of pigmented schwannomas was based on histomorphologic features with negative staining for melan A and positive staining for laminin. Metastasis was found in 3 of 7 cases diagnosed as malignant melanoma; 2 had extensive pulmonary involvement and resulted in euthanasia of the animal; 1 animal developed eyelid and ear lesions that also resulted in euthanasia.

A review of episodes of zinc phosphide toxicosis in wild geese (Branta spp.) in Oregon (2004-2011).

Epizootic mortality in several geese species, including cackling geese (Branta hutchinsii) and Canada geese (Branta canadensis), has been recognized in the Willamette Valley of Oregon for over a decade. Birds are generally found dead on a body of water or are occasionally observed displaying neurologic clinical signs such as an inability to raise or control the head prior to death. Investigation of these epizootic mortality events has revealed the etiology to be accidental poisoning with the rodenticide zinc phosphide (Zn(3)P(2)). Gross and histologic changes are restricted to acute pulmonary congestion and edema, sometimes accompanied by distension of the upper alimentary tract by fresh grass. Geese are unusually susceptible to this pesticide; when combined with an epidemiologic confluence of depredation of specific agricultural crops by rodents and seasonal avian migration pathways, epizootic toxicosis may occur. Diagnosis requires a high index of suspicion, appropriate sample collection and handling, plus specific test calibration for this toxicant. Interagency cooperation, education of farmers regarding pesticide use, and enforcement of regulations has been successful in greatly decreasing these mortality events since 2009.

Serosurvey for antibody to deerpox virus in five cervid species in Oregon, USA.

Five cervid species in Oregon, USA were tested with a serum neutralization assay for antibody to deerpox virus (DPV). None of the 50 elk (Cervus elaphus ssp. roosevelti and nelsonii) had detectable antibody. Prevalence of antibody to DPV in the remaining species was: 52% (n=55) in black-tailed deer (Odocoileus hemionus columbianus), 32% (n= 59) in mule deer (O. hemionus hemionus), and 36% (n=50) in Columbian white-tailed deer (O. virginianus leucurus), with an overall antibody prevalence of 40.2% (n=164) for Odocoileus spp. Antibody-positive animals were identified throughout the state with no statistically significant differences among geographic regions. No statistically significant gender or age-related differences in antibody prevalence were demonstrated at either the genus or species level. This serosurvey indicates that exposure to DPV is common in Odocoileus populations in Oregon. Given the low rates of observed DPV-related disease, this high antibody prevalence suggests a pathogen of low virulence.

Investigation of koi herpesvirus latency in koi.

Koi herpesvirus (KHV) has recently been classified as a member of the family of Alloherpesviridae within the order of Herpesvirales. One of the unique features of Herpesviridae is latent infection following a primary infection. However, KHV latency has not been recognized. To determine if latency occurs in clinically normal fish from facilities with a history of KHV infection or exposure, the presence of the KHV genome was investigated in healthy koi by PCR and Southern blotting. KHV DNA, but not infectious virus or mRNAs from lytic infection, was detected in white blood cells from investigated koi. Virus shedding was examined via tissue culture and reverse transcription-PCR (RT-PCR) testing of gill mucus and feces from six koi every other day for 1 month. No infectious virus or KHV DNA was detected in fecal secretion or gill swabs, suggesting that neither acute nor persistent infection was present. To determine if KHV latent infections can be reactivated, six koi were subjected to a temperature stress regime. KHV DNA and infectious virus were detected in both gill and fecal swabs by day 8 following temperature stress. KHV DNA was also detectable in brain, spleen, gills, heart, eye, intestine, kidney, liver, and pancreas in euthanized koi 1 month post-temperature stress. Our study suggests that KHV may become latent in leukocytes and other tissues, that it can be reactivated from latency by temperature stress, and that it may be more widespread in the koi population than previously suspected.

Results of total DNA measurement in koi tissue by Koi Herpes Virus real-time PCR.

Koi Herpes Virus (KHV) has been classified recently as a member of the Alloherpesviridae within the Herpesvirales order (Waltzek et al., 2005). Although one of the unique features of Herpesviridae, the sister family of Herpesvirales, is latent infection, it has not been demonstrated consistently that KHV of Alloherpesviridae can cause latent infection and be reactivated from latency. To investigate if KHV genomic DNA is present in koi exposed to KHV infection, 10 healthy fish were investigated from a koi population with a history of a KHV outbreak. No gross lesions or microscopic changes were observed at necropsy or by histological examination. No infectious virus was isolated from either the blood plasma or tissues. However, KHV DNA was detected in the white blood cells of nine of the ten fish by real-time PCR and PCR-Southern blot. KHV DNA was also detected in the brain, eye, spleen, gills hematopoietic kidney, trunk kidney, and intestine of nine of the ten fish by PCR-Southern blot. Interestingly, KHV DNA was also detected in the intestinal contents from seven of ten koi. Portions of major capsid gene DNA, amplified from two of the ten koi WBCs, were found to be identical to KHV-U. This study demonstrated that KHV genomic DNA can be detected in normal koi exposed previously to KHV and suggests that KHV becomes latent in fish.

Reduction of herpes simplex virus type-2 replication in cell cultures and in rodent models with peptide-conjugated morpholino oligomers.

BACKGROUND: Genital herpes, caused by herpes simplex virus type-2 (HSV-2), is a recurrent, lifelong disease affecting tens of millions of people in the USA alone. HSV-2 can be treated therapeutically with acyclovir (ACV) and its derivatives; however, no treatment can prevent HSV reactivation. Novel topical anti-HSV microbicides are much needed to reduce HSV-2 transmission and to treat primary or reactivated infections, especially for ACV-resistant strains. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are single-stranded DNA analogues that enter cells readily and can reduce target gene expression through steric blockage of complementary messenger RNA (mRNA). METHODS: We investigated the antiviral activities of PPMOs targeted to the translation start-site regions of the mRNA for two HSV-2 immediate early genes, immediate early protein (ICP)0 and ICP27, and two early genes, unique long gene (UL)30 and UL39. RESULTS: In cell cultures, PPMOs targeting ICP0 or ICP27 mRNA were found to be highly effective against two strains of HSV-2, one of which was ACV-resistant. In vivo, daily topical applications of up to 1 mM ICP27 PPMO caused no gross or microscopic damage to the genital tract of uninfected BALB/c mice or cotton rats. Cotton rats receiving topical application of ICP27 PPMO 24 h after HSV-2 inoculation showed a reduction in genital lesions and a 37.5% reduction in mortality at 14 days post-infection. Mice receiving topical application of 100 µM of an ICP27 and ICP0 PPMO combination before HSV-2 inoculation had no detectable viral replication in the genital tract at 3-5 days post-infection. CONCLUSIONS: These results demonstrate that topically applied PPMOs hold promise as candidate antiviral microbicides against HSV-2 genital infection.

Experimental deerpox infection in black-tailed deer (Odocoileus hemionus columbianus).

The pathogenic potential of deerpox virus was investigated via an experimental study utilizing seven black-tailed deer fawns (Odocoileus hemionus) between June and August of 2007. Successful transmission was achieved via intracutaneous and intravenous routes, and by commingling an uninoculated animal with experimentally infected fawns. One fawn became depressed and reluctant to eat but systemic clinical signs in the other fawns were confined to mild transient pyrexia. Typical multifocal poxviral cutaneous lesions of erythema, papules, pustules, ulceration, and crusting were observed. Two locally extensive ulcerative lesions also developed at inoculation sites. Oral lesions were seen in one commingled fawn, with some palatine epithelial cells containing intracytoplasmic inclusions. Microscopic cutaneous lesions included epithelial cell hyperplasia with hydropic change, intraepithelial pustules, erosions, folliculitis, and dense leukocytic dermal infiltrates. Transmission was confirmed by one or more of virus isolation, polymerase chain reaction or serum neutralization tests. Significant internal lesions were not seen at necropsy.

Inhibition of HSV-1 ocular infection with morpholino oligomers targeting ICP0 and ICP27.

Alternative therapies are needed for HSV-1 infections in patients refractory to treatment with Acyclovir (ACV) and its derivatives. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA analogues that enter cells readily and reduce target gene expression through steric blockage of complementary RNA. When applied before or soon after infection PPMO targeting the translation-start-site regions of HSV-1 ICP0 or ICP27 mRNA reduced HSV-1 plaque formation by 70-98% in vitro. The ICP0 PPMO also reduced ACV-resistant HSV-1 (strain 615.9) plaque formation by 70-90%, while an equivalent dose of ACV produced only 40-50% inhibition when the treatment was applied between 1 and 3hpi. Seven daily topical treatments of 100microg ICP0 PPMO caused no gross or microscopic damage to the corneas of uninfected mice. Topical application of 10microg ICP0 PPMO to the eyes of HSV-1 infected mice reduced the incidence of eye disease by 37.5-50% compared to controls. This study demonstrates that topically applied PPMO holds promise as an antiviral drug candidate against HSV-1 ocular infection.

Prevalence of Baylisascaris procyonis in raccoons (Procyon lotor) in Portland, Oregon, USA.

We investigated the prevalence of Baylisascaris procyonis in raccoons living in the metropolitan area of Portland, Oregon, USA, in order to assess the potential public health risk involved in the transmission of B. procyonis to humans and companion animals. Sixty-nine euthanized raccoons were collected from Portland wildlife-control agencies. Infection with B. procyonis was determined through the harvesting of adult worms from raccoon intestines during necropsy and by fecal analysis using modified double-centrifugation technique with a sugar-flotation solution. Fifty-eight percent of sampled raccoons were found to be infected with B. procyonis. Juveniles represented a greater percentage (64%) of raccoons captured by wildlife-control agents and were found to have the highest prevalence (70%) and heavier adult worm burdens (mean=35 worms). No gender bias was evident. This is one of the few studies of Baylisascaris prevalence in the Pacific Northwest, and it demonstrates that there is a high prevalence of B. procyonis in raccoons inhabiting the Portland area. This factor should be considered in raccoon relocation and management. The data also suggest that juvenile raccoons are the major potential source of B. procyonis contamination in the Portland community and may merit special attention to minimize their interaction with humans.

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.

Bilateral uric acid nephrolithiasis and ureteral hypertrophy in a free-ranging river otter (Lontra canadensis).

We report the first case of uric acid nephrolithiasis in a free-ranging river otter (Lontra canadensis). A 7 yr old male river otter collected from the Skagit River of western Washington (USA) had bilateral nephrolithiasis and severely enlarged ureters (one of 305 examined [0.33%]). The uroliths were 97% uric acid and 3% protein. Microscopic changes in the kidney were confined to expansion of renal calyces, minor loss of medullary tissue, and multifocal atrophy of the cortical tubules. No inflammation was observed in either kidney or the ureters. The ureters were enlarged due to marked hypertrophy of smooth muscle plus dilation of the lumen. Fusion of the major calyces into a single ureteral lumen was several cm distal to that of two adult male otters used as histopathologic control specimens. This case report is part of a large contaminant study of river otters collected from Oregon and Washington. It is important to understand diseases and lesions of the otter as part of our overall evaluation of this population.

Dietary CLA alters yolk and tissue FA composition and hepatic histopathology of laying hens.

The effect of dietary CLA along with n-3 PUFA on yolk FA profile and hepatic lipid accumulation was investigated. Laying hens (n = 40) were randomly assigned to four experimental diets containing 0, 0.5, 1.0, or 2.0% CLA. Menhaden oil was used as the source of n-3 PUFA. Dietary CLA did not affect the total lipid content of egg yolk (P > 0.05). The amounts of CLA isomers (cis-9 trans-11, trans-10 cis-12) in the egg yolk were proportional to the levels of CLA in the diet (P < 0.05). The total CLA content in the egg yolk was 0, 0.97, 2.4, and 5.3 wt%, respectively (P < 0.05). Addition of CLA resulted in an increase in saturated FA (P < 0.05) with a concomitant reduction in monounsaturated FA (P < 0.05) in the yolk, liver, abdominal fat, breast, and thigh muscle. No difference in saturated and monounsaturated FA content in heart and spleen tissue was noted. Dietary CLA at all concentrations resulted in an increase (P < 0.05) in the total number of fat vacuoles and lipid infiltration in hepatocytes. The number of cells with 75% or higher lipid vacuolation in the cytoplasm was also increased (P < 0.05) by 2.0% CLA. Dietary CLA at 0.5% levels resulted in an increase (P < 0.05) in the total lipid content of hepatic tissue. The total lipid content in leg muscle was lower (P < 0.05) in CLA-fed birds. However, no effect of CLA on lipid content of breast muscle, heart, spleen and adipose tissue was observed (P > 0.05). The current study used CLA in a FFA form. The effects of using CLA in other form such as TG on avian hepatic tissue need to be investigated.