Polioencephalomyelitis in Domestic Swine Associated With Porcine Astrovirus Type 3.

In the past decade, different members of the genus Mamastrovirus have been associated with outbreaks of neurologic disease in humans, cattle, sheep, mink, and, most recently, porcine astrovirus 3 (PoAstV3) in swine. We performed a retrospective analysis of 50 cases of porcine neurologic disease of undetermined cause but with microscopic lesions compatible with a viral encephalomyelitis to better understand the role and pathogenesis of PoAstV3 infection. Nucleic acid was extracted from formalin-fixed paraffin-embedded (FFPE) tissue for reverse transcription quantitative polymerase chain reaction (RT-qPCR) testing for PoAstV3. In addition, 3 cases with confirmed PoAstV3-associated disease were assayed by RT-qPCR to investigate PoAstV3 tissue distribution. PoAstV3 was detected in central nervous system (CNS) tissue via RT-qPCR and in situ hybridization in 13 of 50 (26%) FFPE cases assayed. PoAstV3 was rarely detected in any tissues outside the CNS. Positive cases from the retrospective study included pigs in various production categories beginning in 2010, the earliest year samples were available. Based on these results, PoAstV3 appears to be a recurring putative cause of viral encephalomyelitis in swine that is rarely detected outside of the CNS at the time of clinical neurologic disease, unlike other common viral causes of neurologic disease in swine.

Outbreak of H5N2 highly pathogenic avian Influenza A virus infection in two commercial layer facilities: lesions and viral antigen distribution.

The largest outbreak of highly pathogenic avian Influenza A virus (HPAIV) infection in U.S. history began in December 2014 resulting in the euthanasia of millions of birds and collateral economic consequences to the U.S. poultry industry. We describe 2 cases of H5N2 HPAIV infection in laying hens in Iowa. Following a sharp increase in mortality with minimal clinical signs, 15 dead birds, from 2 unrelated farms, were submitted to the Iowa State University Veterinary Diagnostic Laboratory. Common lesions included diffuse edema and multifocal hemorrhage of the comb, catarrhal exudate in the oropharynx, and multifocal tracheal hemorrhage. Less common lesions included epicardial petechiae, splenic hemorrhage, and pancreatic necrosis. Influenza A virus nucleoprotein was detected by immunohistochemistry in multiple cell types including ependymal cells, the choroid plexus, neurons, respiratory epithelium and macrophages in the lung, cardiac myocytes, endothelial cells, necrotic foci in the spleen, Kupffer cells in the liver, and necrotic acinar cells in the pancreas. Real-time polymerase chain reaction and sequencing confirmed H5N2 HPAIV with molecular characteristics similar to other contemporary U.S. H5N2 HPAIVs in both cases.

Clinical investigation into feed-related hypervitaminosis D in a captive flock of budgerigars (Melopsittacus undulatus): morbidity, mortalities, and pathologic lesions.

The Blank Park Zoo began suffering mortalities in the spring of 2012 within a flock of 229 captive budgerigars (Melopsittacus undulatus) housed in an interactive public-feeding aviary. Clinical signs in affected birds included weakness, posterior paresis, inability to fly, or acute death. Gross and microscopic lesions were not initially apparent in acutely affected deceased birds. Many birds had evidence of trauma, which is now hypothesized to have been related to the birds' weakness. Investigation into the cause(s) of morbidity and mortality were complicated by the opening of a new interactive enclosure. For this reason, environmental conditions and husbandry protocols were heavily scrutinized. Microscopic examination of dead budgies later in the course of the investigation revealed mineralization of soft tissues consistent with hypervitaminosis D. Pooled serum analysis of deceased birds identified elevated vitamin D3 levels. Vitamin D3 analysis was performed on the feed sticks offered by the public and the formulated maintenance diet fed to the flock. This analysis detected elevated levels of vitamin D3 that were 22.5-times the manufacturer's labeled content in the formulated diet. These findings contributed to a manufacturer recall of more than 100 formulated diets fed to a wide variety of domestic and captive wild animal species throughout the United States and internationally. This case report discusses the complexities of determining the etiology of a toxic event in a zoologic institution.

Rapid detection of terbufos in stomach contents using desorption electrospray ionization mass spectrometry.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an emerging analytical technique that permits the rapid and direct analysis of biological or environmental samples under ambient conditions. Highlighting the versatility of this technique, DESI-MS has been used for the rapid detection of illicit drugs, chemical warfare agents, agricultural chemicals, and pharmaceuticals from a variety of sample matrices. In diagnostic veterinary toxicology, analyzing samples using traditional analytical instrumentation typically includes extensive sample extraction procedures, which can be time consuming and labor intensive. Therefore, efforts to expedite sample analyses are a constant goal for diagnostic toxicology laboratories. In the current report, DESI-MS was used to directly analyze stomach contents from a dog exposed to the organophosphate insecticide terbufos. The total DESI-MS analysis time required to confirm the presence of terbufos and diagnose organophosphate poisoning in this case was approximately 5 min. This highlights the potential of this analytical technique in the field of veterinary toxicology for the rapid diagnosis and detection of toxicants in biological samples.

Isolation and characterization of porcine epidemic diarrhea viruses associated with the 2013 disease outbreak among swine in the United States.

Porcine epidemic diarrhea virus (PEDV) was detected in May 2013 for the first time in U.S. swine and has since caused significant economic loss. Obtaining a U.S. PEDV isolate that can grow efficiently in cell culture is critical for investigating pathogenesis and developing diagnostic assays and for vaccine development. An additional objective was to determine which gene(s) of PEDV is most suitable for studying the genetic relatedness of the virus. Here we describe two PEDV isolates (ISU13-19338E and ISU13-22038) successfully obtained from the small intestines of piglets from sow farms in Indiana and Iowa, respectively. The two isolates have been serially propagated in cell culture for over 30 passages and were characterized for the first 10 passages. Virus production in cell culture was confirmed by PEDV-specific real-time reverse-transcription PCR (RT-PCR), immunofluorescence assays, and electron microscopy. The infectious titers of the viruses during the first 10 passages ranged from 6 × 10(2) to 2 × 10(5) 50% tissue culture infective doses (TCID50)/ml. In addition, the full-length genome sequences of six viruses (ISU13-19338E homogenate, P3, and P9; ISU13-22038 homogenate, P3, and P9) were determined. Genetically, the two PEDV isolates were relatively stable during the first 10 passages in cell culture. Sequences were also compared to those of 4 additional U.S. PEDV strains and 23 non-U.S. strains. All U.S. PEDV strains were genetically closely related to each other (≥99.7% nucleotide identity) and were most genetically similar to Chinese strains reported in 2011 to 2012. Phylogenetic analyses using different genes of PEDV suggested that the full-length spike gene or the S1 portion is appropriate for sequencing to study the genetic relatedness of these viruses.

Emergence of Porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences.

During the 10 days commencing April 29, 2013, the Iowa State University Veterinary Diagnostic Laboratory received the first 4 of many submissions from swine farms experiencing explosive epidemics of diarrhea and vomiting affecting all ages, with 90-95% mortality in suckling pigs. Histology revealed severe atrophy of villi in all segments of the small intestines with occasional villus-epithelial syncytial cells, but testing for rotaviruses and Transmissible gastroenteritis virus (Alphacoronavirus 1) were negative. Negative-staining electron microscopy of feces revealed coronavirus-like particles and a pan-coronavirus polymerase chain reaction (PCR) designed to amplify a conserved region of the polymerase gene for all members in the family Coronaviridae produced expected 251-bp amplicons. Subsequent sequencing and analysis revealed 99.6-100% identity among the PCR amplicons from the 4 farms and 97-99% identity to the corresponding portion of the polymerase gene of Porcine epidemic diarrhea virus (PEDV) strains, with the highest identity (99%) to strains from China in 2012. Findings were corroborated at National Veterinary Services Laboratories using 2 nested S-gene and 1 nested N-gene PCR tests where the sequenced amplicons also had the highest identity with 2012 China strains. Whole genome sequence for the virus from 2 farms in 2 different states using next-generation sequencing technique was compared to PEDV sequences available in GenBank. The 2013 U.S. PEDV had 96.6-99.5% identity with all known PEDV strains and the highest identity (>99.0%) to some of the 2011-2012 Chinese strains. The nearly simultaneous outbreaks of disease, and high degree of homology (99.6-100%) between the PEDV strains from the 4 unrelated farms, suggests a common source of virus.

Experimental co-infection of pigs with Bovine viral diarrhea virus 1 and Porcine circovirus-2.

The role of Bovine viral diarrhea virus (BVDV) in the development of Porcine circovirus-2 (PCV-2)-associated disease (PCVAD) was investigated in 2 experimental studies. In the first, separate groups of germ-free pigs were inoculated with filtered tissue homogenate (from diseased pigs) containing PCV-2b + BVDV-1-like virus (group 1), PCV-2a + BVDV-1-like virus (group 4), BVDV-1-like virus only (group 3), or PCV-2b + BVDV-1-like virus following a BVDV vaccination protocol (group 2). This last group was used to test if BVDV vaccination would prevent clinical PCVAD in this model. Many of the inoculated pigs had mild multisystemic inflammation consistent with classic PCVAD. One vaccinated, dually inoculated pig had acute respiratory distress followed by death at 21 days postinfection. Lesions in this pig resembled the severe form of PCVAD observed in the field since the fall of 2004, suggesting a role of ruminant pestiviruses and/or vaccination in the development of this disease. In the second study, cesarean-derived, colostrum-deprived pigs were inoculated with PCV-2b and a cytopathic strain of BVDV-1 (cpBVDV-NADL) either alone or in combination. Clinical signs of PCVAD were seen in a single animal inoculated only with PCV-2b. This pig had growth retardation followed by acute respiratory distress leading to death 30 days postinfection. Pulmonary lesions in this animal were similar to those seen in the pig that died in the first study. Infection with cpBVDV-NADL did not enhance PCV-2b replication or lesion formation.

Genotyping of Porcine teschovirus from nervous tissue of pigs with and without polioencephalomyelitis in Indiana.

Porcine teschovirus (PTV) was isolated in cell culture and/or demonstrated by polymerase chain reaction in samples of brain and/or spinal cord in pigs in Indiana during the 2002-2007 period. Testing was initiated on pigs originating from populations exhibiting nervous clinical disease and/or pigs with microscopic lesions in central nervous tissues, indicating viral encephalitis and/or myelitis. Virus was demonstrated in pigs with and without lesions as well as with and without nervous clinical disease. Nucleotide sequence analysis of the 5'-nontranslated region of the viral genome revealed that these isolates had low-level genetic heterogeneity but were homologous to porcine PTV serotype 1 (PTV-1). These findings indicate that low-to-moderate virulence strains of PTV with some homology to PTV-1 are endemic in many swineherds of Indiana and are associated with subclinical and clinical nervous disease in weaned pigs.

Infection of cesarean-derived colostrum-deprived pigs with porcine circovirus type 2 and Swine influenza virus.

Porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are important pathogens for porcine respiratory disease complex, which is economically significant worldwide. The pathogenesis of PCV2-SIV coinfection is unknown. In this study, we focused on establishing a challenge model for PCV2 to determine whether SIV influences PCV2 replication and increases the severity of PCV2-associated disease. Cesarean-derived colostrum-deprived pigs were inoculated intratracheally with cell culture medium only (negative control group), PCV2 only, or PCV2 followed 1 wk later with SIV H1N1. Two pigs from each group were necropsied at 12, 21, 28, and 35 d after inoculation. Coinfection with SIV did not increase the number of PCV2 genomic copies in serum or target tissues or the severity of microscopic lesions associated with PCV2 in lung or lymph node. The antibody titer to PCV2 did not differ significantly between PCV2-SIV- and PCV2-infected groups. In conclusion, SIV H1N1 did not influence PCV2 replication in dually infected pigs in this study.

Vascular-associated lymphoid tissue in swine (Sus scrofa).

Focal accumulations of mononuclear cells in the arterial wall of healthy humans at predilection sites for atherosclerotic lesions have been described as 'vascular-associated lymphoid tissue' (VALT). Here we investigated whether pigs (Sus scrofa), a commonly used animal model for studying cardiovascular disease, have VALT. Samples of major arteries were collected from 10 conventional crossbred pigs (age, 2 to 24 mo) and processed for routine light microscopy, immunohistochemistry, and immunofluorescence. Single or small aggregates of mononuclear cells were noted in the intima and occasionally the inner portion of the tunica media and adventitia at branching sites. The infiltrating cells were primarily CD3+CD4+ T cells, with some macrophages. No CD8+ T cells were present. Infiltrating leukocytes and overlying endothelial cells frequently expressed major histocompatibility class II molecules. Two Ossabaw pigs on low-fat diet had similar leukocytic aggregates at locations where animals of the same breed but fed a high-fat and high-cholesterol diet developed atherosclerotic lesions. Further, the densities of CD3+ T lymphocytes and in these areas were decreased in 2 sedentary and 2 exercised Ossabaw pigs on an atherogenic diet compared with conventional crossbred and Ossabaw pigs on a normal diet. This study shows that focal aggregates of lymphocytes occur in the vasculature of pigs at locations predisposed to development of atherosclerotic lesions. These cellular aggregates are similar to the structures described as VALT in human arteries and reinforce the value of the pig as a model for the study of human cardiovascular disease.

Prevalence and characterization of bovine viral diarrhea virus in the white-tailed deer population in Indiana.

Bovine viral diarrhea (BVD) is one of the economically important diseases of cattle. For many years, different types of vaccines have been commercially available, yet this disease is hard to control in high-density population areas. Detection and isolation of bovine viral diarrhea virus (BVDV) from any potential reservoir is vital, especially when considering virus eradication from a herd or locale. One potential source is wild ruminants. Ear notches and lymph nodes were collected from the wild population of white-tailed deer (Odocoileus virginianus) during deer hunting season in Indiana and tested for BVDV with a commercial BVD antigen capture enzyme-linked immunosorbent assay. Two samples out of 745 collected samples were positive, and subsequently cp and ncp BVDV was isolated from 1 ear notch and 1 lymph node. These isolates were genotyped as type 1a and 1b based on sequence analysis of the 5' untranslated region (UTR). The results of the present study indicate that the prevalence of BVDV in the white-tailed deer population of Indiana is about 0.3%. Wild ruminants infected with BVDV should be taken into consideration during an eradication program of BVDV from the livestock population.

Toxoplasmosis in a woodchuck (Marmota monax) and two American red squirrels (Tamiasciurus hudsonicus).

Toxoplasmosis was diagnosed in a woodchuck (Marmota monax) and 2 American red squirrels (Tamiasciurus hudsonicus). The woodchuck was euthanized by a wildlife rescue organization in New York after progressive clinical signs of head tilt, circling, and rapid weight loss. Necropsy examination revealed acute subdural hemorrhage over the right cerebral hemisphere. Histologic lesions included meningoencephalitis, myocarditis, and hepatitis. Protozoal cysts were present in affected and unaffected neuroparenchyma. The squirrels were found dead, emaciated, and moderately infested with fleas near a park in northern Indiana. In both squirrels, the lungs were consolidated with numerous nodules up to 2 mm in diameter. Histologically, pneumonia and encephalitis were associated with intracellular and free protozoa. Additional histologic lesions included multifocal lymphoplasmacytic encephalitis with intralesional protozoa in both squirrels. The protozoa were positive with Toxoplasma gondii-specific immunohistochemistry and had ultrastructural features consistent with T. gondii in both squirrels. A real-time polymerase chain reaction test using T. gondii-specific probes demonstrated protozoal DNA in the lung, brain, and kidney of the squirrels and in the brain and heart of the woodchuck. To the authors' knowledge, this is the first report of toxoplasmosis in woodchucks or American red squirrels. Because rodents are common near urban settlements, this finding underscores their role as important intermediate hosts for T. gondii.

Carcinoma in a mixed mammary tumor in a llama (Lama glama).

A 13-year-old female llama was presented to the referring veterinarian for swelling and firmness of the right rear mammary gland, for a duration of 2 months, which had been unresponsive to antibiotics. A formalin-fixed wedge biopsy specimen from the affected quarter was submitted to Purdue University Animal Disease Diagnostic Laboratory for histopathology. Histopathologic examination revealed tubulopapillary acinar or solid nest-like clusters of neoplastic epithelial cells surrounded by whorls and sheets of proliferative myoepithelial cells. Histologic criteria for malignancy observed in neoplastic epithelial cells included marked cellular and nuclear atypia, high mitotic index, and numerous bizarre mitoses. The presence of osseous metaplasia in the proliferative mesenchymal component justified classification as a mixed tumor. Positive immunohistochemical staining of neoplastic epithelial cells with anticytokeratin antibody, and proliferative spindloid cells with antiviemtin and antismooth muscle actin antibodies supported the histopathologic diagnosis. The llama was in good health after about 1 year of initial presentation, and metastasis to regional lymph nodes was not reported. Mammary neoplasia is rare in camelids. To the authors' knowledge, this is the first report of a carcinoma in a mixed mammary tumor in a llama.

Muscular pseudohypertrophy (steatosis) in a bovine fetus.

Muscular pseudohypertrophy was diagnosed in the cervical musculature of a full-term crossbred Simmental fetus delivered by fetotomy. Only head and cervical regions were submitted for pathologic examination; the rest of the fetal body was reportedly normal. The neck musculature of the fetus was markedly deformed by 23 cm and 18 cm in diameter, firm, spherical masses that consisted of enlarged and pale left splenius and right serratus ventralis cervicis muscle, respectively, covered by intact skin. Additionally, lipomatous masses were present within the cervical vertebral canal, compressing the spinal cord. Microscopically, the prominent muscular enlargement was due to massive adipose and fibrous connective tissue replacement of atrophic muscle. Focal myelodysplasia and astrocytosis affecting the grey matter was detected in the mid-cervical region of the spinal cord, accompanied by degeneration in the ascending and descending tracts of the remaining cord segments. Abnormal spinal cord development as a result of severe spinal cord compression by the lipomatous masses within the spinal canal leading to replacement of muscle by fat and fibrous tissue was considered to be the cause of the muscular malformation in this fetus.

Congenital lobar emphysema and tension pneumothorax in a dog.

Congenital lobar emphysema (CLE) and tension pneumothorax (TPT) are rarely reported in dogs. A case of CLE of the right middle lung lobe predisposing to air trapping, alveolar hyperinflation, and pleural rupture resulting in fatal spontaneous TPT in a 6-month-old mixed breed dog is described. The unique alteration of "bloat line" was observed in this case in addition to compressive atelectasis of all other lung lobes and lack of negative pressure within the thoracic cavity, signifying markedly elevated intrathoracic pressure. Bronchial cartilage hypoplasia and bronchiectasis were confirmed microscopically, which likely led to abnormal dynamic collapse of bronchi during expiration, consequentially leading to increased intrapulmonary pressure, bullous emphysema, and pleural rupture resulting in TPT. TPT consequent to CLE may therefore be considered one of the potential causes of sudden death in young dogs without overt clinical illness.

Catheter related bacteremia in rats: A preliminary report on the effect of methylene blue coated catheter.

The safety and efficacy of methylene blue (MB) coated indwelling jugular vein/cranial vena cava catheter made up of polyurethane material was tested in a rat model, receiving bacterial culture suspension of Pseudomonas aeruginosa, and Staphylococcus aureus. Daily blood samples were collected from the catheter and peripheral vein for bacterial culture. The clinical parameters (rectal temperature, respiratory rate, total white blood cell count, and loss in body weight) were not different between the groups. All the rats became bacteremic with similar changes in the number of colony forming units in the catheter and peripheral samples. Histopathological lesions were not different between the groups. The findings suggest that rats receiving MB coated catheters behaved similar to non-coated catheters. Based on the results it can be concluded that for this type of gross contamination, catheter coating alone may not eliminate infection/bacteremia.

Porcine circovirus type 2 (PCV2) causes apoptosis in experimentally inoculated BALB/c mice.

BACKGROUND: We have previously described microscopic and electron microscopic alterations in lymphoid organs of PCV2 inoculated mice as apoptosis. In this study we wanted to investigate the molecular pathogenetic mechanism of PCV2-induced apoptosis. Eight-week old BALB/c mice were either sham inoculated (control mice) or inoculated intraperitoneally (ip) and intranasally (in) with a single (sPCV mice) or multiple (mPCV mice) doses of PCV2. Four control mice and 4 sPCV mice were sacrificed 7, 14, 28 and 42 days post inoculation (PI). All 4 mPCV mice were sacrificed 42 days PI. Following necropsy, immunohistochemistry for caspase 3 and in-situ TUNEL assay were performed on sections of spleen, lymph nodes, thymus and ileum from control, sPCV and mPCV mice. In addition, total RNA was extracted from spleens of control, sPCV and mPCV mice for simultaneous detection and semiquantitation of bcl-2 homologues and various caspase mRNAs using a multiprobe RNase protection assay system. RESULTS: PCV2 replicated and was associated with apoptosis in spleens, lymph nodes and Peyer's patches of infected BALB/c mice. Upregulation of caspase 1, 2, 3, 6, 7, 8, 11 and 12 and upregulation for the transcripts of apoptosis inhibitors bcl-2, bcl-w and bcl-X and apoptosis promoters' bax, bak and bad was detected in spleens of sPCV and mPCV mice, but not control mice. Apoptosis was further confirmed by light and electron microscopic morphology as well as by positive TUNEL assay and detection of activated caspase 3. PCV2 nucleic acid was detected by in-situ hybridization in the nuclei and cytoplasm of such apoptotic cells. CONCLUSION: The data presented here support the hypothesis that PCV2 induces apoptosis mediated through the activation of caspases 8 and 3 in the spleens of infected mice.

Sequence analysis of old and new strains of porcine circovirus associated with congenital tremors in pigs and their comparison with strains involved with postweaning multisystemic wasting syndrome.

The entire genomes of 7 isolates of porcine circovirus (PCV) from pigs with congenital tremors (CT), type A2, or postweaning multisystemic wasting syndrome (PMWS) were cloned and sequenced. One isolate (CT-PCV-P7) originated from the late 1960s from a neonatal pig with CT, type A2. Two recent PCV isolates (CT-PCV-P5, CT-PCV-P6) were from 2 affected neonatal pigs, from different farms, with unrelated outbreaks of CT; type A2. Four isolates (PMWS-PCV-P1, PMWS-PCV-P2, PMWS-PCV-P3, PMWS-PCV-P4) originated from pigs with PMWS from 4 different farms. A comparative analysis of these PMWS and PCV isolates demonstrated 99% sequence identity with each other, and over 96% sequence identity with previously sequenced PCV2 isolates. The CT-PCV-P5 and CT-PCV-P6 isolates, however, shared 99% of the same identity with each other, and interestingly also with PMWS PCV isolates. There were no consistent genomic differences between PMWS and recent CT isolates. The CT-PCV-P7 showed 98% identity similarity to PK-15-derived PCV1 and demonstrated only 72% identity similarity to either CT-PCV-P5 or CT-PCV-P6. Phylogenetic analysis confirmed that the old isolate (CT-PCV-P7), and the new isolates (CT-PCV-P5, CT-PCV-P6, PMWS-PCV-P1, PMWS-PCV-P2, PMWS-PCV-P3, PMWS-PCV-P4) were correctly classified as PCV1 and PCV2, respectively.