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Viral vectors serve as promising tools for the development of novel multivalent or multipathogen vaccines. Poliovirus (of the family Picornaviridae) vectors offer the advantages of small genome size, ease of manipulation, inherent stability in the intestinal tract, and induction of potent mucosal immunity through oral administration. Porcine teschovirus (PTV), also belonging to Picornaviridae, generally causes asymptomatic infections in pigs. PTV’s wide tissue tropism suggests that it can act as a potential vector for vaccine development; however, no infectious PTV cDNA clone has been reported yet. In this study, infectious PTV cDNA was cloned and recombinant porcine teschovirus (rPTV) was constructed with a unique XhoI site introduced into 2A, as well as substitution of the G–H loop sequence “RNNQIPQDF” of VP1 by an 8-histidine marker which helps to differentiate it from the parental PTV. Subsequently, the coding sequence of a small fluorescent protein, iLOV, was incorporated into the XhoI site to rescue the recombinant rPTV-iLOV virus, allowing for direct visualization of the viral infection. These rescued viruses were replication-competent and antigenically identical to the parental virus, but showed attenuation due to an impaired self-cleaving function caused by the insertion of iLOV into the 2A protease site, as assessed by a double reporter expressing system. This rescued recombinant virus shows potential for the development of attenuated vaccines with few safety concerns and may serve as an important tool to visually study virus–cell interactions.

Bluetongue is an arthropod-borne disease in domestic and wild ruminants caused by bluetongue virus (BTV), and it leads to great economic loss worldwide. Previous studies showed that BTV in ruminants in Taiwan was often subclinical infection. The aim of this study was to determine the current status (years 2016–2017) of BTV infection in ruminants in Taiwan, to compare it to the results of a large-scale study conducted in the year 2003, and to investigate whether new viral strains exist. Competitive ELISA tests of serum samples for anti-BTV-VP7 group-specific antibody revealed seropositive rates of 26.7% in cattle by head, similar to 32.7% in the year 2003, suggestive of a BTV-vector-host (cattle) dynamic balance. In goats, the seropositive rate was 18.6%, slightly increased from 8.2% in the year 2003, suggestive of a slow but active infection taking place. This notion was supported by the detection of VP1 gene nucleic acid from whole blood in six out of 29 seropositive goats by reverse transcription–polymerase chain reaction. However, no new virus strain was isolated from embryonating chicken embryos (ECEs) inoculation. Alignment of VP7 amino acid sequences revealed that Taiwan and Japan isolates possessed three specific amino acids on sites No. 82 (arginine), No. 328 (aspartate), and No. 336 (glutamine), which are different from many countries. In a three-dimensional model, these amino acids were located closely on the middle lateral surface of VP7 trimers. Since VP7 is a major outer protein engaged in entry into insect cells and a strong T cell response inducer, these differences likely indicate the result of positive selection of local vectors and hosts in Taiwan.

Summary A 16-year-old male Russian blue cat was presented with acute onset of paraparesis of the forelimbs that progressed to tetraparesis. Neurological examination revealed non-ambulatory tetraparesis with decreased postural reactions in all four limbs. Magnetic resonance imaging revealed multifocal nerve root swelling on the right at C6/C7 and C7/T1, while ultrasonography demonstrated swelling of the right brachial plexus. To understand the cause of the nerve swelling, the right musculocutaneous nerve arising from the brachial plexus and the pectoralis muscle were biopsied. Histologically, there was evidence of neurolymphomatosis (neurotropic lymphoma) with Wallerian degeneration and denervation atrophy of myofibres. The neoplastic lymphoid cells expressed CD79a, CD20 and CD56. Based on these findings, a diagnosis of B-cell neurolymphomatosis was made. Expression of CD56, synonymous with neural cell adhesion molecule, is rare in B-cell lymphomas and has not been reported in feline B-cell lymphomas or feline neurolymphomatosis. CD56 expression was suspected to have played an important role in neurotropism of the neoplastic cells in this case.

Bluetongue is an infectious viral hemorrhagic disease of domestic and wild ruminants that has a considerable economic impact on domestic ruminants. There are currently at least 29 serotypes of bluetongue virus (BTV) in the world. Noteworthily, the pathogenesis among BTV serotypes is different, even in the same animal species. In this study, BTV2/KM/2003 and BTV12/PT/2003 were used to investigate the differential immunological effects on bovine peripheral blood mononuclear cells (PBMCs). The BTV viral load and the expression of cytokine messenger RNA (mRNA) in PBMCs were measured by fluorescence-based real-time reverse-transcription PCR (qRT-PCR). The immunofluorescence assay (IFA) was applied to detect BTV signals in monocyte-derived macrophages (MDMs). The SWISS-MODEL and IL-4pred prediction tools were used to predict the interleukin 4 (IL-4)-inducing peptides in BTV-coat protein VP2. Synthetic peptides of VP2 were used to stimulate PBMCs for IL-4-inducing capability. This study demonstrated that the cytokine profiles of BTV-induced PBMCs were significantly different between BTV2/KM/2003 and BTV12/PT/2003. BTV2 preferentially activated the T helper 2 (Th2) pathway, represented by the early induction of IL-4, and likely fed back to inhibit the innate immunity. In contrast, BTV12 preferentially activated the innate immunity, represented by the induction of tumor necrosis factor -&alpha, (TNF-&alpha, ) and interleukin 1 (IL-1), with only minimal subsequent IL-4. The BTV nonstructural protein 3 antibody (anti-BTV-NS3) fluorescent signals demonstrated that monocytes in PBMCs and MDMs were the preferred targets of BTV replication. Bioinformatics analysis revealed that the capability to induce IL-4 was attributed to the tip region of the VP2 protein, wherein a higher number of predicted peptide segments on BTVs were positively correlated with the allergic reaction reported in cattle. Synthetic peptides of BTV2-VP2 induced significant IL-4 within 12&ndash, 24 h post-infection (hpi) in PBMCs, whereas those of BTV12 did not, consistent with the bioinformatics prediction. Bovine PBMCs and synthetic peptides together seem to serve as a good model for pursuing the BTV-induced IL-4 activity that precedes the development of an allergic reaction, although further optimization of the protocol is warranted.

Nephroblastomas are uncommon embryonal tumors in dogs. We report herein a blastema-predominant nephroblastoma with gingival metastasis in an 8-y-old Miniature Pinscher dog. Histologically, the mass was composed mainly of blastemal elements with minor epithelial and mesenchymal differentiation. Metastatic masses in the gingiva had histologic and immunohistochemical features similar to those of the primary renal nephroblastoma. Neoplastic cells were extensively positive for both vimentin and PAX8, and scattered positive for cytokeratin. Using the clinical staging of human Wilms tumor, we staged our case as stage IV with

A 10-year-old male Fox Terrier presented with two descended testes of different sizes. The left testis was normal, while the right one was atrophic. The tunica albuginea (TA) of the right testis was distributed with brown spots of less than 1[Formula: see text]mm in diameter, and was microscopically determined to be ectopic testis which consists of Leydig cell adenoma, primitive seminiferous tubules (STs), intratubular seminoma, and reduplication of arterioles. At the junction of the TA with the spermatic cord, units of testis primordia were present. The STs of the remaining atrophic (not ectopic) right testis were populated not by germ cells but by ciliated pseudostratified cells mimicking those of the epididymis, as supported by the immunohistochemistry results. During the adaptation process, multiple smaller tubules combined into fewer but larger less convoluted tubules. This is a case of concurrent ectopic testis and arterial malformation in the TA with hypoxia, subsequent testicular atrophy, and epididymal hyperplasia in a dog.

In this study, acute myocardial injuries or necrosis were experimentally induced in calves and pigs by ligation of either the left anterior descending coronary artery or left circumflex branch for 30[Formula: see text]min without reperfusion. Various antibodies directed to structural and functional proteins of the sarcomere, as well as activated proteinases, were employed in immunohistochemistry to compare for their potentials to detect early myocardial injury. For comparison, the histological criteria (designated as “Method A”) of cardiomyocyte necrosis such as nuclear pyknosis, sarcoplasmic fragmentation, flocculation, and/or the presence of a contraction band, and inflammatory infiltration were also scored. Additional criteria (designated “Method B”) of changes in late reversible stage of cell injury such as irregular nuclear shape or hyperchromasia, sarcomplasmic hypereosinophilia with either swelling or atrophy in diameter, and perinuclear vacuolation likely of swollen organelles, were also scored. In this setting, telethonin (T-cap), cardiac troponin I (cTnI), the current gold standard, and Method B, were superior to others in detecting ligation-induced ischemic injury. Other markers were either less specific, or less sensitive, or inconclusive for the current application. In conclusion, telethonin may serve as an early immunohistochemical marker in ligation-induced ischemic myocardial injury due to a combination of biomechanical stress, hypoxia, and possibly additional factor as matrix metalloproteinase activation.

Classical swine fever virus (CSFV) shares high structural and antigenic homology with bovine viral diarrhea virus (BVDV) and border disease virus (BDV). Because all three viruses can infect swine and elicit cross-reactive antibodies, it is necessary to differentiate among them with regard to serological diagnosis of classical swine fever. To understand the mechanism of cross-reactivity, it is important to define common or specific epitopes of these viruses. For this purpose, epitope mapping of six monoclonal antibodies (mAbs) was performed using recombinant expressed antigenic domains of CSFV and BDV E2 proteins. One CSFV-specific conformational epitope and one CSFV and BDV common epitope within domain B/C of E2 were identified. Site-directed mutagenesis confirmed that residues G725 and V738/I738 of the CSFV-specific epitope and P709/L709 and E713 of the second epitope are important for mAbs binding. Infection of CSFV in porcine cells was significantly reduced after pre-incubation of the cells with the domain B/C of E2 or after pre-incubation of CSFV with the mAbs detecting domain B/C. 3D structural modeling suggested that both epitopes are exposed on the surface of E2. Based on this, the identified epitopes represent a potential target for virus neutralization and might be involved in the early steps of CSFV infection.

A 22-year-old gelding Thoroughbred horse had been in poor physical condition with a prolapsed penis. On necropsy, numerous white nodules/plaques were found on the lungs, omentum, and multiple lymph nodes, as well as several ulcerations and cauliflower-like growths throughout the glans penis to the internal fold. Histologically, the white nodules/plaques were composed of similar neoplastic squamous cells, which were immunostained positive for cytokeratin (CK), CK 5/6, and P63. The cauliflower-like growths were diagnosed as squamous papilloma, inverted papilloma, and penile squamous cell carcinoma (PSCC). It is speculated that the lung metastasis and peritoneal carcinomatosis in this case are derived from the PSCC. The lesion tissues from the penis and omentum were characterized negative, by polymerase chain reaction, for Equus caballus papillomavirus 2 (EcPV2). This finding suggests that the penile lesions may have been irrelevant to the EcPV2, further implying that multiple pathogeneses exist for the development of an equine PSCC.

A 5-year-old male captive corn snake ( Pantherophis guttatus) with caudal coelomic swelling was admitted for surgical treatment. Laparotomy revealed a 5 × 4 × 2.5 cm, firm, expansile, irregularly shaped mass arising from the middle portion of the right kidney with a mild lobulated pattern and mottled white-to-tan. Microscopically, the mass was composed of numerous bizarre angulated tubules of polygonal neoplastic cells separated by a scirrhous stroma with remarkable heterophilic infiltrates. The neoplastic cells were nonciliated and mucin secreting, with abundant brightly eosinophilic cytoplasm. There were marked cellular and nuclear atypia, frequent cell individualization, and stromal invasion, indicative of malignant behavior, which was confirmed by metastasis to the left kidney 1.5 months postoperatively. Both neoplastic epithelial cells and mesenchymal cells contributing to the scirrhous stroma had variable immunopositivity for pan-cytokeratin. The neoplasm was considered a renal adenocarcinoma resembling human collecting duct carcinoma.

Porcine teschoviruses (PTVs) belong to the genus Teschovirus within the family Picornaviridae. PTVs are universal contaminants in pig herds in endemic and multi-infection statuses. Previous research has demonstrated PTV antigens and nucleic acid in renal glomeruli and tubular epithelia, suggesting the possibility that PTVs might be shed and transmitted via urine. The study aimed to demonstrate, in the context of pathogenesis, the presence of PTVs in the urine of naturally infected pigs. Viral loads of fluid and tissue samples quantified by an established qRT-PCR showed detection rates of 100% by head and in urine, feces, plasma and nasal swabs, and 38% in kidney. As predicted, PTVs were present in urine at 10(4.02 ± 1.45) copies/100 μl volume, equivalent to 17% of that in plasma. No significant differences were observed between healthy and culled pigs or among the 7 sampled herds. The presence of PTVs in urine was further substantiated by molecular serotyping. In particular, PTV-10 was identified in the urine of 3 piglets from 3 separate herds, consistent with the most prevalent serotype found in this study, and in plasma. The urine mixes with feces to form slurry making it easier for PTV to spread and contaminate the environment.

Bluetongue is a fatal viral disease in ruminants and has serious economic impacts on the livestock industry. Interactions between bluetongue virus (BTV) and immune cells are interesting because of the unique scenarios in each combination of animal species/breed and viral virulence/serotype. This study investigated the immune response in bovine peripheral blood mononuclear cells (PBMC) infected by the BTV2 Taiwan strain. The replication of the virus was limited in monocytes and monocyte-derived macrophages (MDM), and lymphocytes were less permissive. The cytokine mRNA of IL-4 in PBMC was expressed earlier and in greater quantities than that of innate immunity (TNFα, IL-1β) and cell mediated immunity (CMI) (IFNγ), and the IL-4 protein was stably present in the culture medium until 72 h post-infection (hpi). Even in MDM reconstituted with autologous lymphocyte (MDM-Lymphocyte), the IL-4 still had high mRNA expression level. The level of IgE antibody also increased at 24-72 hpi, suggestive of the engagement of type I hypersensitivity in the pathogenesis. The anti-viral activity contained in the culture supernatant was transferrable to recipient infected PBMC from other cows. However, in infected MDM largely free of lymphocytes, mRNA expressions of IL-1β, TNFα and IL-12p40 were normally expressed from 6 to 48 hpi, supporting the notion that IL-4 elaborated by lymphocytes in PBMC mediated the inhibition of both innate immunity and CMI to BTV2. The sum of responses subsequent to the early IL-4 expression likely constitutes part of the unique scenario in the current BTV2-Cow experimental combination biased toward Th2 response.

In Taiwan, the prevalent CSFV population has shifted from the historical genotype 3.4 (94.4 strain) to the newly invading genotype 2.1 (TD/96 strain) since 1996. This study analyzed the competition between these two virus genotypes in dual infection pigs with equal and different virus populations and with maternally derived neutralizing antibodies induced by a third genotype of modified live vaccine (MLV), to simulate that occurring in natural situations in the field. Experimentally, under various dual infection conditions, with or without the presence of maternal antibodies, with various specimens from blood, oral and fecal swabs, and internal organs at various time points, the TD/96 had consistently 1.51&minus, 3.08 log higher loads than those of 94.4. A second passage of competition in the same animals further widened the lead of TD/96 as indicated by viral loads. The maternally derived antibodies provided partial protection to both wild type CSFVs and was correlated with lower clinical scores, febrile reaction, and animal mortality. In the presence of maternal antibodies, pigs could be infected by both wild type CSFVs, with TD/96 dominating. These findings partially explain the CSFV shift observed, furthering our understanding of CSFV pathogenesis in the field, and are helpful for the control of CSF.

Plasmacytoid and rhabdoid variants of urothelial carcinomas (UCs) of the urinary bladder have been described in humans with plasma cell–like or rhabdoid cellular appearance and aggressive clinical outcome. Canine UC of the bladder is generally classified as papillary/nonpapillary and infiltrating/noninfiltrating with limited information regarding other histological patterns. We report 3 cases of UC of the urinary bladder showing a unique discohesive cellular morphology with malignant behavior resembling the human plasmacytoid and rhabdoid variants of UC, which may raise some difficulties in diagnosis. Epithelial-mesenchymal transition and reduced E-cadherin expression were revealed by immunohistochemistry in 2 cases, possibly explaining the discohesive and invasive behavior of the tumor cells. The findings broaden the morphological spectrum as well as the distinct clinical features of canine UC of the urinary bladder.

Supplemental material, DS1_JDVI_10.1177_1040638718762560 for A blastema-predominant canine renal nephroblastoma with gingival metastasis: case report and literature review by Bo Chen, Wen-Ta Li and Fun-In Wang in Journal of Veterinary Diagnostic Investigation

Bluetongue (BT), an arthropod-borne viral disease, is caused by the bluetongue virus (BTV), belonging to the genus Orbivirus of the family Reoviridae. Most species of ruminants are susceptible to BTV, but most infections go subclinical. These 'reservoir hosts' may potentially further increase the viral transmission and expansion of the disease; thus, detection of subclinical infection is important. To detect the BTV, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed using primers targeted to six regions of the segment 5 (NS1) gene of the BTV2/KM/2003. The assay was completed in 1 h at a temperature 65°C, and the products were specifically digested with MboII enzyme presented in the target region. The in vitro sensitivity of the RT-LAMP was 100 copies, characterized by a qRT-PCR. The RT-LAMP did not cross-react with four tested common ruminant infectious agents, namely foot and mouth disease virus, goat pox virus, bovine herpesvirus 1, and Clostridium perfrigens. The RT-LAMP was applied to whole blood samples from 15 clinically healthy dairy cattle, and was able to detect BTV from 3/15 animals, and in particular 1 of the 3 animals was seronegative by cELISA. Positive RT-LAMP samples were reproducible. This RT-LAMP provides a simple, efficient, and sensitive method to specifically detect BTV and is suitable for the screening of field samples with a potential to pick up subclinical infection. The alignments of the outer primer region indicated matches of > 85% with 18 out of 26 BTV serotypes, implying its potential as universal primers.

Porcine reproductive and respiratory syndrome virus (PRRSV) was first identified in Taiwan in 1991, but the genetic diversity and evolution of PRRSV has not been thoroughly investigated over the past 20 years. The aim of this study was to bridge the gap in understanding of its molecular epidemiology. A total of 31 PRRSV strains were collected and sequenced. The sequences were aligned using the MUSCLE program, and phylogenetic analysis were performed by the maximum-likelihood method and the neighbor-joining method using MEGA 5.2 software. In the early 1990s, two prototype strains, WSV and MD001 of the North American genotype, were first identified. Over the years, both viruses evolved separately. The population dynamics of PRRSV revealed that the strains of the MD001 group were predominant in Taiwan. Evolution was manifested in changes in the nsp2 and ORF5 genes. In addition, a suspected newly invading exotic strain was recovered in 2013, suggesting that international spread is still taking place and that it is affecting the population dynamics. Overall, the results provide an important basis for vaccine development for the control and prevention of PRRS.

A 11-year-old female Koala showed anorexia, lethargy and weakness. Hematology, blood chemistry and urinalysis were non-specific. Computed tomography (CT) revealed ascites and masses within the abdomen, and the volumes of some masses increased 20% during a 10-week period. At necropsy, there was 115 mL of clear ascites, and visceral lesions were located mainly in the spleen and liver. The spleen was diffusely enlarged by infiltrative white firm neoplastic tissue with irregularly protuberant nodules, of 0.2 to 5 cm, that had totally effaced the parenchyma. The liver contained multiple random scattered softer nodules, of 0.2 to 2.0 cm, some of which were protruding. One nodule of < 1 cm was noted on the mesentery. Histologically, the neoplasm was composed of a low cellularity of elongated fusiform cells distributed amongst an abundant collagenous matrix. Tumor cells were stained positive intracytoplasmically for vimentin and negative for α-smooth muscle actin. This fibrosarcoma (FS) likely originated from the spleen, with secondary metastasis to the liver, from which via transcoelomic implantation spread to the diaphragm and mesentery. This is considered a rare report of primary splenic FS in a captive koala.

In industrial swine production, several important viral diseases are frequently complicated with bacterial infections, suggestive of virus–bacteria synergism. Although most virus-induced leukocyte dysfunctions are attributed to monocytes and lymphocytes, the role of polymorphonuclear cells (PMNs, mainly neutrophils) should not be excluded, because they are the first leukocytes to respond to inflammation. Previous studies indicated that pseudorabies virus (PrV) blocked the indirect recruitment of PMNs by inhibiting the production of interleukin-8 by primed peripheral blood mononuclear cells (PBMCs) and also reduced the phorbol 12-myristate 13-diacetate (PMA)-induced oxidative burst (OB) in PMNs. This study used PrV as a model to explore other possibilities of virus-induced PMN dysfunctions in pigs. The goals were: (1) to develop assay for simultaneous evaluation of phagocytosis and OB to Actinobacillus pleuropneumoniae (APP) and (2) to evaluate whether PrV could affect these responses to APP in an ex vivo setting. Approximately 12% of the PMNs underwent phagocytosis, as indicated by the presence of intracellular propidium iodide(PI)-labeled APP (PI-APP), and 32% of the PMNs launched OB to APP, as detected by dihydrorhodamine (DHR). Approximately 86% of those OB responsive cells did not contain engulfed PI-APP, and the OB was presumably induced by the bacteria lipopolysaccharide (LPS). Only 5 ± 1% of the cells underwent simultaneous phagocytosis and OB. Under certain conditions, PrV reduced the non-phagocytosis-associated OB by approximately 40% (p < 0.05), while the phagocytosis was unaffected overall. These results suggest that PrV may interfere with bacteria LPS-mediated signaling, suggesting a possibility of PrV-APP synergism in causing pneumonia.

A mass mortality event of captive Hong Kong warty newts Paramesotriton hongkongensis with non-granulomatous necrotic lesions occurred in Taipei Zoo, Taiwan, in 2014. Clinically, the sick newts were lethargic and often covered with water mold Saprolegnia sp. on the skin of the body trunk or extremities. Predominant pathological findings were multifocal non-granulomatous necrotic lesions in the liver, spleen, and kidneys and severe skin infection with Saprolegnia sp., with deep invasion and involvement of underlying muscles. The possibility of ranavirus infection was ruled out by negative PCR results. Unexpectedly, abundant intralesional acid-fast positive bacilli were found in the necrotic lesions of the liver, spleen, and kidney in all 14 sick newts. PCR targeting the hsp65, ITS region, and partial 16S rRNA genes was performed, and the sequence identity from amplified amplicons of hsp65 and partial 16S rRNA genes was 100% identical to that of the corresponding gene fragment of Mycobacterium marinum. Further molecular investigations demonstrated that the current M. marinum was a mycolactone-producing mycobacterium with the presence of esxA/esxB genes. Mycolactone is a plasmid-encoded, immunosuppressive, and cytotoxic toxin. The possible immunosuppression phenomenon characterized by systemic non-granulomatous necrotic lesions caused by M. marinum and the unusual deep invasive infection caused by water mold might be associated with the immunosuppressive effect of mycolactone. Therefore, it should be noted that non-granulomatous necrotic lesions in amphibians can be caused not only by ranavirus infection but also by mycobacteriosis.

Classical swine fever (CSF) is an acute viral disease that has high contagion and high mortality in domestic pigs and wild boars. In Taiwan, classical swine fever virus (CSFV) of subgroup 3.4 (Native strain; 94.4/IL/94/TWN) was almost 100% isolated before 1993. In 1994, Subgroup 2.1 (Exotic strain; TD/96/TWN) was first detected, and since a severe outbreak in 1995, it has dominated the field. There is a concern that the Lapinized Philippines Coronel (LPC) of genotype 1.1, widely used in Taiwan, may not completely protect animals from the currently prevalent genotype 2.1 CSFV in the field. In this study, pigs were immunized by LPC vaccine and E2 glycoprotein expressed from cloned genes of LPC, TD/96, and 94.4 viruses, respectively before challenge with genotypically homologous viruses. The viral distributions and intensity after challenge were analyzed by using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). Both LPC vaccine and E2 glycoproteins provided significant protection potency from CSFV. However, the LPC vaccine antigen was noted, although to a significantly milder degree (p < 0.05) than in the positive control, in lymphoid follicles of the lymph node and in various organs after the vaccinated animals were challenged with virulent ALD strain. Tissues from all experimental groups were tested negative by RT-PCR. In conclusion, the LPC attenuated virus and E2 glycoproteins offer protection against their genotypically homologous viruses. A change of vaccine seed to genotype 2.1 may be an option to combat the currently prevalent genotype 2.1 CSFVs in the field.

This study characterized how porcine teschovirus (PTV) contributed to causing nonsuppurative encephalitis in the endemic field situation which was mostly subacute to chronic. Tissues from 57 encephalitic pigs, in the years 2005–2008, were randomly selected for detection of PTV antigens by immunohistochemistry. In only 3/57 (5.3%) pigs PTV signal was detected in brain cells of noninflammatory sites. On the other hand, PTV signals were present at significant rates in visceral and lymphoid organs. Spleen (33.3%), lung (24.6%) and kidney (14%) were among the higher, while those of lymphoid organs were 17.5%–26.3%, and intestines were 21.1%–24.6%. Nursery pigs not only comprised a bulk of the samples (n = 26), but also had a higher signal detection rate of 53.8% (by head) and average numbers of 2.65 positive organs per animal. Cellular tropism and antigen localization of PTVs are closely related to viremia, fecal and urinal sheddings, reinfection and recirculation of PTV within the herd.

Porcine teschoviruses (PTVs) belong to the genus Teschovirus within the family Picornaviridae. PTVs are universal contaminants in pig herds in endemic and multi-infection status. To further the understanding of PTV pathogenesis in endemically infected pigs, a set of samples was studied by real time reverse transcription PCR (qRT-PCR) to quantitate viral loads in tissues and by in situ hybridization (ISH) to locate PTV signals in target cells, both targeting the 5'-NTR. cRNA of PTV-1 and PTV-7, in vitro transcribed from cloned fragments of 5'-NTR of 2 viruses, was used to construct standard curves and to run parallel in qRT-PCR, which had detection limits of 10(1) copies/per reaction, with a linearity in between 10(1) and 10(7) copies/per reaction and correlation coefficients of 0.997-0.9988. The qRT-PCR specifically amplified RNA from PTV-1 to -11, while excluding those of Sapelovirus, PEV-9 and PEV-10. Inguinal lymph node (LN) had the highest viral load of all (assuming 100%), followed by ileac LN (89-91%), tonsil (66-68%), ileum (59-60%), spleen (38-40%), and kidney (30-31%), with the least in brain (22.9%) of the inguinal LN. The 22.9% load in brain was higher than that anticipated from a simple fecal-oral-viremia operative model. The results suggested in addition that intranasal infection and retrograding axonal infection from the tonsils were equally operative and significant. ISH revealed PTV signals in a wider variety of tissue cell types than before. PTV signals were noted most impressively in neurons of the cerebral cortex and hippocampus and in the dark zone of the germinal center and adjacent paracortex of regional LN. Multiple operative models indicated that PTVs seemed to have no difficulty invading the brain. The key to whether encephalitis would ensue resided in the animal's immune status and topographic differences of neurons' susceptibilities to PTVs. When common co-infected agents are present, as is typical in the field, PTVs may synergize in causing diseases.

Porcine teschoviruses (PTVs) belong to the genus Teschovirus within the family Picornaviridae. Hitherto, PTVs have had 13 serotypes associated with a variety of clinical diseases. The virulent PTV-1 strains were associated with highly fatal, nonsuppurative encephalomyelitis of pigs (Teschen disease) in the 1930-1950s. Today, less virulent Talfan strains of PTV-1 are more widespread, and PTVs have contaminated swine herds worldwide (endemic or enzootic) together with a variety of common swine pathogens (multi-infection status). The aim of this study was to investigate the extent to which PTVs play a role in causing diseases in the field, under the endemic and multi-infection situation, when most pigs in the herds are infected and immune. Based on the fecal-oral model of pathogenesis, a set of 15 organs were collected from 30 culled post-weanling piglets of 4-8 weeks old. For nested RT-PCR targeted on the 5'-NTR, the PTV detection rate was 96.7% (by heads), confirming the endemic status, and infection was most commonly detected in the intestines (averaged 61%) and lymphoid organs (averaged 59%), followed by visceral organs (averaged 37%) and the CNS (different parts varied from 17 to 47%). The correlation of PTVs detected by nested RT-PCR and a histological lesion were analyzed by Chi-square test showing that in the field situation only non-suppurative encephalitis in the caudal part of the brain (P=0.054) may be marginal significantly attributed to infection by PTVs. By genotyping based on partial VP1 sequences, 5 serotypes, namely PTV-1, -4, -6, -7, and -11, were identified, with some animals having two serotypes co-existed in different organs.

An ~21-year-old female Barbary sheep ( Ammotragus lervia) died spontaneously following a lengthy episode of difficulty in walking. An ~6 × 3 × 3 cm, unilocular cystic growth was found in the cranioventral thorax. The fibrotic cystic wall, lined by a single layer of flattened to cuboidal epithelial cells, was invaginated and partially encircled solid masses of fusiform neoplastic cells with multiple intratumoral cystic structures. The fusiform neoplastic cells were intensely positive for cytokeratin (CK) and partially positive for α–smooth muscle actin and vimentin, but negative for thyroid transcription factor–1 (TTF-1) and neuron-specific enolase (NSE). The intratumoral cysts were lined by CK-positive but TTF-1– negative, NSE-negative, flattened, cuboidal to columnar epithelial cells, suggestive of cystically dilated medullary duct epithelium-derived structures. Based on the location and histopathologic findings of the growth, concurrent spindle-cell thymoma and thymic cysts was diagnosed. We also discuss the correlation between thymic cysts and thymoma and review the literature of thymomas in ovine and wildlife species.

Classical swine fever (CSF), an economically important and highly contagious disease of pigs, is caused by classical swine fever virus (CSFV). In Taiwan, CSFVs from field outbreaks belong to two distinct genotypes. The historical genotype 3.4 dominated from the 1920s to 1996, and since 1996, the newly invading genotype 2.1 has dominated. To explain the phenomenon of this virus shift in the field, representative viruses belonging to genotypes 2.1 and 3.4 were either inoculated alone (single infection) or co-inoculated (co-infection), both in vivo and in vitro, to compare the virus replication and pathogenesis. In pigs co-infected with the genotype 2.1 TD/96/TWN strain and the genotype 3.4 94.4/IL/94/TWN strain, the newly invading genotype 2.1 was detected earlier in the blood, oral fluid, and feces, and the viral loads were consistently and significantly higher than that of the historical genotype 3.4. In cell cultures, the ratio of secreted virus to cell-associated virus of the genotype 2.1 strain was higher than that of the genotype 3.4 strain. This study is the first to demonstrate a possible explanation of virus shift in the field, wherein the newly invading genotype 2.1 replicates more efficiently than did genotype 3.4 and outcompetes the replication and pathogenicity of genotype 3.4 in pigs in the field.

The characteristics of 163 spontaneous neoplasms diagnosed in 150 necropsied zoo mammals, birds, and reptiles at Taipei Zoo during 1994-2003 were analyzed. Histopathology and immunohistochemistry were employed to classify the tumor types. A total of 2657 necropsied zoo animals, including 1335 mammals, 873 birds and 449 reptiles led to the diagnosis of tumor in 8.1% (108/1335), 4.2% (37/873) and 1.1% (5/449) of cases, respectively. The most predominant type of tumors in mammals was mammary gland tumors (12.0%, 13/108), followed by uterine smooth muscle tumors (10.2%, 11/108), lymphosarcoma (9.3%, 10/108), hepatocellular carcinoma (8.3%, 9/108), and cutaneous squamous cell carcinoma (6.5%, 7/108). The avian neoplasm with the highest incidence was lymphosarcoma (35.1%, 13/37). Five individual neoplasms were found in different reptile species. The overall incidence of malignant tumors (63.8%, 104/163) was greater than that of benign tumors (36.2%, 59/163). Immunohistochemistry characterization of these tumors revealed a histogenesis which is similar to that seen in domestic animals and humans.

BTV2/KM/2003 and BTV12/PT/2003 are the first identified bluetongue viruses in Taiwan. The prototype virus BTV2/KM/2003 was previously characterized in various respects as low virulent. In the present study, nucleotide sequences of the ten genome segments and their coding regions of the Taiwan strains were determined and analyzed. The two strains had >96.8% nucleotide and >97.9% deduced amino acid identities to each other, except for the VP2 genes. Their genome sequences, except for NS1 and VP2 genes, clustered overall in the Asian lineage, and were closely related to strains from China, India, Indonesia, and Japan. The phylogenetic trees and nucleotide identities of six BTV genes were suggestive of the geographical origin of the bluetongue virus strains analyzed, with a few exceptions. To examine which genes better distinguished strains from different origins (topography), the distribution of and the levels of differences in nucleotide identities were analyzed, revealing that VP3, NS2, and NS3 genes were more suitable for topotyping of BTVs. Analysis of ratios of non-synonymous/synonymous substitutions (dN/dS values) between putative ancestry and their descendant strains suggested that most BTV genes evolved under a negative selection, whereas the VP7 gene evolved under positive selection, and its non-synonymous substitutions accumulated more rapidly in strains from the Mediterranean region.

Bluetongue is an arthropod-borne viral disease affecting domestic and wild ruminants. Taiwan, with the Tropic of Cancer crossing through it, was considered free of bluetongue virus (BTV) before 2001. The goals of this study are to identify the serotype and phylogeny of Taiwan BTV isolates and to understand the serological status and chronology of BTV infection. Analysis of the S10 gene segment revealed that Taiwan BTV isolates are closely related to Chinese strains. Seropositive results were found in 32.7% of the cattle and 8.2% of the goats by head, and 90.7% of the cattle herds and 28.9% of the goat flocks. Anti-BTV antibodies have existed in goat sera since 1989 and in bovine sera since 1993, and over the years, the seropositive rates in rapidly urbanized districts have decreased, most likely due to the loss of vector habitats. Seropositive rates for sheep were variable, due to a small sample size and a small sheep population. Thus far, all natural BTV infections have been subclinical, consistent with experimental sheep inoculation, revealing that the Taiwan isolate is of low virulence.

Taiwan had been declared rabies-free in humans and domestic animals for five decades until July 2013, when surprisingly, three Formosan ferret badgers (FB) were diagnosed with rabies. Since then, a variety of wild carnivores and other wildlife species have been found dead, neurologically ill, or exhibiting aggressive behaviors around the island. To determine the affected animal species, geographic areas, and environments, animal bodies were examined for rabies by direct fluorescent antibody test (FAT). The viral genomes from the brains of selected rabid animals were sequenced for the phylogeny of rabies viruses (RABV). Out of a total of 1016 wild carnivores, 276/831 (33.2%) Formosan FBs were FAT positive, with occasional biting incidents in 1 dog and suspected spillover in 1 house shrew. All other animals tested, including dogs, cats, bats, mice, house shrews, and squirrels, were rabies-negative. The rabies was badger-associated and confined to nine counties/cities in sylvatic environments. Phylogeny of nucleoprotein and glycoprotein genes from 59 Formosan FB-associated RABV revealed them to be clustered in two distinct groups, TWI and TWII, consistent with the geographic segregation into western and eastern Taiwan provided by the Central Mountain Range and into northern rabies-free and central-southern rabies-affected regions by a river bisecting western Taiwan. The unique features of geographic and genetic segregation, sylvatic enzooticity, and FB-association of RABV suggest a logical strategy for the control of rabies in this nation.

A 9-year-old, female Mongrel dog was presented for posterior hindlimb weakness, inability to stand, and pain in the lumbosacral and pelvic regions. Radiography revealed a lytic lesion extending from L5 to L6 to the ilium. At necropsy, an 8 − 2 to 3.2 − 3 cm, irregular, white, firm mass was identified extending from the left dorsolateral aspect of the L6 vertebrae to the sacrum, crossing the sacroiliac joint to the ilium, and reaching the acetabulum without affecting the joint cartilage. Tumor masses were also present bilaterally near the costochondral junction of several ribs. White, soft nodules were present in the heart, pericardium, lungs, spleen, and kidneys as well. Histologically, osteolysis with disruption of the cortical bone and reactive bone with the presence of multinucleated osteoclasts was noted. Neoplastic cells consisted of variable, small basophilic round cells (SBRC) with very scant cytoplasm, larger polygonal cells with abundant eosinophilic cytoplasm, and vacuolated cells resembling adipocytes. Within the marrow cavity, vacuolated cells with necrosis predominated, whereas in periosteal areas, polygonal and vacuolated cells that were mixed with a lower percentage of SBRC were more common. In the lungs and heart, SBRC predominated, and in the spleen, polygonal cells were more numerous. Tumor cells stained positive for vimentin and S-100 and stained negative for CD99, neuron-specific enolase, synaptophysin, chromogranin A, cytokeratins, desmin, myoglobin, and actin. This tumor most likely arose from the marrow cavity of the L6 and later invaded through the vertebral body into adjacent vertebrae and various visceral sites.

Bovine ephemeral fever (BEF), a vector-borne disease of cattle, is caused by the Ephemerovirus of the family Rhabdoviridae. In the past 40 years, Taiwan has had seven BEF epizootics, and we have previously reported the first five. This study summarizes the 2001 and 2002 epizootics; conducted case-control serologic studies on 10 herds involved in the 2001 epizootic; determined whether the recent BEF viruses have varied significantly; and discusses the relationship between epizootic patterns and possible variant BEF viruses. For mature cows that had received at least 2 doses of vaccine before the study, a negative correlation between the prevaccinated (the 3rd dose and after) serum neutralization antibody (SNA) titers and their postvaccinated peak rates was found. When prevaccinated SNA levels were ator = 32, their postvaccinated SNA levels increased significantly faster (P0.01) than for those ator = 32. The glycoprotein gene of isolates from 1999, 2001, and 2002 had a 99.2-99.9% homology, without consistent amino acid variations in the neutralization sites. Phylogenetic analysis of Taiwanese isolates revealed 2 distinct clusters, the 1983-1989 and 1996-2002 isolates. Cross-neutralization tests confirmed the glycoprotein gene sequence analysis results. In conclusion, annual boosters at SNA levels32, at more than 2 doses, or at intervals shorter than 6 months are not advisable. The occurrence of frequent small epizootics implies the dominance of BEF virus over host immunity, but not a variant virus.

Industrial swine production is affected by several serious viral diseases, such as pseudorabies, hog cholera, porcine reproductive and respiratory syndrome, which are frequently complicated with the increased incidence of bacterial complications such as Actinobacillus pleuropneumoniae (APP). This clinical observation is suggestive of a virus-bacteria synergism on the pathogenesis. One hypothesis is that viruses induce polymorphonuclear cell (PMNs, primarily neutrophils) dysfunction resulting in defective antibacterial resistance. The purpose of this study was to use the pseudorabies virus (PrV) as a model to explore the possibility of virus-induced PMN dysfunctions in pigs. The goals were to evaluate, in ex vivo settings, the oxidative burst (OB) function of pig PMNs, and to evaluate whether PrV could affect these responses to APP. We found that PrV served as a mild OB stimulant (2-fold) to pig PMNs, which also launched a significant burst to phorbol 12-myristate 13-diacetate (PMA; 61-fold), to non-opsonized, heat-killed and formaldehyde-fixed APP (8-fold), and to normal pig serum-opsonized APP (34-fold). Interestingly, the PMA-induced OB could be reduced 50-70% by preincubating PMNs with PrV, and the critical target was not likely the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase itself. Inactivated PrV was as efficient as viable PrV at exerting the inhibitory effect. On the other hand, PrV exerted a primarily additive effect on APP-induced OB, when the cytotoxic effect of APP on PMNs was avoided. The current finding suggests the possibility that activated PMNs are susceptible to PrV-induced dysfunction, and that the PrV-APP synergism may require upstream stimuli of PMNs to be initiated.

The aim of this study was to determine the chronic toxicity of a mixture of chlorinated alkanes and alkenes (CA) consisting of chloroform, 1,1-dichloroethane, 1,1-dichloroethylene, 1,1,1-trichloroethane, trichloroethylene, and tetrachloroethylene. These chlorinated organic solvents were present in the underground water near an electronic appliances manufactory in Taoyuan, Taiwan. Male and female weanling ICR mice were treated with low-, medium-, and high-dose CA mixtures in drinking water for 16 and 18 mo, respectively. A significant number of male mice treated with the high-dose CA mixture developed tail alopecia and deformation, which was not prominent in CA-treated female mice. Medium- and high-dose CA mixtures induced marginal increases of liver and lung weights, blood urea nitrogen, and serum creatinine levels in male mice. In female mice, the high-dose CA mixture increased liver, kidney, and uterus and ovary total weights, without affecting serum biochemistry parameters. CA mixtures had no effects on the total glutathione content or the level of glutathione S-transferase activity in the livers and kid- neys of male and female mice. Treatments with CA mixtures produced a trend of increasing frequency of hepatocelluar neoplasms in male mice, compared to male and female controls and CA-treated female mice. The high-dose CA mixture induced a significantly higher incidence of mammary adenocarcinoma in female mice. The calculated odds ratios of mammary adenocarcinoma in female mice induced by low-, medium-, and high-dose CA mixtures were 1.14, 1.37, and 3.53 times that of the controls, respectively. The low-dose CA mixture induced a higher incidence of cysts and inflammation in and around the ovaries. This study has demonstrated that the CA mixture is a potential carcinogen to male and female mice. These animal toxicology data may be important in assessing the health effects of individuals exposed to the CA mixture.

A 14-year-old male Chapman's zebra had been treated for a cervical granulomatous cellulitis for 3 months prior to sudden death associated with myocardial fibrosis. Incidental findings at necropsy included multiple 3–8-cm unilocular cysts in the liver and lungs. Cysts contained either a clear fluid with or without hydatid sand or a gelatinous substance with foci of mineralization. Light and scanning electron microscopic examination of the hydatid sand revealed free protoscolices as well as intact and ruptured brood capsules with protoscolices attached to the germinal membrane. The protoscolices had 2 rows of 36–38 rostellar hooks with a length of 25–30 μm. The cyst wall consisted of inner germinal, intermediate laminated, and outer adventitial layers. Hydatidosis caused by Echinococcus granulosus was diagnosed based on the unilocular cysts, multiple protoscolices formed in a brood capsule, typical trilayered cyst wall, and herbivorous intermediate host. This is the first reported case of animal hydatidosis in Taiwan. The infection is thought to have been established 12 years ago in South Africa prior to importation.

Swine neutrophils were quantitatively examined for the direct and indirect migratory responses to Actinobacillus pleuropneumoniae (APP) in vitro and the effects of pseudorabies virus (PrV), frequently co-infecting with APP, were also observed. About 30% of swine neutrophils responded to viable APP, while 3.2% of the neutrophils responded to 0.1% casein which served as the control. The migration of APP was not affected by preincubation of neutrophils with PrV, which inhibited the random migration. When the random migration was normalized to 1, the chemotactic indices for APP, opsonized-APP and casein were 64, 70 and 8.5, respectively. Heat-killed APP or E. coli lipopolysaccharide stimulated the production of interleukin-8 activity by adherent peripheral blood mononuclear cells (PBMC). Preincubation of PBMC with PrV inhibited the production of neutrophil attractant activity when stimulated with heat-killed APP. The results suggested that the direct chemotaxis of neutrophils to viable APP might contribute to early infiltration in Actinobacillus pleuropneumonia, and that PrV might inhibit indirect recruitment of neutrophils to infected lungs by compromising the functions of PBMC.