Amount of skin shrinkage affecting tumor versus grossly normal marginal skin of dogs for cutaneous mast cell tumors excised with curative intent.

OBJECTIVE To assess differences in skin shrinkage between grossly visible tumor and grossly normal marginal skin of dogs for cutaneous mast cell tumors (MCTs) excised with curative intent and to determine an equation to estimate postexcisional gross tumor margins from preexcisional measurements and vice versa. SAMPLE 19 cytologically confirmed and surgically excised cutaneous MCTs obtained from dogs. PROCEDURES Tumors were measured in craniocaudal and dorsoventral directions before excision, immediately after excision, and after fixation in formalin. Both grossly visible tumor and surrounding grossly normal skin that comprised the surgical margin were measured at each time point. Percentage of shrinkage was compared among time points and between the tumor and surrounding grossly normal skin. Patient and histopathologic variables were correlated to skin shrinkage. RESULTS Overall shrinkage was 17.70%. The amount of shrinkage within the grossly visible tumor (4.45%) was less than that within the surrounding grossly normal skin (24.42%). Most of the shrinkage occurred immediately after excision. There was no effect of age, sex, completeness of excision, or degree of edema. Accuracy of an equation to estimate postexcisional margins from preexcisional measurements was only 18.4%. CONCLUSIONS AND CLINICAL RELEVANCE Grossly evident MCTs of dogs shrunk less than did the grossly normal surrounding skin. Although an equation to estimate postexcisional margins from preexcisional measurements could be derived, it likely would need to contain additional variables not included in the study reported here. Until such an equation exists, care must be used when extrapolating surgical margins from histologic margins and vice versa.

The impact of finasteride and dutasteride treatments on proliferation, apoptosis, androgen receptor, 5α-reductase 1 and 5α-reductase 2 in TRAMP mouse prostates.

BACKGROUND: Previously, we studied the effect of finasteride- or dutasteride-containing diets in male C57BL/6 TRAMP x FVB mice. Pre (6 weeks of age) and post (12 weeks of age) groups received finasteride or dutasteride to determine the efficacy of these pharmaceuticals on prostate cancer (PCa) development in male C57BL/6 TRAMP x FVB mice. Post-Dutasteride treatment was more effective than Pre-Dutasteride treatment, and dutasteride treatments were more effective than finasteride treatments in decreasing prostatic intraepithelial neoplasia (PIN) progression and PCa development. Finasteride and Pre-Dutasteride treatments significantly decreased high-grade PIN incidence, but increased poorly differentiated PCa incidence. In this study, molecular changes in prostates of these mice were characterized in an effort to elucidate the discordant response in Pre-Dutasteride and finasteride groups, and determine why Post-Dutasteride treatment was more effective. METHOD/PRINCIPAL FINDINGS: Ki-67 (proliferation marker) and androgen receptor (AR) protein, apoptotic DNA fragmentation (TUNEL assay), 5α-reductase 1 (5αR1) and 5α-reductase 2 (5αR2) mRNA were quantified in male TRAMP mice prostate tissues with genitourinary weight < 1 and > 1 gram. Overall, proliferation and AR were decreased and apoptosis was increased in most tumors versus prostate epithelium and hyperplasia. Proliferation and AR were increased notably in hyperplasia versus prostate epithelium and tumor. There were no clear trends or differences in 5α-reductase 1 and 5α-reductase 2 levels between large and small tumors. The discordant response in Pre-Finasteride and Pre-Dutasteride groups may be due to upregulated 5αR1 levels in large versus small tumors. It is not clear what the mechanism is for the different response in the Post-Finasteride group. Post-Dutasteride treatment was more effective than Pre-Dutasteride treatment in decreasing 5αR1 in large tumors. Therefore, this may be why this treatment was more effective in decreasing PIN progression and PCa development. CONCLUSION: The effect of finasteride and dutasteride on these biomarkers did not clearly elucidate their mechanism of action, but tumor 5αR1 levels were significantly positively correlated with adjusted prostate severe lesion score.

5α-reductase 1 mRNA levels are positively correlated with TRAMP mouse prostate most severe lesion scores.

BACKGROUND: The contribution of 5α-reductase 1 and 5α-reductase 2 to prostate cancer development and progression is not clearly understood. TRAMP mice are a common prostate cancer model, in which 5α-reductase 1 and 5α-reductase 2 expression levels, along with prostate lesions scores, have not been investigated at different time points to further understand prostate carcinogenesis. METHOD/PRINCIPAL FINDINGS: To this end, 8-, 12-, 16-, and 20-week-old male C57BL/6TRAMP x FVB mice prostate most severe and most common lesion scores, 5α-reductase 1 and 5α-reductase 2 in situ hybridization expression, and Ki-67, androgen receptor, and apoptosis immunohistochemistry levels were measured. Levels of these markers were quantified in prostate epithelium, hyperplasia, and tumors sections. Mice developed low- to high-grade prostatic intraepithelial neoplasia at 8 weeks as the most severe and most common lesions, and moderate- and high-grade prostatic intraepithelial neoplasia at 12 and 16 weeks as the most severe lesion in all lobes. Moderately differentiated adenocarcinoma was observed at 20 weeks in all lobes. Poorly differentiated carcinoma was not observed in any lobe until 12-weeks-old. 5α-reductase 1 and 5α-reductase 2 were not significantly decreased in tumors compared to prostate epithelium and hyperplasia in all groups, while proliferation, apoptosis, and androgen receptor were either notably or significantly decreased in tumors compared with prostate epithelium and hyperplasia in most or all groups. Prostate 5αR1 levels were positively correlated with adjusted prostate most severe lesion scores. CONCLUSION: Downregulation of androgen receptor and 5α-reductase 2, along with upregulation of 5α-reductase 1 in tumors may promote prostatic intraepithelial neoplasia and prostate cancer development in TRAMP mice.

Experimental acute infection of alpacas with Bovine viral diarrhea virus 1 subgenotype b alters peripheral blood and GALT leukocyte subsets.

Bovine viral diarrhea virus (BVDV) is a pathogen in cattle and alpacas ( Vicugna pacos), causing acute and persistent BVDV infections. We characterized the effect of acute BVDV infection on the immune system of alpacas by determining lymphocyte subpopulations in peripheral blood and gut-associated lymphoid tissues (GALT) as well as serum interferon levels. Alpacas were experimentally infected with BVDV-1b (strain CO-06). Peripheral blood leukocytes were isolated at 0, 3, 6, and 9 d postinfection (dpi), and leukocytes of GALT at 9 dpi, and evaluated using flow cytometry. Serum interferon levels were determined daily. Flow cytometric analyses of peripheral blood leukocytes showed a significant decrease in CD4+, CD8+, and αß T-lymphocytes at 3 dpi. CD8+ lymphocytes were significantly increased, and activated lymphocytes were significantly decreased in the C3-stomach region in BVDV-infected alpacas. Serum interferon concentrations significantly increased in BVDV-infected alpacas at 3-6 dpi, peaking at 3 dpi. Our study confirms that BVDV can be a primary acute pathogen in alpacas and that it induces an interferon response and alters leukocyte subset populations. The changes in the proportion of T-lymphocytes during the early stages of BVDV infection may result in transient immunosuppression that may contribute to secondary bacterial and viral infections, similar to cattle.

Widespread detection and characterization of porcine parainfluenza virus 1 in pigs in the USA.

Porcine parainfluenza virus 1 (PPIV1) was first identified in 2013 in slaughterhouse pigs in Hong Kong, China. Here, two near-complete genomes were assembled from swine exhibiting acute respiratory disease that were 90.0-95.3% identical to Chinese PPIV1. Analysis of the HN gene from ten additional PPIV1-positive samples found 85.0-95.5% identity, suggesting genetic diversity between strains. Molecular analysis identified 17 out of 279 (6.1%) positive samples from pigs with respiratory disease. Eleven nursery pigs from a naturally infected herd were asymptomatic; however, nasal swabs from six pigs and the lungs of a single pig were quantitative reverse transcriptase (qRT)-PCR positive. Histopathology identified PPIV1 RNA in the nasal respiratory epithelium and trachea. Two serological assays demonstrated seroconversion of infected pigs and further analysis of 59 swine serum samples found 52.5% and 66.1% seropositivity, respectively. Taken together, the results confirm the widespread presence of PPIV1 in the US swine herd.

Clinical and histologic description of ocular anatomy in captive black-tailed prairie dogs (Cynomys ludovicianus).

OBJECTIVE: To describe the clinical and histologic ocular anatomy of the black-tailed prairie dog (PD). ANIMALS STUDIED: Seventeen captive black-tailed PDs (11 males and six females), ranging in age from approximately 4 months to 4.5 years. PROCEDURES: Complete ocular examinations, including slit-lamp biomicroscopy, direct and indirect ophthalmoscopy, were performed under isoflurane anesthesia. The globes (n = 2) of one black-tailed PD were harvested immediately after euthanasia and processed after formalin fixation. Staining with hematoxylin-eosin, cytokeratin AE1/AE3, glial fibrillary acidic protein, chromogranin A, claudin-5, smooth muscle actin, and vimentin was performed for light microscopic evaluation. RESULTS: A thick mucinous precorneal tear film was present on the ocular surface. A vestigial nictitating membrane was identified in the medial canthus area. The limbus was heavily pigmented, the iris was a dark homogenous brown, and the pupil was round. Funduscopically, there was no tapetum lucidum, the retinal vascular pattern was holangiotic, and a horizontally elongated optic disk was visualized. The most common ocular abnormalities were acquired eyelid margin defects, present in seven eyes of six black-tailed PDs (35.3%). On histologic examination, the retina was asymmetric, thicker below the optic disk and thinner above it. CONCLUSIONS: The black-tailed PD fundus is atapetal with a holangiotic retinal vessel pattern and a horizontally elongated optic disk. Acquired lesions of the peri-ocular and eyelid region were the most common documented abnormality. Unique anatomic features of the globe and adnexa were confirmed with histologic and immunohistochemical analysis.

Description of a novel fatigue syndrome of finished feedlot cattle following transportation.

Ensuring appropriate animal welfare is a high priority for the beef industry, and poorly defined abnormalities in the mobility of cattle at abattoirs have gained considerable attention recently. During the summer of 2013, abattoirs throughout the United States reported concerns about nonambulatory or slow and difficult to move cattle and cattle that sloughed hoof walls. This report describes various cattle that developed these mobility problems soon after arrival at an abattoir. Affected cattle had various clinical signs including tachypnea with an abdominal component to breathing, lameness, and reluctance to move. Some cattle sloughed 1 or more hoof walls while in lairage pens and were euthanized. Other cattle recovered after being rested overnight. Affected cattle had serum lactate concentration and creatine kinase activity increased from reference ranges. Histologic findings included diffuse necrosis of the epidermal laminae with degenerate collagen and perivascular infiltration of neutrophils in the underlying deep dermis, and were similar for digits that had and had not sloughed the hoof wall. With the exception of the sloughed hoof walls, the clinical signs and serum biochemical abnormalities observed in affected cattle were similar to those observed in pigs with fatigued pig syndrome, and we propose that fatigued cattle syndrome be used to describe such cattle. Although anecdotal evidence generated concern that cattle fed the ß-adrenergic receptor agonist zilpaterol hydrochloride were at greater risk of developing mobility problems, compared with cattle not fed zilpaterol, this condition is likely multifactorial. Strategies to prevent this condition are needed to protect the welfare of cattle.

Comparative virulence of wild-type H1N1pdm09 influenza A isolates in swine.

In 2009, a novel swine-origin H1N1 (H1N1pdm09) influenza A virus (IAV) reached pandemic status and was soon after detected in pigs worldwide. The objective of this study was to evaluate whether differences in the HA protein can affect pathogenicity and antigenicity of H1N1pdm09 in swine. We compared lung pathology, viral replication and shedding and the antigenic relationships of four wild-type H1N1pdm09 viruses in pigs: one human (CA/09) and three isolated in swine after the pandemic (IL/09, IL/10, and MN/10). The swine strains were selected based upon unique amino acid substitutions in the HA protein. All selected viruses resulted in mild disease and viral shedding through nasal and oral fluids, however, viral replication and the degree of pathology varied between the isolates. A/Swine/IL/5265/2010 (IL/10), with substitutions I120M, S146G, S186P, V252M, had lower viral titers in the lungs and nasal secretions and fewer lung lesions. The other two swine viruses caused respiratory pathology and replicated to titers similar to the human CA/09, although MN/10 (with mutations D45Y, K304E, A425S) had lower nasal shedding. Swine-adapted H1N1pdm09 have zoonotic potential, and have reassorted with other co-circulating swine viruses, influencing the evolution of IAV in swine globally. Further, our results suggest that amino acid changes in the HA gene have the potential to alter the virulence of H1N1pdm09 in swine. Importantly, the limited clinical signs in pigs could result in continued circulation of these viruses with other endemic swine IAVs providing opportunities for reassortment.

Efficacy of Type 2 PRRSV vaccine against Chinese and Vietnamese HP-PRRSV challenge in pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant reproductive losses in the sow herd and respiratory disease in growing pigs. The virus belongs to the family Arteriviridae and there are two major genotypes. Type 1 is represented by Lelystad virus, the European prototype virus, and Type 2 is represented by the North American prototype virus, VR-2332. Depending on husbandry, immune status of the herd, and virulence of the isolate, the severity of disease and magnitude of economic loss can be variable. Vaccine use is not always successful indicating a lack of cross-protection between vaccine strains and circulating wild-type viruses. To date, there is no clear method to demonstrate if a vaccine confers protection against a specific isolate except for empirical animal studies. In 2006, a new lineage of Type 2 PRRSV emerged in Chinese swine herds that were suffering dramatic losses resulting in those viruses being described as "Highly Pathogenic PRRSV" (HP-PRRSV). Experimental reproduction of severe disease with HP-PRRSV isolates and virus derived from HP-PRRSV clones demonstrated the causal role of this virus. Recently, partial heterologous protection has been reported for Type 1 and Type 2 attenuated PRRSV vaccines against challenge by different Chinese HP-PRRSV isolates providing some hope for reducing economic loss. This paper reports the efficacy of a commercially available Type 2 attenuated vaccine in young pigs against heterologous challenge with a Chinese and Vietnamese HP-PRRSV isolate. When compared to unvaccinated pigs, vaccination decreased the length of viremia and viral titer, diminished the time of high fever and reduced macroscopic lung scores following homologous and heterologous PRRSV challenge. These results demonstrate the potential use of vaccine as an aid in the control of HP-PRRSV outbreaks.

Divergent immune responses and disease outcomes in piglets immunized with inactivated and attenuated H3N2 swine influenza vaccines in the presence of maternally-derived antibodies.

Live-attenuated influenza virus (LAIV) prime-boost vaccination previously conferred protection against heterologous H3N2 swine influenza challenge, including in piglets with maternally derived antibodies (MDA). Conversely, a whole-inactivated virus (WIV) vaccine was associated with enhanced disease. This study was aimed at identifying immune correlates of cross-protection. Piglets with and without MDA received intramuscular adjuvanted WIV or intranasal LAIV, and were challenged with heterologous H3N2. WIV induced cross-reactive IgG, inhibited by MDA, and a moderate T cell response. LAIV elicited mucosal antibodies and T cells cross-reactive to the heterologous challenge strain. The presence of MDA at LAIV vaccination blocked lung and nasal antibody production, but did not interfere with T cell priming. Even without mucosal antibodies, MDA-positive LAIV vaccinates were protected, indicating a likely role for T cells. Based on the data, one LAIV dose can induce cell-mediated immunity against antigenically divergent H3N2 influenza virus despite passive antibody interference with humoral immune responses.

Distribution of lymphoid depletion and viral antigen in alpacas experimentally infected with Bovine viral diarrhea virus 1.

It was hypothesized that acute postnatal Bovine viral diarrhea virus 1 (BVDV-1) infection leads to leukopenia and lymphoid depletion of gut-associated lymphoid tissues similar to acute disease in calves. The objectives of the current study were to characterize the pathologic effects, viremia, viral shedding, and viral antigen deposition in 6-24-month-old, acutely infected alpacas following experimental infection with noncytopathic BVDV-1 subgenotype 1b (BVDV C0-6). The BVDV-1 isolate was obtained from a cria with naturally occurring persistent infection. Lymphocytopenia occurred 3-7 days postinfection, with a 50% reduction in peripheral lymphocytes in infected alpacas. Depletion of B-cell populations in gut-associated lymphoid tissues was evident microscopically. Populations of T cells in parafollicular zones and in nodular aggregates along the superficial submucosa remained intact. The BVDV antigen was deposited most consistently in submucosal gastrointestinal aggregated lymphoid tissues of ileum, proximal colon, and stomach compartment three. Viral antigen was more variably evident in other lymphoid tissues. Antigen distribution correlated well with histologic lesions in gastrointestinal aggregated lymphoid tissues, confirming the role of virus in lymphoid depletion. Nasal shedding was detected in all challenged alpacas on day 6 and in 4 out of 12 challenged alpacas on day 9. Viremia was present as early as day 3, and present in all challenged alpacas on days 5, 6, 7, and 9 postchallenge. Lymphocytopenia and depletion of gastrointestinal aggregated lymphoid tissues associated with acute BVDV-1 infection likely results in immune compromise and is expected to exacerbate concurrent infections even though uncomplicated BVDV-1 infection was clinically unapparent.

Preventive and therapeutic efficacy of finasteride and dutasteride in TRAMP mice.

BACKGROUND: The prostate cancer prevention trial (PCPT) and Reduction by dutasteride of Prostate Cancer Events (REDUCE) trial found that 5α-reductase (5αR) inhibitors finasteride and dutasteride respectively, decreased prostate cancer prevalence but also increased the incidence of high-grade tumors. 5αR2 is the main isoenzyme in normal prostate tissue; however, most prostate tumors have high 5αR1 and low 5αR2 expression. Because finasteride inhibits only 5αR2, we hypothesized that it would not be as efficacious in preventing prostate cancer development and/or progression in C57BL/6 TRAMP x FVB mice as dutasteride, which inhibits both 5αR1 and 5αR2. METHOD/PRINCIPAL FINDINGS: Six-week-old C57BL/6 TRAMP x FVB male mice were randomized to AIN93G control or pre- and post- finasteride and dutasteride diet (83.3 mg drug/kg diet) groups (n =30-33) that began at 6 and 12 weeks of age, respectively, and were terminated at 20 weeks of age. The pre- and post- finasteride and dutasteride groups were designed to test the preventive and therapeutic efficacy of the drugs, respectively. Final body weights, genitourinary tract weights, and genitourinary tract weights as percentage of body weights were significantly decreased in the Pre- and Post-dutasteride groups compared with the control. The Post-dutasteride group showed the greatest inhibition of prostatic intraepithelial neoplasia progression and prostate cancer development. Surprisingly, the Post-dutasteride group showed improved outcomes compared with the Pre-dutasteride group, which had increased incidence of high-grade carcinoma as the most common and most severe lesions in a majority of prostate lobes. Consistent with our hypothesis, we found little benefit from the finasteride diets, and they increased the incidence of high-grade carcinoma. CONCLUSION: Our findings have commonalities with previously reported PCPT, REDUCE, and the Reduction by dutasteride of Clinical Progression Events in Expectant Management (REDEEM) trial results. Our results may support the therapeutic use of dutasteride, but not finasteride, for therapeutic or preventive use.

Experimental infection of United States swine with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus.

The pathogenesis of Type 2 highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in 10-week old swine in the United States was investigated. rJXwn06, rescued from an infectious clone of Chinese HP-PRRSV, replicated in swine with at least 100-fold increased kinetics over U.S. strain VR-2332. rJXwn06 caused significant weight loss, exacerbated disease due to bacterial sepsis and more severe histopathological lung lesions in pigs exposed to HP-PRRSV than to those infected with VR-2332. Novel findings include identification of bacterial species present, the degree of thymic atrophy seen, and the inclusion of contact animals that highlighted the ability of HP-PRRSV to rapidly transmit between animals. Furthermore, comprehensive detailed cytokine analysis of serum, bronchoalveolar lavage fluid, and tracheobronchial lymph node tissue homogenate revealed a striking elevation in levels of cytokines associated with both innate and adaptive immunity in HP-PRRSV infected swine, and showed that contact swine differed in the degree of cytokine response.

Analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a major pathogen of swine worldwide. Emergence in 2006 of a novel highly pathogenic PRRSV (HP-PRRSV) isolate in China necessitated a comparative investigation into the host transcriptome response in tracheobronchial lymph nodes (TBLN) 13 days post-infection with HP-PRRSV rJXwn06, PRRSV strain VR-2332 or sham inocula. RNA from each was prepared for next-generation sequencing. Amplified library constructs were directly sequenced and a list of sequence transcripts and counts was generated using an RNAseq analysis pipeline to determine differential gene expression. Transcripts were annotated and relative abundance was calculated based upon the number of times a given transcript was represented in the library. RESULTS: Major changes in transcript abundance occurred in response to infection with either PRRSV strain, each with over 630 differentially expressed transcripts. The largest increase in transcript level for either virus versus sham-inoculated controls were three serum amyloid A2 acute-phase isoforms. However, the degree of up or down-regulation of transcripts following infection with HP-PRRSV rJXwn06 was greater than transcript changes observed with US PRRSV VR-2332. Also, of 632 significantly altered transcripts within the HP-PRRSV rJXwn06 library 55 were up-regulated and 69 were down-regulated more than 3-fold, whilst in the US PRRSV VR-2332 library only 4 transcripts were up-regulated and 116 were down-regulated more than 3-fold. CONCLUSIONS: The magnitude of differentially expressed gene profiles detected in HP-PRRSV rJXwn06 infected pigs as compared to VR-2332 infected pigs was consistent with the increased pathogenicity of the HP-PRRSV in vivo.

Intranasal vaccination with replication-defective adenovirus type 5 encoding influenza virus hemagglutinin elicits protective immunity to homologous challenge and partial protection to heterologous challenge in pigs.

Influenza A virus (IAV) is widely circulating in the swine population and causes significant economic losses. To combat IAV infection, the swine industry utilizes adjuvanted whole inactivated virus (WIV) vaccines, using a prime-boost strategy. These vaccines can provide sterilizing immunity toward homologous virus but often have limited efficacy against a heterologous infection. There is a need for vaccine platforms that induce mucosal and cell-mediated immunity that is cross-reactive to heterologous viruses and can be produced in a short time frame. Nonreplicating adenovirus 5 vector (Ad5) vaccines are one option, as they can be produced rapidly and given intranasally to induce local immunity. Thus, we compared the immunogenicity and efficacy of a single intranasal dose of an Ad5-vectored hemagglutinin (Ad5-HA) vaccine to those of a traditional intramuscular administration of WIV vaccine. Ad5-HA vaccination induced a mucosal IgA response toward homologous IAV and primed an antigen-specific gamma interferon (IFN-γ) response against both challenge viruses. The Ad5-HA vaccine provided protective immunity to homologous challenge and partial protection against heterologous challenge, unlike the WIV vaccine. Nasal shedding was significantly reduced and virus was cleared from the lung by day 5 postinfection following heterologous challenge of Ad5-HA-vaccinated pigs. However, the WIV-vaccinated pigs displayed vaccine-associated enhanced respiratory disease (VAERD) following heterologous challenge, characterized by enhanced macroscopic lung lesions. This study demonstrates that a single intranasal vaccination with an Ad5-HA construct can provide complete protection from homologous challenge and partial protection from heterologous challenge, as opposed to VAERD, which can occur with adjuvanted WIV vaccines.

Strain-dependent effects of PB1-F2 of triple-reassortant H3N2 influenza viruses in swine.

The PB1-F2 protein of the influenza A viruses (IAVs) can act as a virulence factor in mice. Its contribution to the virulence of IAV in swine, however, remains largely unexplored. In this study, we chose two genetically related H3N2 triple-reassortant IAVs to assess the impact of PB1-F2 in virus replication and virulence in pigs. Using reverse genetics, we disrupted the PB1-F2 ORF of A/swine/Wisconsin/14094/99 (H3N2) (Sw/99) and A/turkey/Ohio/313053/04 (H3N2) (Ty/04). Removing the PB1-F2 ORF led to increased expression of PB1-N40 in a strain-dependent manner. Ablation of the PB1-F2 ORF (or incorporation of the N66S mutation in the PB1-F2 ORF, Sw/99 N66S) affected the replication in porcine alveolar macrophages of only the Sw/99 KO (PB1-F2 knockout) and Sw/99 N66S variants. The Ty/04 KO strain showed decreased virus replication in swine respiratory explants, whereas no such effect was observed in Sw/99 KO, compared with the wild-type (WT) counterparts. In pigs, PB1-F2 did not affect virus shedding or viral load in the lungs for any of these strains. Upon necropsy, PB1-F2 had no effect on the lung pathology caused by Sw/99 variants. Interestingly, the Ty/04 KO-infected pigs showed significantly increased lung pathology at 3 days post-infection compared with pigs infected with the Ty/04 WT strain. In addition, the pulmonary levels of interleukin (IL)-6, IL-8 and gamma interferon were regulated differentially by the expression of PB1-F2. Taken together, these results indicate that PB1-F2 modulates virus replication, virulence and innate immune responses in pigs in a strain-dependent fashion.

Evaluation of submucosally injected polyethylene glycol-based hydrogel and bovine cross-linked collagen in the canine urethra using cystoscopy, magnetic resonance imaging and histopathology.

OBJECTIVE: To (1) investigate the tissue response to a novel urethral bulking agent, polyethylene glycol carboxymethyl cellulose hydrogel (PEG-CMC) injected submucosally in the canine urethra and (2) compare PEG-CMC with bovine collagen (BC), the current standard for urethral bulking. STUDY DESIGN: Experimental study. ANIMALS: Purpose-bred female hound dogs (n = 8). METHODS: Standardized submucosal urethral injections of BC and PEG-CMC were performed in 8 female dogs. Injection sites were evaluated by cystoscopy on days 0 (n = 8), 30 (n = 4), and 90 (n = 4), magnetic resonance imaging on days 0 (n = 8), 30 (n = 8), and 90 (n = 4) and by histopathology on days 30 (n = 4) and 90 (n = 4). RESULTS: Both PEG-CMC and BC were detectable on MRI as hyperintense foci on T2-weighted images. Grossly, PEG-CMC formed more prominent blebs than BC. On follow-up cystoscopic examination, 6/8 PEG-CMC injection needle tracts were visible, and 3 of these sites had mucosal erosions. Histopathologic scores for foreign body reaction and inflammation were significantly higher for PEG-CMC compared with BC (P < 0.005). BC elicited a lymphoplasmacytic reaction whereas PEG-CMC incited a granulomatous response. CONCLUSIONS: The overall physical characteristics and histologic response associated with PEG-CMC support its use as a urethral bulking agent; however, the current formulation needs to be adjusted for clinical use.

Restored PB1-F2 in the 2009 pandemic H1N1 influenza virus has minimal effects in swine.

PB1-F2 is an 87- to 90-amino-acid-long protein expressed by certain influenza A viruses. Previous studies have shown that PB1-F2 contributes to virulence in the mouse model; however, its role in natural hosts-pigs, humans, or birds-remains largely unknown. Outbreaks of domestic pigs infected with the 2009 pandemic H1N1 influenza virus (pH1N1) have been detected worldwide. Unlike previous pandemic strains, pH1N1 viruses do not encode a functional PB1-F2 due to the presence of three stop codons resulting in premature truncation after codon 11. However, pH1N1s have the potential to acquire the full-length form of PB1-F2 through mutation or reassortment. In this study, we assessed whether restoring the full-length PB1-F2 open reading frame (ORF) in the pH1N1 background would have an effect on virus replication and virulence in pigs. Restoring the PB1-F2 ORF resulted in upregulation of viral polymerase activity at early time points in vitro and enhanced virus yields in porcine respiratory explants and in the lungs of infected pigs. There was an increase in the severity of pneumonia in pigs infected with isogenic virus expressing PB1-F2 compared to the wild-type (WT) pH1N1. The extent of microscopic pneumonia correlated with increased pulmonary levels of alpha interferon and interleukin-1ß in pigs infected with pH1N1 encoding a functional PB1-F2 but only early in the infection. Together, our results indicate that PB1-F2 in the context of pH1N1 moderately modulates viral replication, lung histopathology, and local cytokine response in pigs.

Vaccination with NS1-truncated H3N2 swine influenza virus primes T cells and confers cross-protection against an H1N1 heterosubtypic challenge in pigs.

The diversity of contemporary swine influenza virus (SIV) strains impedes effective immunization of swine herds. Mucosally delivered, attenuated virus vaccines are one approach with potential to provide broad cross-protection. Reverse genetics-derived H3N2 SIV virus with truncated NS1 (NS1Δ126 TX98) is attenuated and immunogenic when delivered intranasally in young pigs. We analyzed T-cell priming and cross-protective efficacy in weanling piglets after intranasal inoculation with NS1Δ126 TX98 versus wild type TX98. In vivo replication of the truncation mutant was minimal compared to the wild type virus. T-cell responses were greater in magnitude in pigs infected with the wild type virus in in vitro restimulation assays. According to the expression of activation marker CD25, peripheral T cell recall responses in NS1Δ126 TX98 infected pigs were minimal. However, intracellular IFN-γ data indicate that the attenuated virus induced virus-specific CD4(+)CD8(-), CD4(+)CD8(+), CD4(-)CD8(+), and γδ T cells within 28 days. The IFN-γ response appeared to contract, as responses were reduced at later time points prior to challenge. CD4(+)CD8(+) cells isolated 5 days after heterosubtypic H1N1 challenge (day 70 overall) showed an elevated CD25 response to virus restimulation. Pigs previously infected with wild type TX98 were protected from replication of the H1N1 challenge virus. Vaccination with NS1Δ126 TX98 was associated with significantly lower levels of Th1-associated cytokines in infected lungs but provided partial cross-protection against the H1N1 challenge. These results demonstrate that NS1Δ SIV vaccines can elicit cell-mediated cross-protection against antigenically divergent strains.

Effect of the viral protein N(pro) on virulence of bovine viral diarrhea virus and induction of interferon type I in calves.

OBJECTIVE: To characterize the influence of the viral protein N(pro) on virulence of bovine viral diarrhea virus (BVDV) and on type I interferon responses in calves. ANIMALS: 10 calves, 4 to 6 months of age. PROCEDURES: BVDV virulence and type I interferon responses of calves (n = 5) infected with a noncytopathic BVDV with a deleted N(pro) were compared with those of calves (5) infected with a noncytopathic BVDV with a functional N(pro). Rectal temperatures, clinical signs, platelet counts, and total and differential WBC counts were evaluted daily. Histologic examinations and immunohistochemical analyses of tissues were conducted to assess lesions and distribution of viral antigens, respectively. Serum type I interferon concentrations were determined. RESULTS: Calves infected with N(pro)-deleted BVDV developed leukopenia and lymphopenia, without developing increased rectal temperatures or lymphoid depletion of target lymphoid organs. There was minimal antigen deposition in lymphoid organs. Calves infected with N(pro) BVDV developed increased rectal temperatures, leukopenia, lymphopenia, and lymphoid depletion with marked BVDV antigen deposition in lymphatic tissues. Interferon type I responses were detected in both groups of calves. CONCLUSIONS AND CLINICAL RELEVANCE: Deletion of N(pro) resulted in attenuation of BVDV as evidenced by reduced virulence in calves, compared with BVDV with a functional N(pro). Deletion of N(pro) did not affect induction of type I interferon. The N(pro)-deleted BVDV mutant may represent a safe noncytopathic virus candidate for vaccine development.