Invasive Microsporum canis causing rhinitis and stomatitis in a cat.

Microsporum canis is a pathogenic fungus that typically causes dermatophytosis in cats. This report describes a cat with a Microsporum canis infection causing invasive fungal rhinitis that extended through the hard palate, resulting in adjacent stomatitis. Treatment with itraconazole and terbinafine resolved the infection.

PD-L1 expression is increased in monocyte derived dendritic cells in response to porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus infections.

Host immune system suppression is thought to be crucial in the development of porcine circovirus associated diseases (PCVAD). Many immune suppressive mechanisms have been studied in cases of PCVAD, however, the role of programmed death ligand-1 (PD-L1) during porcine circovirus type 2 (PCV2) infection and PCVAD development has yet to be determined. PD-L1 has become an important research target because of its ability to interfere with effective T-cell activity and proliferation during the course of an immune response. In this study, porcine monocyte derived dendritic cells (MoDC) were infected with different combinations of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) and evaluated for expression levels of PD-L1, as well as the expression levels of swine major histocompatibility complexes 1 and 2 (SLA-1 and SLA-2) as a measure of MoDC stimulatory capacity. PD-L1 expression levels were also tested in MoDCs after treatment with interferon alpha (IFN-α) and beta (IFN-ß). The results showed that the expression levels of PD-L1 were increased in PCV2-infected MoDCs, as well as in PCV2 and PRRSV co-infected MoDCs. The MoDCs infected with PRRSV only also showed a strain-dependent increase in PD-L1 expression. Both IFN-α and IFN-ß treatment also increased the expression levels of PD-L1 in MoDCs. SLA-1 and 2 expression levels were increased by PCV2 infection, and altered in the PRRSV, and PCV2+PRRSV co-infected MoDCs in a strain-dependent manner. These results indicate a potential immuno-suppressive role for dendritic cells during PCV2 infection and the development of PCVAD and will be helpful in more fully elucidating the underlying mechanisms leading to clinical PCVAD.

The PD-L1/CD86 ratio is increased in dendritic cells co-infected with porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus, and the PD-L1/PD-1 axis is associated with anergy, apoptosis, and the induction of regulatory T-cells in porcine lymphocytes.

Porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome virus (PRRSV) continue to have a negative economic impact on global swine production operations. Host immune modulations that potentiate disease during PCV2 and/or PRRSV infections are important areas of ongoing research. In this study, we evaluated the expression levels of PD-L1, CD86, and IL-10 in order to phenotype dendritic cells following viral infection with PCV2b and/or PRRSV. The results showed that the inhibitory marker PD-L1 was significantly increased in monocyte derived dendritic cells (MoDC) in both singular PCV2 infection and PCV2/PRRSV co-infections. MoDC expression of stimulatory marker CD86 was significantly increased during singular PCV2 infections, while it was significantly decreased in the treatment groups co-infected with both PCV2 and PRRSV. IL-10 production was highest among MoDCs that were co-infected with PCV2 and PRRSV. These results indicate that dendritic cells develop a regulatory phenotype following PCV2/PRRSV co-infections. We further investigated the role of the PD-L1/PD-1 axis in lymphocyte anergy, apoptosis, and the induction of regulatory T-cells in porcine mononuclear cell populations. Lymphocyte populations with normal PD-1 expression had higher percentages of anergic, apoptotic lymphocytes and CD4(+)CD25(HIGH)FoxP3(+) regulatory T-cells when compared to a PD-1 deficient lymphocyte population. These results implicate the PD-L1/PD-1 axis in negative regulation of lymphocyte responses in pigs.

Effects of a dietary antioxidant blend and vitamin E on fatty acid profile, liver function, and inflammatory response in broiler chickens fed a diet high in oxidants.

The aim of the current study was to determine the effects of a dietary antioxidant blend and vitamin E on fatty acid profile, inflammatory response, and liver function. Cobb 500 male broilers (n = 1,200, d 0) were randomly distributed into 6 treatments with 10 replicate floor pens. Treatments included (1) a high-oxidant diet, with vitamin E at 10 IU/kg, 3% oxidized oil, 3% polyunsaturated fatty acids (PUFA) source (HO); (2) the HO diet with vitamin E at 200 IU/kg (VE); (3) the HO diet with an antioxidant blend at 135 mg/kg (AOX); (4) the HO diet with both vitamin E at 200 IU/kg and an antioxidant blend at 135 mg/kg (VE+AOX); (5) standard control (SC); and (6) a positive control, which was the SC diet with an antioxidant blend at 135 mg/kg. The concentrations of 20:4, 20:5, 22:5, 22:6, and all the n-3 fatty acids were greater in the abdominal fat of HO, VE, AOX, and VE+AOX birds than SC and positive control birds on d 21 and 42 (P < 0.001). Compared with HO treatment, AOX and VE+AOX preserved the deposition of PUFA better (P < 0.001). The HO birds had greater concentrations of aspartate aminotransferase on d 21 and 42, and γ-glutamyl transferase on d 21, whereas AOX and VE+AOX chickens had restored γ-glutamyl transferase concentration (P < 0.01). The inflammation scores of abdominal fat of AOX and VE+AOX birds were lower than the HO on d 21 (P < 0.001). Compared with SC, the VE and VE+AOX birds exhibited greater vacuole scores on d 21 and 42 (P < 0.01). The lower vacuoles score in SC was associated with a greater expression of peroxisome proliferator activated receptor -γ and -α (P < 0.05). The expression of inflammatory genes in the liver did not differ among treatments. In conclusion, the AOX and AOX+VE diets were effective in preserving PUFA in the abdominal fat, moderately improved liver function, and reduced inflammation in fat.

Experimental comparison of hemolytic and nonhemolytic Ornithobacterium rhinotracheale field isolates in vivo.

Ornithobacterium rhinotracheale (ORT) is a nonhemolytic, gram-negative, pleomorphic, rod-shaped bacterium that causes upper and lower respiratory tract disease in poultry. Recently, hemolytic strains of ORT have been isolated with increasing frequency from field outbreaks. A study was conducted to determine whether the hemolytic phenotype is associated with any change in virulence. Briefly, 225 turkey poults, vaccinated against hemorrhagic enteritis at 4 wk of age, were randomly divided into nine replicates housed in separate rooms: three sham treatment controls (25 poults/replicate), three challenged with a nonhemolytic (NH) field isolate (24 poults/replicate), and three challenged with a hemolytic (H) field isolate (24 poults/replicate). Nine days postvaccination, poults were inoculated intratracheally with either 0.2 ml sterile phosphate-buffered saline (PBS), 2 x 10(8) colony-forming units (CFU) of the NH isolate in 0.2 ml PBS, or 2 x 10(8) CFU of the H isolate in 0.2 ml PBS. Serum and body weights were obtained at 0, 7, 14, and 21 days postinoculation (dpi). Tissues were taken for culture and histopathology from five randomly selected poults/replicates at 7, 14, and 21 dpi. When compared with poults inoculated with the H isolate or controls, those inoculated with the NH isolate showed a highly significant depression in weight gain at 7 dpi. NH poults also had significantly higher levels of antibody against ORT at 14 and 21 dpi. Reisolations decreased over time and, by 21 dpi, only the NH phenotype could be found. Based on a Likert-type scale, poults inoculated with the NH isolate had significantly higher histopathologic lesion scores in lung tissue at 7, 14, and 21 dpi. Results suggest that nonhemolytic field isolates are more virulent then hemolytic ones. These findings are unusual because hemolytic phenotypes are often more virulent in other bacterial species.

Co-infection of porcine dendritic cells with porcine circovirus type 2a (PCV2a) and genotype II porcine reproductive and respiratory syndrome virus (PRRSV) induces CD4(+)CD25(+)FoxP3(+) T cells in vitro.

Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases in the global swine industry. Porcine circovirus type 2 (PCV2) is the primary causative agent, however co-infection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV) is often required to induce the full spectrum of clinical PCVAD. While the specific mechanisms of viral co-infection that lead to clinical disease are not fully understood, immune modulation by the co-infecting viruses likely plays a critical role. We evaluated the ability of dendritic cells (DC) infected with PRRSV, PCV2, or both to induce regulatory T cells (T(regs)) in vitro. DCs infected with PCV2 significantly increased CD4(+)CD25(+)FoxP3(+) T(regs) (p<0.05) and DCs co-infected with PRRSV and PCV2 induced significantly higher numbers of T(regs) than with PCV2 alone (p<0.05). Cytokine analysis indicated that the induction of T(regs) by co-infected DCs may be dependent on TGF-ß and not IL-10. Our data support the immunomodulatory role of PCV2/PRRSV co-infection in the pathogenesis of PCVAD, specifically via T(reg)-mediated immunosuppression.

High hydrostatic pressure processing of murine norovirus 1-contaminated oysters inhibits oral infection in STAT-1(-/-)-deficient female mice.

We have previously demonstrated that high pressure processing (HPP) is effective in preventing in vitro replication of murine norovirus strain 1 (MNV-1), a human norovirus surrogate, in a monocyte cell line following extraction from MNV-1-contaminated oysters. In the present study, the efficacy of HPP to prevent in vivo replication within mice fed HPP-treated MNV-1-seeded oyster extracts was evaluated. Oyster homogenate extracts seeded with MNV-1 were given 5-min, 400-MPa (58,016-psi) treatments and orally gavaged into immunodeficient (STAT-1(-/-)) female mice. Mice orally gavaged with HPP-treated MNV-1 showed significant (P ≤ 0.05) weight loss leading to enhanced morbidity, whereas those given 100 and 200 PFU of HPP-treated MNV-1 were comparable to uninfected controls. MNV-1 was detected, via real-time PCR, within the liver, spleen, and brain of all mice fed non-HPP-treated homogenate but was not detected in the tissues of mice fed HPP-treated homogenates or in uninfected control mice. Hepatocellular necrosis and lymphoid depletion in the spleen were observed in non-HPP-treated MNV-1 mice only. These results clearly show that HPP prevents MNV-1 infection in vivo and validates that viral inactivation by HPP in vitro is essentially equivalent to that in vivo. Further, the data suggest that HPP may be an effective food processing intervention for norovirus-contaminated shellfish and thus may decrease risk to both immunocompromised and immunocompetent individuals who consume shellfish.

A modified live PRRSV vaccine and the pathogenic parent strain induce regulatory T cells in pigs naturally infected with Mycoplasma hyopneumoniae.

The lack of heterologous protection by porcine reproductive and respiratory syndrome virus (PRRSV) vaccines is currently a major problem in the field. Heterologous protection by PRRS vaccines depends on the ability of the vaccine to induce an interferon gamma (IFN-γ) response. One mechanism by which the virus evades the immune system is by activating regulatory T cells (T(regs)), resulting in induction of interleukin 10 rather than IFN-γ. Our hypothesis that current PRRS vaccines do not differ from pathogenic strains in the ability to induce T(regs) was tested by inoculating three groups of pigs with two pathogenic viruses and an attenuated vaccine strain and evaluating the number of T(regs) in peripheral blood mononuclear cells. Before inoculation, the pigs, although vaccinated became infected naturally with Mycoplasma hyopneumoniae before shipment to our research facility. Our results show that the PRRSV vaccine strain and parent strain are equally able to induce T(regs) in pigs naturally infected with M. hyopneumoniae. Pigs in the vaccine and PRRSV groups had higher lung lesion scores than pigs in the control groups. The results suggest that the exacerbation M. hyopneumoniae respiratory disease may be due to the ability of PRRSV vaccination and viral infection to induce regulatory T cells.

Generation of antibody- and B cell-deficient pigs by targeted disruption of the J-region gene segment of the heavy chain locus.

A poly(A)-trap gene targeting strategy was used to disrupt the single functional heavy chain (HC) joining region (J(H)) of swine in primary fibroblasts. Genetically modified piglets were then generated via somatic cell nuclear transfer (SCNT) and bred to yield litters comprising J(H) wild-type littermate (+/+), J(H) heterozygous knockout (±) and J(H) homozygous knockout (-/-) piglets in the expected Mendelian ratio of 1:2:1. There are only two other targeted loci previously published in swine, and this is the first successful poly(A)-trap strategy ever published in a livestock species. In either blood or secondary lymphoid tissues, flow cytometry, RT-PCR and ELISA detected no circulating IgM(+) B cells, and no transcription or secretion of immunoglobulin (Ig) isotypes, respectively in J(H) -/- pigs. Histochemical and immunohistochemical (IHC) studies failed to detect lymph node (LN) follicles or CD79α(+) B cells, respectively in J(H) -/- pigs. T cell receptor (TCR)(ß) transcription and T cells were detected in J(H) -/- pigs. When reared conventionally, J(H) -/- pigs succumbed to bacterial infections after weaning. These antibody (Ab)- and B cell-deficient pigs have significant value as models for both veterinary and human research to discriminate cellular and humoral protective immunity to infectious agents. Thus, these pigs may aid in vaccine development for infectious agents such as the pandemic porcine reproductive and respiratory syndrome virus (PRRSV) and H1N1 swine flu. These pigs are also a first significant step towards generating a pig that expresses fully human, antigen-specific polyclonal Ab to target numerous incurable infectious diseases with high unmet clinical need.

The open reading frame 3 (ORF3) of porcine circovirus type 2 (PCV2) is dispensable for virus infection but evidence of reduced pathogenicity is limited in pigs infected by an ORF3-null PCV2 mutant.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases (PCVAD) in pigs. The open reading frame (ORF) 3 of PCV2 reportedly induces apoptosis and is associated with PCV2 pathogenicity. In this study, we first created an ORF3-null PCV2 mutant (muPCV2) by site-directed mutagenesis and demonstrated that the dimerized plasmid DNA of muPCV2 clone is infectious when injected intramuscularly (I.M.) into pigs. Subsequently, by using a well-characterized pig model we compared the pathogenicity of the muPCV2 and the wildtype PCV2. Thirty-one pigs were divided into 3 groups of 11, 10, and 10 each: group 1 pigs were each inoculated I.M. with PBS buffer as negative controls, group 2 pigs each with 200 microg of muPCV2 infectious DNA clone, and group 3 pigs each with 200 microg of wildtype PCV2 infectious DNA clone. Blood was collected prior to inoculation and weekly thereafter, and tested for PCV2 antibodies by ELISA and serum viral DNA loads by quantitative PCR. All pigs were necropsied at 35 days post-inoculation. The results showed that pigs inoculated with muPCV2 had a delayed seroconversion and lower serum viral load. However, there was no significant difference in the average scores of the histological or gross lesions or the amount of PCV2-specific antigen in tissues between wildtype PCV2- and muPCV2-inoculated groups. Thus, the data from this study do not fully support the conclusion of a previous report regarding PCV2 attenuation by abrogation of ORF3 although the results did show that ORF3 is dispensable for PCV2 replication in pigs.

Comparative pathogenesis in specific-pathogen-free chickens of two strains of avian hepatitis E virus recovered from a chicken with Hepatitis-Splenomegaly syndrome and from a clinically healthy chicken.

Avian hepatitis E virus (avian HEV) is the primary causative agent of Hepatitis-Splenomegaly (HS) syndrome in chickens. Recently, a genetically unique strain of avian HEV, designated avian HEV-VA, was recovered from healthy chickens in Virginia. The objective of this study was to experimentally compare the pathogenicity of the prototype strain recovered from a chicken with HS syndrome and the avian HEV-VA strain in specific-pathogen-free chickens. An infectious stock of the avian HEV-VA strain was first generated and its infectivity titer determined in chickens. For the comparative pathogenesis study, 54 chickens of 6-week-old were assigned to 3 groups of 18 chickens each. The group 1 chickens were each intravenously inoculated with 5x10(2.5) 50% chicken infectious dose of the prototype strain. The group 2 received the same dose of the avian HEV-VA strain, and the group 3 served as negative controls. Six chickens from each group were necropsied at 2, 3 and 4 weeks post-inoculation (wpi). Most chickens in both inoculated groups seroconverted by 3wpi, and the mean anti-avian HEV antibody titers were higher for the prototype strain group than the avian HEV-VA strain group. There was no significant difference in the patterns of viremia and fecal virus shedding. Blood analyte profiles did not differ between treatment groups except for serum creatine phosphokinase levels which were higher for prototype avian HEV group than avian HEV-VA group. The hepatic lesion score was higher for the prototype strain group than the other two groups. The results indicated that the avian HEV-VA strain is only slightly attenuated compared to the prototype strain, suggesting that the full spectrum of HS syndrome is likely associated with other co-factors.

Development and validation of a negative-strand-specific reverse transcription-PCR assay for detection of a chicken strain of hepatitis E virus: identification of nonliver replication sites.

As a positive-strand RNA virus, hepatitis E virus (HEV) produces an intermediate negative-strand RNA when it replicates. Thus, the detection of negative-strand viral RNA is indicative of HEV replication. The objective of this study was to develop a negative-strand-specific reverse transcription-PCR (RT-PCR) assay for the identification of extrahepatic sites of HEV replication. Briefly, a 494-bp fragment within the orf1 gene of a chicken strain of HEV (designated avian HEV) was amplified and cloned into a pSK plasmid. A synthetic negative-strand viral RNA was generated from the plasmid by in vitro transcription and was used to standardize the assay. A nested set of primers was designed to amplify a 232-bp fragment of the negative-strand viral RNA. The assay was found to detect up to 10 pg and 10(-5) pg of negative-strand HEV RNA in first- and second-round PCRs, respectively. The standardized negative-strand-specific RT-PCR assay was subsequently used to test 13 conveniently obtained tissue specimens collected sequentially on different days postinoculation from chickens experimentally infected with avian HEV. In addition to the liver, the negative-strand-specific RT-PCR assay identified replicative viral RNA in gastrointestinal tissues, including the colorectal, cecal, jejunal, ileal, duodenal, and cecal tonsil tissues. The detection of replicative viral RNA in these tissues indicates that after oral ingestion of the virus, HEV replicates in the gastrointestinal tract before it reaches the liver. This is the first report on the identification of extrahepatic sites of HEV replication in animals after experimental infection via the natural route. The assay should be of value for studying HEV replication and pathogenesis.

The effects of growth hormone and insulin-like growth factor I on the immune system of aged female monkeys.

The aging process is associated with a significant reduction in circulating GH and insulin-like growth factor I (IGF-I) levels. During this period, immune function also declines. Rodent data suggest that treatment with recombinant human GH (rhGH) or rhIGF-I enhances immune function in normal adult mice. To determine whether rhGH and/or rhIGF-I treatment could improve immune function in aged female rhesus monkeys, we administered rhIGF-I (120 micrograms/kg x day), rhGH (100 micrograms/kg x day), a combination of therapies, or excipient alone by sc infusion using Alzet pumps over a 7-week period. At 28 days, the pumps were replaced, and the animals were bled and immunized with tetanus toxoid. At the end of the 7-week period, the animals were killed. rhGH and rhIGF-I increased serum GH and IGF-I levels, respectively; rhGH and rhIGF-I in combination induced the highest serum IGF-I levels (906 +/- 261 ng/ml vs. control, 185 +/- 36 ng/ml at death). rhGH and rhIGF-I also increased IGFBP-3 levels. rhGH infusion resulted in the most marked changes in lymph node and splenic reactivity, as determined by histological assessment. Lymph nodes from the rhGH-treated animals showed changes from baseline indicating a stimulated reactive state. Both rhGH and rhIGF-I had effects on lymphocyte phenotype, but there were different responses in blood compared to spleen and lymph nodes. In the peripheral blood, the percent B cells and percent CD8 cell count rose after rhIGF-I therapy, with a fall in the CD4/CD8 ratio. In the spleen, on the other hand, the CD4/CD8 ratio nearly doubled (0.33 +/- 0.12 vs. 0.53 +/ 0.12) after rhIGF- I therapy. In the spleen, the combination of rhGH and rhIGF- I increased the percent T cells from 26.7 +/- 2.3 to 42.4 +/- 4.4 and the CD4/CD8 ration to 0.71 +/- 0.11. Both rhGH and rhIGF-I increased in vivo (antibody titer to tetanus toxoid) responses by lymphocytes, and rhGH increased Con-A- induced DNA synthesis in vitro. These results confirm the rodent data showing that rhGH and rhIGF-I cause beneficial changes in immune function and suggest that further investigation is warranted to assess their therapeutic potential.