Bovine respiratory syncytial virus enhances the attachment of Trueperella pyogenes to cells.

In cattle, bovine respiratory syncytial virus (BRSV) is associated with secondary bacterial infections; however, the mechanisms of the interaction between BRSV and bacteria are unclear. Trueperella pyogenes (T. pyogenes) causes pneumonia in cattle and is involved in secondary infections following viral infections. In this study, we evaluated the effect of BRSV infection on the adhesion of T. pyogenes to BRSV-infected cells. BRSV infection significantly enhanced the adhesion of T. pyogenes to cells in a multiplicity of infection- and time-dependent manner. The BRSV-mediated change in the adhesion of T. pyogenes was widely observed in various cell types and bacterial strains. The results from the gentamicin protection assay showed that BRSV infection did not affect the intracellular invasion ability of T. pyogenes. Furthermore, adhesion assays conducted using BRSV G protein-expressing cells and anti-BRSV G antibodies revealed that the increased adhesion of T. pyogenes to cells was mediated by the G protein of BRSV. In addition, immunofluorescence assay revealed the colocalization of BRSV G protein and T. pyogenes. Thus, BRSV infection can potentially lead to bovine respiratory disease complex by promoting the adhesion of T. pyogenes to the infected cells.

Epididymitis with Pasteurella multocida isolated from two male Japanese Black beef calves.

Two male Japanese Black calves developed an enlarged scrotum and testis. Orchiectomy was performed and pus was collected during surgery. After removal of the testis, bacteriological and histopathological examinations were conducted to investigate the cause and confirm the diagnosis. Based on the results obtained, both cases were diagnosed with epididymitis caused by an infection with Pasteurella multocida. This is the first study to show that P. multocida causes epididymitis in male calves. Further studies are required to clarify the details underlying the infection of calves with P. multocida.

Molecular detection of Lotmaria passim in honeybees in Japan.

Crithidia mellificae (C. mellificae) and Lotmaria passim (L. passim) are trypanosomatids that infect Apis mellifera. We analyzed the prevalence of C. mellificae and L. passim in six regions of Japan from 2018 to 2019. The detection rate of C. mellificae was 0.0% in all regions, whereas L. passim was detected in 16.7%-66.7% of the honeybees. L. passim was detected at a significantly lower rate in the Cyugoku-Shikoku region than in other regions. Furthermore, phylogenetic analysis of the internal transcribed spacer 1 (ITS1) locus of related species was performed. All the samples in this study could be assigned to the L. passim clade. This study reveals that L. passim infection is predominantly prevalent in Japan. Further epidemiological surveys are needed to clarify the prevalence of C. mellificae infection in honeybees in Japan.

Transcriptional inhibition of feline immunodeficiency virus by alpha-amanitin.

Alpha-amanitin, one of the amatoxins in egg amanita, has a cyclic peptide structure, and was reported as having antiviral activity against several viruses. We investigated whether α-amanitin has antiviral activity against feline immunodeficiency virus (FIV). FL-4 cells persistently infected with FIV Petaluma were cultured with α-amanitin. Reverse transcriptase (RT) activity in the supernatant of FL-4 cells was significantly inhibited by α-amanitin. In addition, the production of FIV core protein in FL-4 cells was inhibited by α-amanitin when analyzed by western blotting. Furthermore, α-amanitin inhibited the transcription of FIV in real-time RT-PCR. These data suggested that α-amanitin showed anti-FIV activity by inhibiting the RNA transcription level.

Antiviral Effects of Hydroxychloroquine and Type I Interferon on In Vitro Fatal Feline Coronavirus Infection.

Feline infectious peritonitis (FIP) is a viral disease with a high morbidity and mortality by the FIP virus (FIPV, virulent feline coronavirus). Several antiviral drugs for FIP have been identified, but many of these are expensive and not available in veterinary medicine. Hydroxychloroquine (HCQ) is a drug approved by several countries to treat malaria and immune-mediated diseases in humans, and its antiviral effects on other viral infections (e.g., SARS-CoV-2, dengue virus) have been confirmed. We investigated whether HCQ in association with interferon-ω (IFN-ω) is effective for FIPV in vitro. A total of 100 µM of HCQ significantly inhibited the replication of types I and II FIPV. Interestingly, the combination of 100 µM of HCQ and 104 U/mL of recombinant feline IFN-ω (rfIFN-ω, veterinary registered drug) increased its antiviral activity against type I FIPV infection. Our study suggested that HCQ and rfIFN-ω are applicable for treatment of FIP. Further clinical studies are needed to verify the combination of HCQ and rIFN-ω will be effective and safe treatment for cats with FIP.

Identification of an antilymphocyte transformation substance from Pasteurella multocida.

Pasteurella multocida is one of the most important bacteria responsible for diseases of animals. Crude extracts from sonicated P. multocida strain Dainai-1, which is serotype A isolated from bovine pneumonia, were found to inhibit proliferation of mouse spleen cells stimulated with Con A. The crude extract was purified by cation and anion exchange chromatography and hydroxyapatite chromatography. Its molecular weight was 27 kDa by SDS-PAGE and it was named PM27. PM27 was found to inhibit proliferation of mouse spleen cells stimulated with Con A as effectively as did the crude extract; however, its activity was lost after heating to 100°C for 20 min. PM27 did not directly inhibit proliferation of HT-2 cells, which are an IL-2-dependent T cell line, nor did it modify IL-2 production by Con A-stimulated mouse spleen cells. The N-terminal amino acid sequence of PM27 was determined and BLAST analysis revealed its identity to uridine phosphorylase (UPase) from P. multocida. UPase gene from P. multocida Dainai-1 was cloned into expression vector pQE-60 in Escherichia coli XL-1 Blue. Recombinant UPase (rUPase) tagged with His at the C-terminal amino acid was purified with Ni affinity chromatography. rUPase was found to inhibit proliferation of mouse spleen cells stimulated with Con A; however, as was true for PM27, its activity was lost after heating to 100°C for 20 min. Thus, PM27/UPase purified from P. multocida has significant antiproliferative activity against Con A-stimulated mouse spleen cells and may be a virulence factor.

Differential Distribution of Salmonella Serovars and Campylobacter spp. Isolates in Free-Living Crows and Broiler Chickens in Aomori, Japan.

Salmonella and Campylobacter cause foodborne enteritis mainly via the consumption of raw/undercooked contaminated poultry meat and products. Broiler flocks are primarily colonized with these bacteria; however, the underlying etiology remains unclear. The present study was conducted in order to obtain further information on the prevalence and genotypic distribution of Salmonella and Campylobacter in free-living crows and broiler flocks in a region for 2 years, thereby facilitating estimations of the potential risk of transmission of C. jejuni from crows to broiler flocks. Salmonella serovars Bredeney and Derby were isolated from 8 and 3 out of 123 captured crows, respectively, both of which are not common in broiler chickens. Campylobacter were isolated from all 89 crows tested and C. jejuni was prevalent (85 crows). Pulsed field gel electrophoresis showed broad diversity in the crow isolates of C. jejuni. However, 3 crow isolates and 2 broiler isolates showing similar banding patterns were assigned to different sequence types in multi-locus sequence typing. These results indicate that crows do not share Salmonella serovars with broilers, and harbor various genotypes of C. jejuni that differ from those of broiler flocks. Thus, our results indicate that crows are not a potential vector of these bacteria to broiler flocks in this region.

Prevalence of staphylococcal enterotoxins in Staphylococcus pseudintermedius isolates from dogs with pyoderma and healthy dogs.

To investigate the role of staphylococcal enterotoxins (SEs) produced by Staphylococcus pseudintermedius in the pathogenesis of pyoderma, isolates from dogs with pyoderma and healthy dogs were analyzed. According to reverse passive latex agglutination, 14/184 isolates (7.6%) from dogs with pyoderma and 9/87 (10.3%) from healthy dogs produced SEs (SEA, SEC or SED). According to multiplex PCR, 99 isolates (53.7%) from dogs with pyoderma and 97 (90.8%) from healthy dogs possessed one or more se genes. There was no significant difference regarding ses between dogs with pyoderma and healthy dogs. Therefore, SEs may not be a direct virulence factor in pyoderma.

Antimicrobial resistance of Staphylococcus pseudintermedius isolates from healthy dogs and dogs affected with pyoderma in Japan.

Staphylococcus pseudintermedius strains were isolated from healthy dogs and dogs with pyoderma in 2000-2002 and 2009. All the isolates from dogs with pyoderma in 1999-2000 and from healthy dogs in 2000-2002 and 2009 were susceptible to cefalexin and/or other cephalosporins and oxacillin. However, 7.1-12.5 and 11.4% of S. pseudintermedius isolates from dogs with pyoderma in 2009 were resistant to cephalosporins and oxacillin, respectively. All S. pseudintermedius isolates from dogs with pyoderma in 1999-2000 and those from healthy dogs in 2000-2002 were susceptible to fluoroquinolones; however, 50% of the S. pseudintermedius strains isolated from dogs with pyoderma in 2009 and 30% of the S. pseudintermedius strains isolated from healthy dogs in 2009 were resistant to fluoroquinolones. Of the 21 oxacillin-resistant S. pseudintermedius (MRSP) isolates, 11 carried SCCmec type V and 10 carried hybrid SCCmec types II-III. Staphylococcus pseudintermedius strains that were resistant to only one of three fluoroquinolones had a mutation in the quinolone resistance determination region of grlA, whereas S. pseudintermedius strains that were resistant to two or more fluoroquinolones had mutations in the quinolone resistance determination regions of both grlA and gyrA.

Identification and characterization of feline UBE1L gene.

Interferon-stimulated gene 15 (ISG15) is one of the type I interferon-inducible proteins. Addition of ISG15 known as ISGylation is an ubiquitin-like posttranslational modification. Coexpression of ISG15 and ubiquitin-activating enzyme E1-like protein (UBE1L) is required to induce ISGylation. Previously, we identified feline ISG15 gene and found that the capsid protein of feline immunodeficiency virus was ISGylated in vitro by treatment with feline interferon-ω. In this study, we cloned feline UBE1L (FeUBE1L) gene to further study the mechanism of the antiviral activities induced by ISGylation. Sequencing analysis revealed that active sites of FeUBE1L were highly conserved. These data suggest that FeUBE1L has an enzymatic activity. Further, expression of FeUBE1L was induced in feline cell lines by treatment with feline interferon-ω and ovine interferon-τ.

Effect of progesterone on the in vitro response of peripheral blood mononuclear cells stimulated by Escherichia coli in mares.

Escherichia coli(E. coli) isolated from the uterus of a Thoroughbred mare with bacterial endometritis was used to evaluate the effect of progesterone (P(4)) on the immune response of mares. Peripheral blood mononuclear cells (PBMCs) were collected from 10 nonpregnant clinically healthy adult mares (range, 4-12 years) during diestrus, four Thoroughbreds and six Hokkaido native horses. Cell proliferation and expression of cytokine mRNA, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-10, of PBMCs stimulated with E. coli and P(4) were examined in vitro. P(4) was shown to have significantly inhibited E. coli induced proliferation and expression of IFN-γ in PBMCs. These results indicate that P(4) inhibits the immune response to E. coli in mares.

Development of a high-expression system for staphylococcal exfoliative toxin genes.

We constructed a new expression system for staphylococcal exfoliative toxin (ET). The expression vector, pETA-exp2, was constructed based on Bacillus-Escherichia shuttle vector pHY300PLK. The pETA-exp2 vector includes the regulator of the ETA gene (eta), the promoter and Shine-Dalgarno (SD) sequences of eta, a SalI sequence at the end of the signal sequence of eta, a nucleotide sequence encoding mature ETA, an XhoI site, a 6x His sequence just before the stop codon and the end of the transcription sequence of eta. The nucleotide sequences coding for the mature proteins of ETB, ExhA, ExhB, ExhC, ExhD and SHETB were amplified by polymerase chain reaction (PCR) and inserted into pETA-exp2. These recombinant plasmids were transformed into Bacillus megaterium. The major protein in the culture supernatant of the transformant was recombinant ET (rET). The yields of all rETs were high, and all of them showed exfoliative activity in susceptible animals. The antigenicities of rETs and ETs were not distinguishable from each other.

Comparison of the immunosuppressive effects of dexamethasone, flunixin meglumine and meloxicam on the in vitro response of calf peripheral blood mononuclear cells.

This study compared the immunosuppressive effects of dexamethasone (DEX), flunixin meglumine (FLU) and meloxicam (MEL) on the peripheral blood mononuclear cells (PBMCs) of seven healthy Holstein calves in vitro. DEX significantly inhibited lymphocyte proliferation and expression of interferon (IFN)-γ, interleukin (IL)-2 and IL-4 messenger RNA (mRNA) in comparison with FLU and MEL. FLU and MEL dose-dependently inhibited lymphocyte proliferation, but did not significantly reduce mRNA expression. Our in vitro study indicates that steroidal anti-inflammatory drugs (SAIDs) as well as nonsteroidal anti-inflammatory drugs (NSAIDs) have immunosuppressive effects on calf PBMCs. These findings are important for assessing the indications and complications of NSAIDs in calves.

Detection of Staphylococcus hyicus exfoliative toxin genes by dot blot hybridization and multiplex polymerase chain reaction.

We designed a novel DNA probe and novel PCR primer sets for detecting the genes coding for Staphylococcus hyicus (S. hyicus) exfoliative toxin (ET). In dot blot hybridization, the novel DNA probe hybridized with chromosomal DNA of ExhA-, ExhB-, ExhC-, ExhD-, and SHETA-producing strains. This probe also hybridized with the plasmid DNA of a SHETB-producing strain. In Southern blot hybridization, the probe hybridized with a 1.5 kb HindIII fragment of chromosomal DNA from a SHETA-producing strain. The above fragment was cloned into E. coli and the nucleotide sequence of the SHETA gene determined, this gene proved to have almost the same homology (99.6%) as the ExhB gene. It was therefore thought that SHETA is a subtype of ExhB. In multiplex PCR using five primer sets, each gene gave a band distinguishable from the others. This multiplex PCR system has high specificity among the well-known S. hyicus ET genes. Of the 69 known ET-producing S. hyicus strains, 38, 19, 10, 2 and 1 strains have exhB, exhD exhA, shetb and exhC genes, respectively.

Molecular cloning and sequence analysis of feline interferon-stimulated gene 15.

The interferon-stimulated gene 15 (ISG15) is induced by type I interferon (IFN). Recent studies have revealed that like ubiquitin, ISG15 is conjugated with target proteins. In this study, the feline ISG15 (FeISG15) gene was cloned from feline IFNomega (FeIFNomega)-stimulated feline kidney epithelial (CRFK) cells. According to gene sequence results, cDNA was 474bp long and encoded a protein of 157 amino acids. The putative amino acid sequences showed 62.5-72.1% identity with those of other mammalian ISG15s. Similar to human and mouse ISG15, FeISG15 included tandem ubiquitin-like domains; its homology with feline ubiquitin was 36.3-39.5%. The LRLRGG conjugating motif was located only in the carboxyl terminal ubiquitin-like domain. FeISG15 also lacked the carboxyl terminal extension after the LRLRGG motif, which is present in mouse and human ISG15. Recombinant FeISG15 protein was expressed as a His-tagged fusion protein in Escherichia coli and purified by ion-exchange chromatography followed by affinity chromatography. Monoclonal anti-FeISG15 antibodies revealed free FeISG15 and FeISG15 conjugated with target proteins in cells after IFNomega stimulation by Western blotting analysis. Furthermore, mRNA of IFNgamma was detected from peripheral blood mononuclear cells (PBMCs) after stimulation with rFeISG15 extracellularly by RT-PCR. Taken together, these results suggested that FeISG15 had ubiquitin- and cytokine-like activity, as in other species.

Dual-subtype FIV vaccine (Fel-O-Vax FIV) protection against a heterologous subtype B FIV isolate.

Vaccine trials were undertaken to determine whether the Fel-O-Vax FIV, a commercial dual-subtype (subtypes A and D) feline immunodeficiency virus (FIV) vaccine, is effective against a subtype B FIV isolate. Current results demonstrate the Fel-O-Vax FIV to be effective against a subtype B virus, a subtype reported to be the most common in the USA.