Virulence genes and antimicrobial susceptibility in Pasteurella multocida isolates from calves.

A total of 378 isolates of Pasteurella multocida from clinically healthy and diseased calves were characterised for their susceptibility to 9 antimicrobial agents and screened by PCR for the presence of antimicrobial resistance genes and 22 genes virulence-associated, including capsule biosynthesis genes. Of the 378 isolates, 102 (27.0%) were resistant to at least one of the 9 tested antimicrobial agents. Resistance to oxytetracycline (21.7%) was the most frequently observed phenotype among the isolates. The tet(H) gene were the primary determinant detected. The resistance rates for thiamphenicol, ampicillin, kanamycin and florfenicol were 13.2%, 5.8%, 9.0% and 0.5%, respectively. Cefazolin, ceftiofur, cefquinome and enrofloxacin were effective antimicrobial agents, with no resistant isolates emerging over the course of the investigation. Most isolates were identified as capsular type A, only 6.3% belonged to capsular type D and no other capsular type was identified. Four of the virulence-associated genes (pfhA, tadD, tbpA and HAS) exhibited associations to the capsular type, and three (pfhA, tbpA and hgbB) were associated with the disease status of the animals. These virulence genes have been considered as epidemiological markers and are hypothesised to have a strong positive association with the outcome of disease in cattle.

Relationship between serotype and the antimicrobial susceptibility of Mannheimia haemolytica isolates collected between 1991 and 2010.

The antimicrobial susceptibilities and serotype distribution of 310 Mannheimia haemolytica isolates obtained from cattle with bovine respiratory disease during 2002-2010 were investigated. Of the 310 isolates, 198 (63.9%) were resistant to at least one of the 16 tested antimicrobial agents. The resistance rates for ampicillin, amoxicillin, dihydrostreptomycin, kanamycin, oxytetracycline, doxycycline, chloramphenicol, thiamphenicol, nalidixic acid, enrofloxacin, and danofloxacin were 20.3%, 14.5%, 43.5%, 23.5%, 24.8%, 21.9%, 23.2%, 23.9%, 47.1%, 18.7%, and 18.7%, respectively. Almost 90% of the isolates belonged to three serotypes (serotypes A1, A2, and A6), and the relative prevalence of serotype A6 increased significantly over the last decade. Compared with bacteria belonging to other serotypes, bacteria belonging to serotype A6 exhibited a significantly higher antimicrobial resistance rates (χ2 test, p<0.05). The results of this investigation provide useful information for understanding the serotype prevalence and antimicrobial resistance patterns of one of the major bacteriological agents implicated in pneumonic pasteurellosis.

Effects of ribavirin combined with interferon-alpha 2b on viral kinetics during first 12 weeks of treatment in patients with hepatitis C virus genotype 1 and high baseline viral loads.

This study aimed to find how ribavirin increases viral disappearance in patients with hepatitis C virus (HCV) of genotype 1 and high baseline viral loads (>5.0 x 10(5) copies/mL) when given with interferon (IFN). Using the real-time quantitative polymerase chain reaction, we measured serum HCV in 20 patients during the first 12 weeks of therapy with IFN-alpha 2b and ribavirin. Controls were 10 similar patients given IFN-alpha 2b alone. IFN-alpha 2b was given at 6 MU daily for 2 weeks, and then three times weekly. Ribavirin was given at 600 or 800 mg daily. Serum HCV RNA decreased rapidly in the first phase, during the first 24 h of therapy (day 0), and more slowly in the early second phase (days 1-14). The median decrease was by 1.41 and 0.078 log 10/day in these two phases in the combination therapy group, and 0.90 and 0.081 log 10/day in the monotherapy group. The difference between groups in the first phase was not significant (P = 0.24), nor was that in the next phase (P = 0.68). Later in the second phase, between days 14 and 84, the median decrease was larger in the combination therapy group (0.030 log 10/day) than in the monotherapy group (0.015 log 10/day, P = 0.035). In patients with HCV genotype 1 and high viral loads, the effects of ribavirin with IFN-alpha appeared slowly, after the earliest days of treatment. A long-term favourable outcome of combination therapy may be associated with a rapid viral decline in this later phase of therapy.

Molecular typing of Mannheimia (Pasteurella) haemolytica serotype A1 isolates from cattle in Japan.

Pulsed field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD) methods were applied for molecular typing of 130 Mannheimia (Pasteurella) haemolytica serotype A1 isolates obtained from 13 prefectures in Japan. These isolates were divided into 15 ApaI PFGE profiles that formed six distinct clusters (clusters A-F). Fifty-three (40.7%) isolates were classified in cluster B, and 20.0, 13.8, 12.3, 6.9 and 6.1% of isolates were in clusters E, A, F, D and C, respectively. The isolates of cluster B were differentiated into seven subtypes (B1-B7) and subtype B5 contained 63% (34/53) of isolates. RAPD revealed four banding patterns (types I-IV), and among 130 isolates 60.7% (79/130) of isolates were RAPD type I. All of the RAPD type I isolates were grouped into clusters A-C by PFGE. There was no relationship between molecular typing and geographic origin of these isolates. These results indicate that isolates of M. haemolytica A1 strain with various molecular profiles have already spread in Japan and may have caused sporadic infections.

Further development of a recombinant feline herpesvirus type 1 expressing the Gag protein of feline immunodeficiency virus.

We have previously reported the construction of a recombinant feline herpesvirus type 1 (FHV-1), designated C7301ddlTK-gag, expressing the Gag precursor protein of feline immunodeficiency virus (FIV). In this study, we report the construction of a further recombinant FHV-1 (ddlTK(gBp)-gag) which carries an FHV-1 gB promoter sequence upstream of the FIV gag gene of C7301ddlTK-gag. Strong expression of the FIV Gag protein by ddlTK(gBp)-gag was confirmed by immunoblot analyses and enzyme-linked immunosorbent assays. Although C7301ddlTK-gag and ddlTK(gBp)-gag failed to induce anti-FIV Gag antibodies in cats, we confirmed the infectivity and stability of these recombinants in cats.

Membrane fluidity correlates with liver cancer cell proliferation and infiltration potential.

We developed a method to measure membrane fluidity of living cancer cells in two- and three-dimensional cultures, and found that there was a close relationship between the membrane fluidity of cancer cells and their proliferative and infiltrative ability. Membrane fluidity is thus a promising indicator of the probability of cancer recurrence.

Efficient expression of the envelope protein of feline immunodeficiency virus in a recombinant feline herpesvirus type 1 (FHV-1) using the gC promoter of FHV-1.

We constructed two recombinant feline herpesvirus type 1 (FHV-1) expressing the envelope (Env) protein of feline immunodeficiency virus (FIV). One recombinant, designated dlTK-env, has the whole FIV env gene inserted at a thymidine kinase (TK) deletion site. The second recombinant, designated dlTK(gCp)-env, has a cassette containing a partial FIV env gene fused with the signal sequence of the gC protein of FHV-1 (under the control of the gC promoter) inserted at the same site. Growth kinetics of both the recombinants in Crandell feline kidney (CRFK) cells were similar to that of the parent strain of FHV-1. By indirect immunofluorescence assays and immunoblot analyses, we confirmed the expression of the FIV Env protein in CRFK cells infected with both recombinants. Enzyme-linked immunosorbent assays showed that the maximum Env expression level achieved by dlTK(gCp)-env was more than four times higher than that observed for dlTK-env. Flow cytometric analyses revealed that the Env protein produced by both recombinants was efficiently expressed on the cell surface. The dlTK(gCp)-env reported here may thus be a promising candidate for a live recombinant vaccine to protect against FIV infection.

Characterization of feline CD56 molecule expressed on insect cells by the baculovirus expression system.

Recently we cloned 140 kDa form of feline CD56 cDNA. In this study, we expressed the feline CD56 molecule by the baculovirus expression system. We found that the molecule was expressed on the cell surface when examined by the indirect immunofluorescence assay using an anti-human CD56 monoclonal antibody. Immunoblotting analysis revealed that the molecular weight of the major expressed product was 140 kDa. Interestingly we found that the insect cells expressing the feline CD56 molecule aggregated, indicating that the expressed molecule mediates homophilic adhesion.

Molecular cloning and expression of feline CD3epsilon.

The cDNA of feline CD3epsilon, one of the T-cell receptor components, was cloned from a feline T-lymphoblastoid cell line (MYA-1 cells) and peripheral blood mononuclear cells and thymocytes of cats by polymerase chain reaction. Sequencing analysis revealed that the open reading frame of feline CD3epsilon consists of 606 base pairs encoding a predicted molecular mass of 25 kDa transmembrane protein which lacks N-glycosylation site. Comparison of the predicted amino acid sequence of feline CD3epsilon with those of other mammalians' homologues revealed that a relatively low homology was present in the extracellular domain. However, the cytoplasmic domain contained several characteristic motifs highly conserved across the species. These motifs were known to be important for signal transduction upon T-cell activation or endoplasmic reticulum retention. In addition, the feline CD3epsilon protein was expressed in an insect cell line (Sf9) by a baculovirus expression system. The expression was confirmed by indirect immunofluorescence assay and immunoblotting analysis using an anti-human CD3epsilon polyclonal antibody. These results will provide additional information for understanding the feline immune system.

Molecular cloning and sequencing of feline stromal cell-derived factor-1 alpha and beta.

The stromal cell-derived factor-1 alpha and beta (SDF-1 alpha/beta) are the ligands of fusin/CXCR4, the co-receptors of human immunodeficiency virus type 1 and the feline immunodeficiency virus. We cloned the cDNA of feline SDF-1 alpha/beta. The open reading frames of feline SDF-1 alpha/beta were 267/279 base pairs and encoded 89/93 amino acid residues.

Cats are protected against feline immunodeficiency virus infection following vaccination with a homologous AP-1 binding site-deleted mutant.

Following establishment, via the vaginal route, of infection with an AP-1 binding-site deleted mutant (delta AP-1) of feline immunodeficiency virus (FIV), cats were challenged with a homologous intact strain (TM2) of FIV. The cats were observed for 23 weeks to evaluate the efficacy of the delta AP-1 against the homologous TM2 strain challenge. These two viruses were differentiated by Southern blotting after amplification of proviral DNA by semi-nested polymerase chain reaction in DNAs of peripheral blood mononuclear cells and tissues. A TM2-specific band was detected in one cat exposed to but not infected with delta AP-1, but not in two delta AP-1-infected. These results indicate that delta AP-1 could protect against subsequent challenge with homologous FIV TM2 strain.

Construction of a recombinant feline herpesvirus type 1 expressing Gag precursor protein of feline immunodeficiency virus.

We constructed a deletion mutant of feline herpesvirus type 1 (FHV-1) and a recombinant FHV-1. The deletion mutant is the virus with a region (367 bp) deleted from the start codon of thymidine kinase (TK) gene to the SmaI site within the TK gene, and the other is a recombinant FHV-1 expressing Gag protein of feline immunodeficiency virus (FIV), in which a cDNA encoding the Gag protein of FIV was inserted at the TK deletion site of the former deletion mutant. These viruses were designated as C7301ddlTK and C7301ddlTK-gag, respectively. Growth kinetics of these viruses in Crandell feline kidney cells was similar to that of the parent C7301 strain. By immunoblot analysis, C7301 ddlTK-gag was confirmed to express the FIV Gag precursor protein in the cells.

Eight-year observation and comparative study of specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) subtypes A and B: terminal acquired immunodeficiency syndrome in a cat infected with FIV petaluma strain.

Three specific pathogen-free cats experimentally infected with feline immunodeficiency virus (FIV) strains Petaluma, TM1 and TM2, respectively were observed for over 8 years. Without showing any significant clinical signs of immunodeficiency syndrome (AIDS) for 8 years and 4 months of asymptomatic phase, the Petaluma-infected cat exhibited severe stomatitis/gingivitis, anorexia, emaciation, hematological and immunological disorders such as severe anemia, lymphopenia, thrombocytopenia, and decrease of CD4/CD8 ratio to 0.075, and finally died with hemoperitoneum at 8 years and 8 months post-infection. Histopathological studies revealed that the cat had systemic lymphoid atrophy and bone marrow disorders indicating acute myelocytic leukemia (aleukemic type). Plasma viral titer of the cat at AIDS phase was considerably high and anti-FIV antibody titer was slightly low as compared with the other FIV-infected cats. In addition, immunoblotting analysis using serially collected serum/plasma samples of these cats revealed that antibodies against FIV proteins were induced in all the infected cats, however in the Petaluma-infected cat anti-Gag antibodies disappeared during the asymptomatic period. These results suggested that plasma viral load and anti-FIV Gag antibody response correlated with disease progression, and supported FIV-infected cats as a suitable animal model of human AIDS.

Cross virus neutralizing antibodies against feline immunodeficiency virus genotypes A, B, C, D and E.

Feline immunodeficiency virus (FIV) is classified into five genotypes, A, B, C, D and E, based on the phylogenetic analysis of the env V3-V5 region. However, whether there is correlation between phylogenetic and antigenic diversities remains unknown. In this study, we examined the cross virus neutralization of FIV genotypes A through E by sera from cats infected with a single genotype. The results indicated some relationships between phylogenetic genotype and neutralization serotype, and that cross-clade virus neutralization is possible. For example, serum from a cat infected with genotype E virus neutralized all five FIV genotypes. Our results suggest that the FIV subtyping according to the sequence diversity is partially reflected by antigenic diversity and serum neutralization.

Expansion of CD8alpha+beta- cells in cats infected with feline immunodeficiency virus.

CD8+ lymphocytes have been subdivided into CD8alphabeta and CD8alpha alpha populations in the peripheral blood lymphocytes (PBL) of humans and in several animal species but have not yet been investigated in cats. Feline immunodeficiency virus (FIV) causes progressive immunological disorders similar to human AIDS. In this study, we analysed CD8+ cells in PBL of FIV-infected or uninfected cats by two-colour flow cytometric analysis. In specific pathogen-free adult cats, feline CD8alpha+beta(high) cells were observed but CD8alpha+beta- cells were not found in significant numbers. On the other hand, not only CD8alpha+beta(high) but also CD8alpha+beta- and CD8alpha+beta(low) cell populations were observed in cats chronically infected with FIV. The expansion of the CD8beta(low) or CD8beta- subpopulations resulted in the apparent differences in CD4/CD8 ratios depending on the anti-CD8 MAb used. These findings suggest a need to reconsider the CD4/CD8 ratio in studies of FIV infection. Furthermore, we found that the CD8alpha+beta- cell population expressed CD5 at a low level.

Protein-binding properties of the putative AP-1 and ATF sequences in the feline immunodeficiency virus long terminal repeat.

Electrophoresis-mobility-shift assays with nuclear extracts from a feline renal cell line and a T-lymphoblastoid cell line revealed that the AP-1 and ATF sites of feline immunodeficiency virus (FIV) TM2 strain had similar protein-binding properties to those of FIV Petaluma strain and consensus sequences of AP-1 and ATF sites, and that nuclear factors binding to these sites differed between the two cell lines. Cross-competition and gel-supershift assays demonstrated that the AP-1 and ATF sites had similar protein-binding properties. The effects of internal deletions of AP-1 and/or ATF sites on the basal promoter activity were also examined. Although deletion of either site moderately reduced activity, a mutant deleted in both sites had dramatically reduced activity. Therefore, we suggest that these two sites co-operatively regulate transcriptional activity of the promoter.

Phylogenetic analysis of feline immunodeficiency virus isolated from cats in Taiwan.

Feline immunodeficiency virus was isolated from four cats in Taiwan. The isolates were designated TI-1, TI-2, TI-3 and TI-4. Each was isolated from PBMCs following co-cultivation of PBMCs with a feline T-lymphoblastoid cell line (MYA-1 cells). However, the Taiwanese isolates did not grow in a feline kidney cell line (CRFK cells). The nucleotide sequences of the V3-V5 region of the envelope gene of the Taiwanese isolates were determined and compared with those of previously described isolates. Phylogenetic analysis of this region indicates that Taiwanese isolates belong to subtype C.

The effects of treatment with chemical agents or infection with feline viruses on protein-binding properties of the feline immunodeficiency virus long terminal repeat.

The effects of treatment with chemical agents or infection with feline viruses on protein-binding properties of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) were examined by gel-mobility-shift assays using oligonucleotides designed to represent putative AP-1 or ATF motif from the FIV LTR. Infection with FIV led to less nuclear proteins binding to the AP-1 and ATF sites, suggesting that proteins binding to the sites were consumed or suppressed by FIV-replication in FIV-infected cells. Nuclear proteins that bind to the AP-1 or ATF site were examined by using extracts from Crandell feline kidney (CRFK) cells treated with TPA (a phorbol ester; a strong activator of protein kinase C) or forskolin (an inducer of cyclic-AMP), or infection with feline herpesvirus type 1 (FHV-1). Although TPA or forskolin treatment moderately increased the level of both proteins that bound to AP-1 and ATF sites, FHV-1 infection markedly changed the protein-binding patterns of the sites. Furthermore, FHV-1-induced proteins that bind adjacent to the transcriptional initiation site of FIV promoter were also observed in FHV-1-infected CRFK cells, suggesting that the FHV-1-induced-proteins affects the transcription of FIV through the AP-1, ATF and leader sequences.

Phylogenetic analysis of feline immunodeficiency virus isolated from cats in Taiwan.

Feline immunodeficiency virus was isolated from four cats from Taiwan. The isolates were designated TI-1, TI-2, TI-3 and TI-4. Each was isolated from PBMCs following co-cultivation of PBMCs with a feline T-lymphoblastoid cell line (MYA-1 cells). However, the Taiwanese isolates did not grow in a feline kidney cell line (CRFK cells). The nucleotide sequences of the V3-V5 region of the envelope gene of the Taiwanese isolates were determined and compared with those of previously described isolates. Phylogenetic analysis of this region indicates that Taiwanese isolates belong to subtype C.