Efficacy of a new inactivated Chlamydophila felis vaccine in experimentally-infected cats.

A new inactivated and adjuvanted Chlamydophila felis vaccine was developed and its efficacy in cats was compared with that of commercially available inactivated and live vaccines. Two commercial vaccines conferred insufficient immunity on inoculated cats, as evaluated by antibody production and a challenge experiment, whereas cats administered the newly generated vaccine produced high-titre antibodies and acquired sufficient immunity. The cats immunised with the new vaccine revealed no or only mild clinical signs, and no chlamydiae were recovered from their tissue samples after exposure to a virulent C felis. However, they shed chlamydiae in their nasal and conjunctival secretions after challenge, as did those immunised with the commercial vaccines and the non-vaccinated controls. The newly developed vaccine caused no adverse reaction in the inoculated cats. These findings suggest that the new vaccine prepared here may be promising for practical use in controlling C felis infection in cats.

Immunological and genetic characterization of feline caliciviruses used in the development of a new trivalent inactivated vaccine in Japan.

Although vaccination against feline calicivirus (FCV) infection is widespread in Japan, FCV-associated diseases are still a significant problem in cats. Thus, we developed a new trivalent inactivated vaccine, Kyoto Biken Feline-CPR, consisting of three FCV strains; one was the production strain of our previous vaccine, and the others were screened from 60 field isolates obtained between 1998 and 2000 based on cross-neutralization tests. In this report, the three FCV strains used for development of the new vaccine were antigenically and genetically characterized. The three strains were antigenically quite different, as revealed by cross-neutralization tests. Alignment of deduced amino acid sequences of capsid regions A to E revealed that there were marked differences between the strains in both the N- and C ends of region E. Antisera against the three vaccine strains, our new vaccine and 2 commercial vaccines were then evaluated for neutralization with 58 field isolates collected between 2003 and 2006. Rat antisera against the three vaccine strains and a mixture of the 3 strains neutralized 49, 37, 42 and 55 isolates, respectively. Cat antiserum against the new vaccine neutralized 50 (86.2%) isolates, whereas the numbers neutralized by cat antisera against 2 commercial vaccines were 37 (63.8%) and 25 (43.1%). In conclusion, the immunological and genetic properties of the 3 vaccine strains investigated varied widely, and the Kyoto Biken Feline-CPR vaccine may have more potential to meet the antigenic diversity of FCVs spreading throughout Japan.

Genetic and antigenic characterization of newly isolated bovine toroviruses from Japanese cattle.

Torovirus, a member of the Coronaviridae family, is a gastrointestinal infectious agent that has been identified in humans, cattle, pigs, and equines. Toroviruses, except equine torovirus, are difficult to propagate in cell culture; indeed, to date, only the Aichi/2004 strain of bovine torovirus (BToV) has been isolated among the human, bovine, and porcine toroviruses. In the present study, four cytopathogenic BToVs were isolated from diarrheal feces of the cattle using the HRT-18 cell line, and their genetic and antigenic properties were compared. The cytopathogenic features of BToV isolates in HRT-18 cells were similar to those of the Aichi/2004 strain. However, none of the isolates showed cytopathogenic effects in the HRT-18 cells of different origin, suggesting that one significant factor contributing to the cytopathogenicity of BToV depends on properties of the HRT-18 cells themselves. All BToVs isolated were able to agglutinate mouse, but not chicken, erythrocytes, while they lacked receptor-destroying enzyme activity. Analysis of the N terminus of the spike gene showed that three isolates, but not the Gifu-2007TI/E strain, were phylogenetically located in cluster 1 and its analogs and revealed high cross-reactivity with each other, as demonstrated by neutralization (NT) and hemagglutination inhibition (HI) assays. The Gifu-2007TI/E strain was classified close to cluster 2 and exhibited relatively low cross-reactivity with these viruses; however, the difference was not sufficient to classify BToVs into serotypes, suggesting that at least two subtypes distinguishable by the structure of the N terminus of the spike gene and that both NT and HI tests may be exist.

Detection and characterization of bovine torovirus from the respiratory tract in Japanese cattle.

Bovine torovirus (BToV), a member of the Coronaviridae family, is a causative agent of diarrhea in cattle, but it may also possess tropism for the respiratory tract. However, no surveys concerning with the relation between respiratory symptoms and the detection of BToV have been conducted in wide range. Among 311 nasal samples, BToV gene products were detected in seven samples (rBToV-1 to -7) derived only from calves with respiratory symptoms, suggesting that BToV may be a predisposing factor and/or causative agent for bovine respiratory disease. Regarding the degree of similarity between the spike and hemagglutinin-esterase coding regions, the rBToVs showed over 90.8% similarity with one another and 73.5-99.0% similarity with fecal tract-derived BToVs. rBToV-1, -2, and -3 were identical despite their being collected during different seasons; in comparison, rBToV-4 and -5 were distinct despite the fact that they were collected from the same herd, suggesting the existence of diversity among domestic rBToVs. One animal with a BToV-positive nasal sample also shed the virus in its feces, suggesting dual tropisms for BToV.

Identification and characterization of haemagglutinin epitopes of Avibacterium paragallinarum serovar C.

The objectives of this study were to identify haemagglutinin (HA) epitopes of Avibacterium paragallinarum serovar C that are capable of eliciting haemagglutination inhibition (HI) antibody, and to investigate their immunogenic role. Three conformational epitopes were detected on HA by blocking ELISA and immuno-dot blot analysis using a panel of five monoclonal antibodies (MAbs) with HI activity, designated 8C1C, 4G8B, 24E4D, 11E11B, and 10D1A. The minimum DNA regions coding these three epitopes were 3195, 2862, and 807bp in size, and mapped within a gene with 6117bp. Nine DNA fragments of various lengths were prepared, and their recombinant proteins were generated in E. coli. One recombinant protein, designated HPC5.5, was recognized by MAb 8C1C, and had strong ability to adsorb HI antibody to Av. paragallinarum serovar C. Other recombinant proteins designated HPC5.1, HPC4.8, and HPC2.5 did not react with MAb 8C1C and only slightly adsorbed HI antibody. All chickens immunized once with HPC5.5 did not show any typical clinical signs such as nasal discharge or facial edema against challenge inoculation with Av. paragallinarum serovar C. However, HPC5.1, which was recognized by four MAbs (not including MAb 8C1C), showed only partial protective immunity in five of eight immunized chickens. The results suggest that the HA epitope recognized by MAb 8C1C is the major epitope responsible for eliciting HI antibody, and HPC5.5 is a practical candidate protein to develop a new vaccine against avian infectious coryza caused by Av. paragallinarum serovar C.

A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C.

An unknown cytotoxin was identified in the culture supernatant of Clostridium perfringens type C. The cytotoxin, named TpeL, which was purified using mAb-based affinity chromatography, had a lethal activity of 62 minimum lethal dose (MLD) mg(-1) in mice and a cytotoxic activity of 6.2x10(5) cytotoxic units (CU) mg(-1) in Vero cells. The nucleotide sequence of TpeL was determined. The entire ORF had a length of 4953 bases, and the same nucleotide sequence was not recorded in the GenBank/EMBL/DDBJ databases. The molecular mass calculated from the deduced amino acid sequence was 191 kDa, and a signal peptide region was not found within the ORF. The deduced amino acid sequence exhibited 30-39 % homology to Clostridium difficile toxins A (TcdA) and B (TcdB), Clostridium sordellii lethal toxin (TcsL) and Clostridium novyi alpha-toxin (TcnA). The amino acid sequence of TpeL is shorter than these toxins, and the homologous region was located at the N-terminal site. Eighteen strains of C. perfringens types A, B and C were surveyed for the presence of the tpeL gene by PCR. The tpeL gene was detected in all type B (one strain) and C strains (five strains), but not in any type A strains (12 strains). TpeL was detected in culture filtrates of the five type C strains by dot-blot analysis, but not in the type B strain. It was concluded that TpeL is a novel toxin similar to the known large clostridial cytotoxins. Furthermore, the data indicated that TpeL is produced by many C. perfringens type C strains.

Porcine T-cell receptor beta-chain: a genomic sequence covering Dbeta1.1 to Cbeta2 gene segments and the diversity of cDNA expressed in piglets including novel alternative splicing products.

Porcine TCRbeta-chain cDNA clones were isolated from thymic and peripheral blood lymphocytes of piglets. Using these nucleotide sequences, a genomic 18kbp sequence stretch covering Dbeta1 to Cbeta2 gene segments was identified, which revealed that the porcine TCRbeta-chain locus consists of two sets of Dbeta-Jbeta-Cbeta gene groups with each set having a Dbeta gene segment, seven Jbeta gene segments and a down stream Cbeta gene segment composed of four exons. This structure is consistent with other known mammalian TCRbeta-chain loci. With this genomic information, TCRbeta-chain clones from cDNA libraries were analyzed. Sixteen Vbeta gene segments were obtained accompanied by either Dbeta1 or Dbeta2 and by one of the nine Jbeta gene segments. Five different Cbeta cDNA sequences were obtained including four types of Cbeta1 sequences and one type of Cbeta2 sequence. The differences among the Cbeta1 sequences are either allelic polymorphisms or two splice variants, one being a product of exon1 splicing to exon3 (exon2 skipping), and another being an alternative splicing using a splice acceptor site newly discovered inside Cbeta1 exon4. The latter splice acceptor site was also found in human, mouse and horse all giving short cytoplasmic domain with Phe at their C-terminal ends. Other splicing products included trans-splicing of Jbeta2 to Cbeta1, non-functional splicing of two Jbeta gene segments in tandem and a part of Jbeta2.7-Cbeta2 intron to Cbeta2 exon1. Numerous examples of splice variants may suggest the involvement of splicing in generating TCRbeta-chain functional diversity.

Polymerase chain reaction-based genetic typing of Japanese porcine reproductive and respiratory syndrome viruses.

Porcine reproductive and respiratory syndrome viruses (PRRSVs) are classified into 2 distinct genotypes: the North American type and the European type. The Japanese PRRSVs were genotyped by reverse transcriptase-polymerase chain reaction using the reported primer pairs that were either reactive to both types, specific to the North American type or specific to the European type. All the PRRSV genomes from 66 tissue homogenates or sera and 55 infectious viruses were of the North American type, whereas no European-type viral genome was detected. Two PCR primers specific to the North American type showed different detection efficiency. Half of the tissue samples and 15% of the infectious viruses were not detected with one primer pair, although all of them were detected with the other primer pair. Nucleotide sequencing analysis of the forward-and reverse primer-binding sites of the nonreactive viruses indicated that all these viruses had nucleotide mismatches within the 4 bases corresponding to the 3' end of the reverse primer. These mismatches appeared to be responsible for the nonreactivity of the former primers to these viruses.

Comparison of capsid sequences from human and animal astroviruses.

We have sequenced the genomic 3'-end, including the structural gene, of human astrovirus (HAstV) serotype 7 and morphologically related viruses infecting pig (PAstV), sheep (OAstV) and turkey (TAstV-1). These sequences were compared with corresponding astrovirus sequences available in the nucleic acid databases, including sequences of the seven other HAstV serotypes, two other avian astroviruses (TAstV-2 and avian nephritis virus) and astrovirus from cat (FAstV). A 35 nt stem-loop motif near the 3'-end of the genome, previously described as being highly conserved, was present in all of the astroviruses except TAstV-2. In the N-terminal half of the capsid precursor protein, there were several short conserved peptide motifs. Otherwise the capsid proteins of astroviruses infecting different hosts were highly divergent. Calculation of genetic distances revealed that the distance between FAstV and HAstV is comparable to the largest distances between different HAstV serotypes. Higher similarities between the HAstV, FAstV and PAstV capsid sequences suggest interspecies transmissions involving humans, cats and pigs relatively recently in the evolutionary history of astroviruses.