Effect of supplemental vitamin E on antibody titer in Japanese black calves vaccinated against bovine herpesvirus-1.

We investigated the effect of supplemental vitamin E on antibody titer against bovine herpesvirus-1 (BHV-1) in Japanese Black calves after vaccination with modified live virus. Thirty calves kept at the same farm were studied. They were divided into two groups; fifteen calves received 300 IU/day of vitamin E orally from 1 to 3 months of age (VE Group), and the other fifteen calves did not receive vitamin E supplement (Control Group). BHV-1 modified live vaccine was injected twice to all calves when they were 2 and 3 months of age. Following the vaccination, serum vitamin E concentration and neutralizing antibody titer to BHV-1 were measured over time. VE Group showed higher serum vitamin E at 2, 3 and 4 months of age compared to Control Group (P<0.05). The antibody titer in Control Group was the highest at 1 month of age, and it gradually decreased until 4 months of age. VE Group showed increase in antibody titer at 4 months of age resulting in significant difference (P<0.01) from Control Group. This study demonstrated that vitamin E supplementation to Japanese Black calves could increase antibody production after the second modified live BHV-1 vaccination.

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.

Epidemiological analysis of bovine torovirus in Japan.

Bovine torovirus (BToV), a member of the Coronaviridae family, is an established gastrointestinal infectious agent in cattle. No epidemiological research on BToV has been reported from Japan. In this study, we performed a survey to detect BToV in Japan in 2004 and 2005 using 231 fecal samples (167 from diarrheic cattle and 64 from asymptomatic cattle) that were analyzed by nested reverse transcription (RT) PCR using primers located in the consensus sequences of the reported BToV nucleocapsid (N), membrane (M), and spike (S) genes. BToV N, M, and S genes were detected in 6.5% (15/231), 6.1% (14/231), and 5.6% (13/231) of samples by nested-RT-PCR, respectively. In conclusion, detectability was improved compared to the results of the first round of RT-PCR. BToV was detected at a significantly higher rate in diarrheic samples than in asymptomatic samples (14/167 diarrheic samples [8.4%] and 1/64 asymptomatic samples [1.6%]), suggesting that BToV may act as a risk factor for diarrhea in Japanese cattle. The nucleotide sequence of M fragments from the BToV isolates including the newly identified Japanese isolates showed more than 97% identity. A similar degree of homology was observed in the N gene fragment among BToV isolates with the exception of BRV-1 and BRV-2. Domestic samples were classified into three clusters by phylogenetic analysis of the S gene fragment, which were considerably correlated with the geographic origin of the samples. BToV positive areas did not adjoin each other but were spread across a wide range, suggesting that BToV exists conventionally in Japan and is geographically differentiated. We also developed an RFLP method to distinguish these clusters using two restriction enzymes, HaeIII and AccI. This method should be useful for comparing newly acquired BToV-positive samples with the reported BToVs.

Genetic variation and cross-reactivity of Clostridium septicum alpha-toxin.

Clostridium septicum alpha-toxin genes were sequenced with the polymerase chain reaction (PCR) products amplified from DNAs of 25 C. septicum strains, and were classified into 10 patterns. Alpha-toxins were purified from the culture supernatant of four C. septicum strains (strains No. 44, Kagoshima 8, Mie and Tokachi) which were specially chosen from patterns of the deduced amino acid sequences. The molecular weights of the alpha-toxins were not different according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. However, the isoelectric points between the alpha-toxins of No. 44 and Tokachi strains differed markedly. Cross-neutralization tests were performed with purified alpha-toxins and antitoxins in mice and in Vero cells. Each antitoxin showed roughly the same titers against the four alpha-toxins in mice and completely identical titers against these in Vero cells. Calves immunized with toxoid prepared from the culture supernatant of No.44 strain were challenged by exposure to spores of Mie strain. The toxoid conferred protection against the challenge in calves. From these results, although genetic variation has been observed within the C. septicum alpha-toxin gene, C. septicum strains toxoid of strain No.44 induces protective immunity against exposure to C. septicum that produce other subtypes of alpha-toxin containing several different amino acid residues.