Expression of α- and ε-toxin genes in Clostridium perfringens type D vaccine strain in contact with the Caco-2 cell line.

Background: Clostridium perfringens commonly resides in the gastrointestinal tract and can survive in different environmental conditions. This pathogen produces several protein toxins including the potent ε-toxin which is classified as a category B toxin by the Centers for Disease Control and Prevention (CDC). In several studies, the induction of C. perfringens type C or D to produce toxins much more rapidly by close contact of bacteria with Caco-2 cells has been reported. Aims: The effect of close contact of enterocyte-like Caco-2 cells with C. perfringens type D (vaccine strain) on the production time of ε- and α-toxins was studied. Methods: During C. perfringens type D contact with Caco-2 cells for 5 h, ε- and α-toxins expressions (at 0, 2, and 5 h) were evaluated by a quantitative real-time PCR assay. Non-contacted bacteria with cells were included as the negative control in this research. Results: Bacterial contact with the Caco-2 cells induces a significant effect on the mean expression of the ε-toxin gene (etx) (P<0.05). Two h after contact, the highest level of gene expression was detected in the experimental group. Bacterial harvesting time, cell treatment, and their interactions did not affect significantly the mean expression of the α-toxin gene (cpa) (P>0.05). Conclusion: According to the findings of the present study, 2 h of bacterial contact with Caco-2 cells could stimulate etx gene expression in the C. perfringens type D vaccine strain.

Antibody titers against canine distemper virus in unvaccinated rural dogs from Ahvaz, Iran.

The purpose of this study was to evaluate the prevalence of antibodies to Canine Distemper Virus (CDV) in unvaccinated rural dogs without known immunization status and to assess risk factors for infection by means of indirect immunofluorescent assay (IFA), in Ahvaz district, southwest of Iran. Serum samples were randomly collected from 97 healthy dogs 6 months and older from villages around Ahvaz city between 2004 and 2005. Dogs were grouped by age and sex to determine whether these factors were associated with antibody titers, using Fisher's exact test. Seroprevalence to CDV antibodies in these dogs were 17.52% indicating that this virus is present in the ecosystem. Also there is evidence of previous natural exposure to CDV. Prevalence of antibodies to CDV did not differ between sexes or ages. Rural dogs are abundant in the ecosystem of area and interact with other species of wild carnivores and domestic animals in ways that could encourage disease transmission. Therefore the role of rural dogs in the epizootiology of CDV in both urban dogs and wildlife needs to be further explored.

The role of outer membrane proteins of Ornithobacterium rhinotracheale in attachment to chicken tracheal epithelium.

Ornithobacterium rhinotracheale (ORT) infections cause major losses to the poultry industry. In search for factors implicated in the pathogenesis of ORT infections, the role of outer membrane proteins (OMPs) in the interaction of ORT with chicken tracheal epithelium was investigated. For this purpose, immune sera were prepared against total extracted OMPs, whole cell bacteria and three major OMPs of 45, 53 and 70 kDa and used in bacterial adherence inhibition assay. The results showed antibodies against a 53 kDa OMP significantly (p < or = 0.05) inhibited the bacterial adherence to chicken tracheal epithelium up to 78%.

Molecular G typing of bovine rotaviruses in Iran.

Fifty rotavirus-positive feacal samples, selected from 500 ELISA tested diarrheic specimens were used in this study. Viral RNA was extracted from each sample and reveres transcribed to cDNA. The cDNA was then amplified by oligonucleotide primers specific for RNA segment 9, coding for VP7. After the first amplification, PCR products were subjected to a multiplex semi-nested PCR to investigate the presence of bovine rotavirus serotypes: G6, G8 and G10. The results indicated prevalence of 48 and 26% for G6 and G10 serotypes, respectively. Twenty four percent of the samples showed a mix infection by G6 and G10 serotypes and no sample was found positive for the type G8. With the best of our knowledge this is the first report of molecular typing of bovine rotaviruses in Iran.

A survey of the prevalence of infectious bronchitis virus type 4/91 in Iran.

To evaluate the prevalence of infectious bronchitis virus (IBV) type 4/91 in Iran, tracheal swabs from 77 broiler flocks in 16 provinces were collected at the slaughterhouse. Swabs were subjected to RNA extraction and tested by RT-PCR, followed by a type-specific nested PCR. The viral RNA was detected in 33 samples (42.8%) from different provinces. The results indicate a relatively high prevalence of IBV type 4/91 in Iran and necessitate revising the vaccination programme against this disease.

Immunogenicity of E. coli-expressed sigma3 protein of avian reovirus in chickens.

An E. coli-expressed fusion protein was used to study the immunogenicity of avian reovirus sigma3 protein in chickens. The protein induced the production of specific antibodies detectable by immunofluorescence, ELISA and immunoblot. The antibody titre was low as determined by ELISA and was negative by virus neutralization test. However, when tested in passive immunization studies, the chicken antiserum specific to this protein and control antiserum from chickens vaccinated with avian reovirus SI 133 strain showed some protection against virus-induced mortality in 1-day-old chicks. The results thus confirm the importance of humoral immunity against avian reovirus infection and indicate that sigma3 protein may play a role in the induction of protective antibodies.

Evidence for the multimeric nature and cell binding ability of avian reovirus sigma 3 protein.

It has been suggested that avian reovirus sigma 3 protein is analogous to sigma 1 trimer, the mammalian reovirus attachment protein. We have investigated the multimeric nature and cell binding ability of sigma 3 protein. The data presented here demonstrate that sigma 3 protein is a multimer in its undisrupted form as determined by SDS-PAGE in non-dissociating conditions. However, virion-associated sigma 3 protein and COS-7 cell-expressed protein behaved differently in SDS-PAGE, suggesting a need for virus-associated factor(s) to control the multimerization of the protein. The data also show that Escherichia coli expressed sigma 3 fusion protein (sigma 3-MBP) in its multimeric form is capable of attaching to Vero cells. The binding was found to be specific and receptor mediated by the fact that it was inhibited by a monoclonal antibody specific for sigma 3 protein and by competition with avian reovirus particles. As determined by a reverse experiment, sigma 3-MBP was also able to reduce the virus p.f.u. in monolayer cell cultures, indicating the important role of sigma 3 protein in the initiation of virus infection.

Characterization of monoclonal antibodies against avian reovirus strain S1133.

Monoclonal antibodies (MAbs) were produced against a vaccinal S1133 strain of avian reovirus. Characterization of six MAbs in Western blotting, radioimmunoprecipitation and gold immunoelectron microscopy revealed that the MAbs were specific to the outer capsid proteins, mu2/mu2c, sigma2 and sigma3. Two of three MAbs, directed against sigma2 protein, neutralized the virus infectivity in a broadly specific manner, whereas the third had no neutralizing activity. The only MAb which reacted with sigma3 protein showed a type-specific neutralizing activity. Two MAbs recognizing the mu2/mu2c proteins failed to neutralize the virus infectivity.

Cloning, sequencing and expression of the S1 gene of avian reovirus.

The S1 genome segment of avian reovirus strain S1133 was cloned and completely sequenced. The sequence comprised 1636 bp with three distinct open reading frames (ORFs), suggesting the gene was polycistronic in nature. The three ORFs from 5' to 3' were predicted to encode polypeptides of 9.8, 3.8 and 34.9 kDa, respectively. Of the three ORFs, only the third possessed the AUG initiation codon in an optimum context for translation. The third ORF-encoded protein, 326 amino acids in length, was expressed in Escherichia coli and used as antigen in immunoblots. The protein was concluded to be sigma 3 on the basis of its recognition by a chicken anti-reovirus antiserum and due to the fact that a mouse antiserum raised against it recognized specifically the viral sigma 3 polypeptide. Sequence comparison of the avian reovirus S1 gene with its mammalian counterpart did not show any significant similarity between the two. However, amino acid sequence analysis and the predicted existence of a heptapeptide repeat pattern, as well as the relatively high frequency of alpha-helix structures in the amino terminal portion of sigma 3 suggests that this protein is structurally, and probably functionally, related to mammalian reovirus sigma 1 protein.

Interaction of avian reovirus with chicken lymphoblastoid cell lines.

Four chicken lymphoblastoid cell lines were inoculated with avian reovirus strain S1133 and two local isolates, 965 and 615. Of the inoculated cell lines, TLT, a B-cell line, was productively infected with the three viruses as demonstrated by immunofluorescence assay (IFA) and radioimmunoprecipitation assay. A comparative growth curve analysis of the three avian reoviruses was done at 37 degrees and 41 degrees C. Isolate 965 replicated to a higher titre at both temperatures while the replication of S1133 and 615 was found to be inhibited at 41 degrees C. IFA revealed that among the transformed T lymphoblastoid cells used in this study, only MDCC-RP1 was permissive to virus infection with isolate 965, and at 41 degrees C, but not 37 degrees C.