Seyed Ali Pourbakhsh

Molecular typing of Iranian field isolates Mycoplasma synoviae and their differentiation from the live commercial vaccine strain MS-H using vlhA gene

Abstract 1. The single-copy domain of the N-terminal region of the vlhA gene of Mycoplasma synoviae was sequenced, analysed and verified and used to type Iranian field isolates of M. synoviae and the MS-H live vaccine strain. In addition, a restriction fragment length polymorphism (RFLP) method was developed to differentiate between field isolates of Iranian and MS-H vaccine strains. 2. All sequences were analysed and aligned; the percentage similarity of the DNA was calculated and dendrograms were constructed. Based on single nucleotide polymorphism (SNP) that existed in all field isolates in Iran, the PCR-RFLP method allowed the differentiation of all M. synoviae field isolates from the vaccine strain. 3. Using phylogenetic analysis, the isolates were assigned to 8 unique genotypes and, within each group, DNA had a high level of similarity. 4. DNA sequence analysis and PCR-RFLP of the amplicon based on percent similarity and evolutionary relationship appeared to be useful tools for strain differentiation whether M. synoviae clinical isolates from infected chickens were derived from the vaccine strain or wild-type strains. 5. This study confirms the potential value of strain typing for epidemiological purposes and suggests that phylogenetic studies are essential to understand the true relationships between strains.

Experimental study on histopathological changes and tissue tropism of Iranian infectious bronchitis serotype 793/B-like virus in SPF chickens

Avian infectious bronchitis virus (IBV) is prevalent in all countries with intensive poultry flocks. This disease is characterised primarily by respiratory signs, but some IBV strains may also infect other organs such as the intestinal and urogenital tracts. The aim of this study was to characterise the histopathological lesions and tissue tropism of Iranian isolate IR/773/2001(793/B) of avian infectious bronchitis virus in different organs of experimentally infected SPF chickens. Forty-two one-day-old, specific pathogen-free (SPF) chicks were divided randomly into two groups (21 chicks to each group). At the age of 12 days, one group was inoculated intra-ocularly with 10(3) EID 50 of the 793/B isolate, and the other was kept as the control group. Tissue samples were collected at 2, 4, 6, 8, 10 and 12 days post-inoculation (PI). The IBV virus was detected in the caecal tonsils and cloaca from the 2nd to the 12th day PI. The virus was also detected in the kidneys from days 4-10 PI and in the bursa of Fabricius from days 4-12 PI. The virus was detected in the trachea, lungs and thymus. The most obvious histopathological lesions were found in the trachea, kidney, lungs and bursa of Fabricius. Amongst the lymphoid tissues, histopathological changes were found most frequently in the bursa of Fabricius. The results of this study indicated that the 793/B serotype of IBV is unlikely to cause mortality, severe clinical signs or gross lesions in infected chickens, but its replication in some tissues including the bursa of Fabricius could render birds susceptible to other micro-organisms.

Phylogenetic characterization of virulent Newcastle disease viruses isolated during outbreaks in northwestern Iran in 2010

The northwest of Iran shares long borders with three neighboring countries; therefore, it is considered one of the main entry portals of Newcastle disease virus (NDV) into the country. Ten virulent NDVs were recovered from 19 poultry farms of various prefectures in northwestern Iran during Newcastle disease outbreaks in 2010. The isolates were genotypically analyzed using an F-gene-specific reverse transcription polymerase chain reaction (RT-PCR) assay. The amplified F gene (nucleotides 189-1666) sequences of the NDV isolates were compared phylogenetically with those of previously published strains in GenBank. All of the NDV isolates belonged to genotype VIIb and were closely related to some isolates from Iran, Russia, and Sweden. Therefore, it can be postulated that these isolates evolved from previously reported strains. The velogenic viruses carried the motif 112R-R-Q-K-R/F117 at the F0 cleavage site and a unique substitution of 190L→F which had never been reported in any NDV genotype VIIb isolate. They shared high sequence similarity with each other but were distinct from current NDV vaccines and NDV strains reported from other countries. This information is fundamental for improving the efficacy of controlling strategies and vaccine development for NDV.

Phylogenetic analysis of Mycoplasma Synoviae isolated from commercial Iranian chicken farms compared with other GeneBank isolate sequences based on 16S rRNA gene

Mycoplasma synoviae (M. synoviae) is an important avian and extracellular pathogen which causes great economic losses in poultry industry. We have performed a phylogenetic analysis of M. synoviae isolates derived from commercial chicken farms in Iran based on 16S rRNA gene. Partial sequences of 16S rRNA genes obtained from 19 isolates of central region of Iran were amplified by PCR in 207bp fragment, then sequenced and next compared with the 16S rRNA gene of M. synoviae sequences that were available in GeneBank. Sequences were aligned using the ClustalW method, the UPGMA algorithm was used to create the phylogenetic tree. Phylogenetic analysis of these sequences demonstrated that all 19 M. synoviae isolated from Iran were most closely related to sequences of M. synoviae from Brazil. Variations, polymorphisms, and differences between nucleotides of all isolates were observed. The results of this study suggest that the close relationship, different molecular structure and heterogeneity among M. synoviae isolated may be explained by the transmission of mutations and variations between countries due to trading or to the stabilisation of spontaneous mutations because of regional conditions.

Isolation and Detection of Mycoplasma agalactiae from Semen Samples of Goats

Contagious agalactia (CA) is a highly infectious disease of goats and sheep, and is a form of Mycoplasmosis, which is usually enzootic. Since Mycoplasma agalactiae (M. agalactiae) is the main cause of this disease in goats, the aim of this study was to isolate and detect M. agalactiae from semen of goat bucks. Thirty-nine semen samples were collected from goat bulks, and all samples were cultured in PPLO broth medium supplemented for M. agalaciae isolation. The bacteria DNAs were extracted from clinical samples and the PCR assay was applied to detect Mycoplasma genus and M. agalactiae species using specific primers, which amplified a 163bp fragment in 16SrRNA gene and a 375bp fragment in lipoprotein gene. The PCR evaluations were performed for both the clinical samples and the cultures. Out of the 39 samples, 29 (74.3%) of the cultures were shown positive and typical Mycoplasma colonies grew on PPLO agar, which could be considered as the diagnostic method. In addition, 38 (97.4%) samples had positive PCR results for Mycoplasma genus and six (15.3%) of the samples were shown to be positive using PCR for M. agalactiae as the diagnostic method. In the present study, M. agalactiae was detected in semen of goat bulks for the first time in Iran. Therefore, it is recommended to concern semen as one of the significant sources for this pathogen and the possibility for transmission to the female goats through semen is highlighted. Moreover, presence of this microorganism in semen could be involved in infertility of goat population.