[Molecular characterization and phylogenetic study base on nucleocapsid gene of avian infectious bronchitis viruses isolated from broiler farms, 2014-2015]

Background: avian infectious bronchitis [IB] is an economically important poultry disease. The emergence of new infectious bronchitis virus genotypes has complicated IB control programs

Objectives: this is the first comprehensive molecular analysis of the Nucleocapsid [N] gene of Iranian IBVs

Methods: the nucleocapsid gene of ten IBV isolates [which belonges to four different genotypes] was amplified using specific primers. The phylogenetic trees were constructed based on nucleotide and amino acid sequences of "N" gene

Results: IBV genotyping based on "N" gene showed similar IBV classification which was obtained from spike gene analysis and ten isolates belonged to Massachusetts, QX, 793/B and Variant-2 genotypes. Different strains had 89.97- 99.75% homology in their amino acid sequences. The highest nucleotide sequence similarity was observed between IBKG-1 and IBKG-8 [793/B type IBVs], while the lowest was seen between IBKG-5 and IBKG-9 [QX- type and Variant-2 type] IBV isolates. This low similarity is of an interest because the N protein is highly conserved among different IBV strains. "N" Protein structural analysis revealed that the isolates has 8to 10 alpha helices and 6 to 8 beta sheets

Conclusions: the present study provided basic information to develop recombinant nucleocapsid proteins that are applicable in rapid diagnostic tests and ELISA and recombinant vaccines

Molecular characterization of recent Iranian infectious bronchitis virus isolates based on S2 protein gene

Background: avian infectious bronchitis [IB], with avian infectious bronchitis virus [IBV] as the causing agent, is a ubiquitous endemic disease of the chicken with devastating effects on its industry. A viral membrane surface protein called S not only induces neutralizing antibodies but also plays an important role in virus binding and entry to host cells. Technically, S1 protein gene sequencing also helps greatly in IBV genotyping

Objectives: the aim of this study was to characterize Iranian IBV based on S2 gene

Methods: after RT-PCR amplification, the S2 gene of nine Iranian IBV isolates were sequenced and then compared with reference strains

Results: the isolates were classified into genotype I as Massachusetts like IB Vs, genotype VII which clustered into two branches, VIIa [IS-1494 like IB viruses], and VIIb, and was related to QX- like viruses and Genotype VIII as 793/B like IBVs

Conclusions: as far as we know, this is the first S2-based classification study on Iranian IBV isolates providing a firm experimental basis to correlate with genotypic characterization

[Phylogenetic analysis comparing partial S1 gene of avian infectious bronchitis virus to commercial vaccine strains in Iran]

Background:Infectious bronchitis is a highly contagious disease which may cause poor weight gain and low feed efficiency in infected chickens. There are a large number of reported serotypes/genotypes, which makes the control of the disease more difficult through vaccination. However, there are only a few amino acid differences in the S1 protein of vaccine and wild type strains which are responsible for protection

Objectives: The purpose of this study is to compare IBV variants isolated from commercial chicken flocks in Iran with currently used vaccine strains

Methods: The partial S1 gene of the spike protein, covering a hypervariable and constant regions, was amplified and sequenced using conventional RTPCR

Results: Phylogenetic analysis of amino acid sequences revealed that eight of total nine isolates were divergence at least 21.8% from vaccinal Massachusetts serotypes, and six of nine isolates were divergence at least 22.7% from 4/91, and none of the nine isolates were similar to Dutch-type, D274,vaccine serotypes

Conclusions: These findings are essential for continuous surveillance disease control strategies and monitoring of variants, and thus emphasize on the importance of improving the vaccination program in Iran

[Molecular characterization and phylogenetic study based on matrix gene of avian influenza viruses [H9N2] in Iran during 1998-2008]

Background: H9N2 avian influenza viruses [AIV] A have become panzootic in Eurasia over the last decade and have caused several human infections in Iran since 1998 and inactivated vaccine has been used in chickens to control the disease

Objectives: The purpose of this study was to analyze H9N2 viruses that have infected broiler in Tehran Province, Iran between 1998 and 2008 based on Matrix gene

Methods: The complete coding region of Matrix [M] gene from 8 of H9N2 subtype isolated from chicken flocks in Tehran Province during 1998-2007 was amplified and sequenced

Results: Sequence analysis and phylogenetic studies of H9N2 viruses on the basis of data of viruses in this study and other selected strains available in the GenBank were conducted and determined variations among these sequences at different levels. Sequence analysis revealed a large number of similar substitution mutations and close evolutionary relation among sequences of M gene. Phylogenetic analysis showed that all our isolates belonged to the G1-like sublineage. In this study, it was determined that Iran's isolates have been in two separate branches and have the most similarity with Pakistan, United Arab Emirate and occupied Palestine's isolates

Conclusions: The available evidence indicates that M genes of H9N2 circulating in Iran during the past years were not well conserved. Our finding emphasizes the importance of reinforcing AIV surveillance

[Molecular identification of Salmonella Infantis isolated from backyard chickens and detection of their resistance genes by PCR]

This study aims at molecular identification of Salmonella Infantis isolated from backyard chickens and the detection of their antibiotic resistance genes. A total of 46 Salmonella-suspected samples isolated from backyard chickens of northern Iran were collected. Serotyping was done by the traditional method and then confirmed by PCR. Antimicrobial susceptibility of the isolates against 13 antimicrobial agents was determined by the standard disk diffusion method. There were 44 samples identified as Salmonella. Serotyping results showed that all 44 isolates belonged to serogroup C1 and serovar Infantis. The most resistance observed was to tetracycline and doxycycline [100%], chloramphenicol [79%] and florfenicol [72%]. The floR, catI, tetA and tetG genes were used for the detection of florfenicol chloramphenicol and tetracycline resistance. In order to identify the phenotypic resistance in strains which showed resistance genes by PCR, colony PCR and culture on plates each containing antibiotic was performed simultaneously. All the Salmonella Infantis resistant to florfenicol and chloramphenicol harbored floR and catI. None of the Salmonella resistant to tetracycline carried tetA or tetG. The result of colony PCR and culture in antibiotic medium confirmed the results of PCR and indicated phenotypic resistance in these samples.

Construction of an iss deleted mutant strain from a native avian pathogenic Escherichia coli O78: K80 and in vitro serum resistance evaluation of mutant

Colibacillosis, caused by different serotypes of avian pathogenic Escherichia coli [APEC], is one of the important diseases in poultry industry. The isolate O78 is the most prevalent serotype of APEC in Iran. One of the APEC virulence factors, increased serum survival [iss] gene, is related to serum resistance. The usual form of colibacillosis in avian is extraintestinal, and serum resistance is applied one way by APEC to reach internal organs; hence, it appears that the control of colibacillosis in poultry regarding the deletion of iss and the construction of a serum sensitive APEC strain is beneficial. Additionally, the knowledge about APEC serum resistance could be extended using mutant strains. The present study was an attempt to generate an iss mutant strain from native APEC-O78 strain |c|1378 and to study the level of serum resistance of native APEC-O78 strain c1378 in comparison with its mutant [APEC-O78 strain c1378|D|iss]. The lambda red recombinase system was utilized to delete iss gene in native APEC-O78 strain c1378. This strain was first transformed with the plasmid pkD46 to introduce the lambda red recombinase system and then the PCR product with sequence homology to the iss gene and a kanamycin resistance marker was transformed into the APEC-O78 strain c1378. Serum sensitivity of mutant and wild type strain was investigated by microtiter test. The generation of mutant was successful and the iss was replaced with kanamycin resistance cassette. Also, it was observed that the mutant was sensitive to serum. However, serum sensitivity of iss deleted mutant was not statistically different from its parents. Application of lambda red recombination could be a simple and useful technique for production of a precisely defined gene deletion. Also, there may be some genes that compensate the activity of iss gene

Phylogenetic study based on the phosphoprotein gene of Iranian Newcastle disease viruses [NDV] isolates, 2010 -2012

Newcastle disease virus [NDV] is the causative agent of the Newcastle disease [ND], a highly contagious disease in birds that causes significant economic losses to the poultry industry worldwide. ND is endemic in Iran and outbreaks are reported regularly in commercial poultry flocks and different species of birds. The current study was carried out to characterize NDV based on phosphorprotein [P] gene from recent outbreaks in Iran, 2010-2012. The P gene fragment of NDV isolates of five chickens, 1 ostrich, and 1 Pigeon paramyxovirus-1 was obtained by RT-PCR and sequenced. Phylogenetic analysis of sequences revealed that chicken and ostrich NDV isolates were closely related and placed in the genotype VII and Pigeon Paramyxovirus-1 was located in the genotype V. This is the first report of Phosphoprotein gene sequences of NDV strains isolated in Iran. This study will help us to understand the epidemiology and molecular characteristics of Newcastle disease virus in Iran

[Phylogenetic study of Iranian infectious bronchitis virus isolates during 2010-2011 using glycoprotein S1 gene]

Infectious bronchitis is an acute, highly contagious, viral disease of poultry with worldwide distribution, and is continuously evolving through point mutation and recombination of their genome; subsequently the emergence of IBV variants complicates disease control. To investigate genetic characterization of new IBV variants isolated from commercial chicken flocks in Iran collected between 2009 and 2010. The partial S1 gene of the spike protein, covering a hypervariable and constant regions, was amplified and sequenced using conventional RT-PCR. Phylogenetic analysis revealed four viruses designated as Razi-HKM891, Razi-HKM892, Razi-HKM893 and Razi-HKM894. Deduced amino acid sequence comparison with other IBV genotypes, published in the GenBank database, indicated that the isolates Razi-HKM891 and Razi-HKM894 were placed into the pathogenic 793/B serotype. However, the isolates Razi-HKM892 and Razi-HKM893 were different with previously described isolates in Iran. The Razi-HKM893 is closely related to recently published isolates from countries in Middle East and likely indigenous to Iran. These findings is essential for improving the disease control strategies and thus emphasize the importance of continuous surveillance of the disease and of sharing the information to the global scientific community, which would help to fill the epidemiological gaps in the regions and to validate the robustness of diagnostic screening

Molecular Surveillance of Avian Influenza in Bird Parks of Tehran, Iran

Avian influenza [AI] viruses have been isolated from a wide diversity of free-living avian species representing several orders. Since 1998, H9N2 AI outbreaks have been one of the major problems in Iranian poultry industry. In 2006, H5N1 was reported in swans in the north of Iran first, but until now there has been no official report from commercial flocks in Iran. The aim of this study was Molecular Surveillance of Avian Influenza in Bird Parks of Tehran, Iran. In this study, 100 fecal samples from different avian species of Public and Bird Parks [The avian species included Pigeon, Duck, Swan, Parrot, Crow and Sparrow] were collected in Tehran, in the central region of Iran during November and December 2009. RNA extraction and RT-PCR have been done according the WHO Instruction for detection of Influenza Type A. In 14% of samples genetic materials [RNA] were detected. Species including duck and sparrow were positive. This is the first report of AIV detection in this these species in Iran. Due to emergence of new H1N1 influenza and bird flu throughout the world and in regional countries, surveillance programs for monitoring the spread of these viruses need to be redesigned. Surveillance activities for AI in wild birds should be continued to provide further virological [subtype] and epidemiological [Phylogenic Study] information about circulating viruses

[Molecular surveillance of avian influenza virus in domestic ducks: a provincial study]

In the first time, avian Influenza [AI] infection, subtype H9N2, was isolated from chicken in 1988 in Qazvin province and since then has become endemic in Iran. Waterfowls, such as wild ducks, are natural reservoirs for all types of influenza A viruses and cause virus circulation in environment and poultry population. In 2006, Iranian Veterinary Organization confirmed that 135 dead swans in Gilan province were positive for H5N1 avian influenza virus. The purpose of this study was to determine the role of domestic ducks in avian influenza virus circulation [subtypes: H5, H7 and H9] in Gilan province during 2010-2011 through molecular surveillance techniques. 550 cloacal swabs from Mallard and Pekin ducks were tested in rural areas of Shaft and Fouman cities. Meanwhile a breeding farm in Gilan was tested by RT-PCR assay for detection of AI virus subtypes [H5, H7 and H9] according to OIE protocols. We did not detect AI viral RNA in 550 samples which were tested for type A and subtypes H5 and H7. While waterfowls could have a crucial role in emergence of new influenza virus strains, no AI viral RNA mentioned subtypes was detected for the mentioned subtypes. These findings could be due to restrict control programs following 2006 AI outbreak in the mentioned region. However, surveillance programs for monitoring AI viruses need to be continuously performed

Antimicrobial resistance profile and presence of class I integrongs among Salmonella enterica serovars isolated from human clinical specimens in Tehran, Iran

Salmonella is one of the leading causes of food-borne diseases. Increasing occurrence of antimicrobial resistance, especially multidrug-resistance, in Salmonella serovars is a major public health problem worldwide. This study was carried out to detect class I integrons and antibiotic resistance profiles in clinical isolates of Salmonella serovars collected from seven hospitals in Tehran during November 2009 to June 2010. Antibiotic susceptibility profile of 19 antibiotics against 58 Salmonella isolates commonly used in humans was determined using disk diffusion assay. Minimum inhibitory concentration against ceftriaxone and ciprofloxacin was studied. PCR assays were used to detect class I integrons. Among 58 Salmonella isolates, 72.4% were Salmonella enterica serovar Enteritidis, 8.7% were Salmonella enteric serovar Typhimurium and 18.9% were other serovars. Of the total 58 Salmonella serovars, 43 [74.1%] were multidrug resistant and showed resistance to three or more antibiotic families. Class I integrons were identified in 38 [88.3%] MDR Salmonella isolates. Ciprofloxacin minimum inhibitory concentration ranged between 0.125-2 microg/ml for four isolates and other four isolates exhibited resistance to ceftriaxone [MIC 64-256 microg/ml]. The high prevalence of class I integrons was seen in our MDR Salmonella isolates and class I integrons might play an important role in the dissemination of antimicrobial resistance determinants

relation of water contamination and colibacillosis occurrence in poultry farms in Qom province of Iran

Seventy poultry farms' drinking water was tested for Escherichia coli contamination in Qom province in Iran. The cases of colibacillosis from positive farms were also collected and tested. The isolates were examined for serotype, detection of virulence genes by multiplex PCR and antibiotic resistance. Thirty poultry farm water samples were E. coli positive [18.57%], although 13 E. coli isolates were recovered from carcasses of related farms. The isolates belonged to O2 serogroup and one O157, with approximately 29% of the strains being non-typeable. Two isolates from water and carcasses were serotyped O2 and one sample serotyped O157, which needs to be further studied. The PCR method was on the basis on showing virulence genes of espB, stxl, stx2 genes were common in a sample from both water and carcass, although five samples from both water and carcass shared a stxl gene as well. All isolates showed maximum sensitivity and resistance to lincospectine and tetracycline, respectively