survey on antibacterial drug use in broiler chicken farms in Qum province, Iran

Background: Antibacterial drugs have long been used for prevention and treatment of poultry diseases but their misuse or overuse can make adverse effects on public health including occurrence of drug residues in poultry products

Objectives: To assess the frequency and status of antibacterial drug consumption in broiler production farms in Qum province

Methods: In the present survey, Qum province was divided into six regions [north, west, southwest, south, southeast and east] and in total 138 broiler production units [59%] were studied by direct interview using a questionnaire

Results: The present study showed that the most frequently used antimicrobial drugs in broiler farms were sulfamethoxazole+trimethoprim [93.4%] followed by enrofloxacin [60.0%], colistin [49.7%], furazolidone [42.0%], oxytetracycline [17.5%], and chloramphenicol [14.6%]. Mean antibacterial consumption rate during a 42-48 day production period was 3.0+/-0.37 times per farm. A notable finding in this survey was the high percentage of banned drug usage such as furazolidone and chloramphenicol, indicating the ignorance or un-awareness of poultrymen regarding the potential hazards of these drugs on public health

Conclusions: Owing to widespread and frequent usage of antibacterial drugs in broiler farms, all-out actions are needed to be taken in educational, research and administrative fields of veterinary and animal production sectors for rational and responsible use of these drugs in poultry industry

Molecular evaluation of M2 protein of Iranian avian influenza viruses of H9N2 subtype in order to find mutations of adamantane drug resistance

Background: the H9N2 subtype of influenza A viruses is considered to be widespread in poultry industry. Adamantane is a group of antiviral agents which is effective both in prevention and treatment of influenza A virus infections. These drugs inhibit M2 protein ion channel which has role on viral replication

Objectives: the main objective of this study is to evaluate M gene of avian influenza viruses [AIVs] of H9N2 subtype in order to find adamantane drug resistance mutations

Methods: over 100 suspected samples were collected from different geographical regions of Iran during 2012-2013. Samples were injected via allantoic sac of 9-11 day-old chicken embryos. A total of 11 out of 100 were AIV. The H9N2 subtype was confirmed by specific RT-PCR. The RT-PCR was conducted for full length M gene. PCR amplified products were purified and then conducted for commercial direct sequencing. Finally, sequences were checked for possible sites of adamantane resistance mutations

Results: overall, 8 out of 11 viruses harbored the adamantine resistance-associated mutations. Of which, four viruses were isolated in 2013 and four viruses in 2012. Two different resistance- associated mutations were observed during different years

Conclusions: the present study provided clear evidence concerning resistance AIVs of H9N2 subtype that were circulating in Iranian poultry sector. This concern is always present as M segment might be introduced into human influenza viruses by reassortment phenomenon

Genetic and phylogenetic analysis of the ribonucleoprotein complex genes of H9N2 avian influenza viruses isolated from commercial poultry in Iran

The H9N2 subtype of avian influenza viruses [AIVs] has been isolated in multiple avian species in many European, Asian, African and American countries. Since the first outbreak of H9N2 virus in Iran in 1998, this virus has widely circulated throughout the country, resulting in major economic losses in chicken flocks. Several amino acids in the virus ribonucleoprotein [RNP] complex including the nucleoprotein [NP] and polymerase [PB2, PB1 and PA] proteins are associated with host range and virulence. Our aim was to understand the molecular characterization of RNP complex proteins of Iranian H9N2 subtype isolates. The full length nucleotide sequences of RNP complex genes of two strains designated as Ck/IR/ZMT-101/98 and Ck/IR/EBGV- 88/10 were amplified and sequenced. The phylogenetic analysis revealed that both strains were located in different sub-lineages. However, based on the genetic similarities, PB1, PA and NP genes of Ck/IR/EBGV-88/10 strain had a close relationship with a H7N3 subtype strain, isolated from Pakistan. Most positions of RNP proteins contained amino acids typical of avian determinants of host range. The results showed that the Iranian RNP complex genes have undergone genetic reassortment. Continuous AIV monitoring in poultry industry would help to obtain more information about genetic variation of H9N2 viruses and possible emergence of virulent and/or pandemic viruses

Molecular characterization of non-structural gene of H9N2 subtype of avian influenza viruses isolated from broiler chickens in Iran

The H9N2 subtype of avian influenza viruses [AIVs] have spread in Asia and Middle East countries and have become a serious threat to poultry industry in Iran. Characterization of genes of H9N2 subtype involving in pathogenicity and diagnosis are crucial in control of avian influenza outbreaks. The Nonstructural [NS] gene and its protein products [NS1 and NS2] are important as diagnostic marker, life cycle and pathogenicity of AIVs. The NS gene of five strains, isolated from 1998 to 2010, were completely sequenced and analyzed. All of the examined strains were composed of 890 nucleotides with 230 amino acids. In this regard, only two Iranian strains from GeneBank had 217 amino acids in NS 1 protein. All Iranian H9N2 strains subdivided into two distinct sublineages including I and II. Comparative analysis of NS genes of Iranian strains showed that since 2003, they might have originated from Pakistan H7N3 strains; whereas from 2008 they could be originated from Pakistan H9N2 strains. Although the low pathogenic H9N2 subtype has been permanently circulating from 1998 to the present in Iran, phylogenetic analysis of NS genes revealed that sublineage II has circulated more in poultry industry of Iran. These epidemio-logically variations could be related to vaccination pressure due to massive vaccination or NS gene reassortment in rural and backyard chickens

Molecular analysis of the nucleocapsid gene and 3[1] untranslated region of two infectious Bronchitis Virus field isolates from Iranian poultry farms

Infectious bronchitis [IB] is an economically important disease of chickens. Due to the emergence of new variants of infectious bronchitis virus [IBV], the control of IB has become a serious problem for the poultry industry worldwide. In the present study, the nucleocapsid gene [N] and 3' untranslated region [UTR] of two IBVs isolated from Iranian poultry farms were sequenced and compared with other IBV strains. Based on nucleotide identity, the N gene and 3' UTR sequences of Iranian IBVs showed 90% similarity to the commonly used IBV vaccine strains, H52 and HI20. However, based on phylogenetic analyses, Iranian IBVs were found to cluster separately from the IBV vaccine strains used in Iran as well as other IBVs isolated in China, Australia and the United States. It was concluded that IBVs circulating in Iran are genetically distinct from IBV vaccine strains that have been used in Iran for many years. Therefore, it is important to develop a new vaccine based on these newly identified strains for controlling IB in Iranian poultry farms

Assessment of a rapid immunochromatographic assay for the detection of avian influenza viruses

Rapid spreading of the low pathogenic avian influenza virus [AIV] caused by the H9N2 subtype and the highly pathogenic AIV caused by H5N 1 have caused serious economic losses in the poultry industries of Asia. Therefore, the early detection of AIVs is crucial for the control of the disease. In the present study, the applicability of a rapid immunochromatographic [RIC] assay, which specifically detected type A antigens of AIVs, was evaluated. This assay detected H9N2 viruses at 10[3.2] ELD[50]/ml and H5, H7 and H9 antigens at 128 HA titers, but did not react with other respiratory viruses. The assessment of cloacal swab samples prepared from 1 to 10 d post-inoculation [PI] revealed that the first positive samples were detectable on day 2 and 3 PI, and the last positive samples were detectable on day 10 and 9 PI, by the virus isolation [VI] and RIC assays, respectively. Collectively, the relative specificity, sensitivity, positive predictive value, negative predictive value, accuracy and correlation rate of the RIC and VI assays, were 100%, 71.5%, 100%, 78.5%, 0.86, and 0.98, respectively. There was also a good correlation [K> 0.81] between the results of the haemagglutination [HI], VI and RIC assays of cloacal/tracheal swab samples that were obtained from broiler flocks involved with viral respiratory diseases. Overall, RIC showed a low sensitivity and high specificity for the rapid diagnosis of H9N2 isolates in both experimental and clinical infections

[Characterization and phylogenetic analysis of neuraminidase gene in isolated Avian Influenza viruses of H[9]N[2] subtype from Iran]

Avian Influenza [AI] is a rival respiratory disease of poultry industry that cause great economic losses in worldwide. In this study, the neuraminidase [NA] gene of two H[9]N[2] avian influenza virus [AIV] strains [A/Chicken/Iran/ZMT-101 / 1998 and A/Chicken/Iran/NGV-1/2006], isolated from AI infected poultry farms in 1998 and 2006, were cloned and sequenced. Amino acids at hemadsorbing [HB] site of these isolates showed some differences between them and also with other Iranian isolates. No insertion or deletion or shortening in the stalk region of the NA gene were observed. Phylogenetic analysis showed that N[2] gene of these strains, belonged to the same A/Qail/Hong Kong/ G1/ 97-like virus sublineage. However, these strains were allocated in two different groups in the same sublineage. These results indicate that N2 gene and protein sequences of the recently isolated H[9]N[2] stain [NG-1/2006] have substantial variation compared to the previous Iranian reported H[9]N[2] strain [ZMT-101/1998]. These changes may cause some new biologic and immunologic characteristics for AIVs, such as reduction of vaccination efficacy in immunized chickens. Therefore, evaluation of important genes and protective efficacy of used H[9]N[2] vaccine strain used in Iran would be crucial

Comparison between RT-PCR and viral solation for detection OFH9 subtype of avian influenza viruses

During 2004-2006, a total number of 22 tissue samples obtained from poultry flocks suspected to respiratory diseases submitted to avian virology laboratory of faculty of veterinary medicine, were prepared for avian influenza virus [AIV] isolation in embryonated chicken eggs, according to the standard method. RT-PCR was established on tissue samples using specific primers for H9 gene. 3.5 Amplified PCR products were 435 bp in length. Analytical sensitivity of the RT-PCR was 10 EID50 and sensitivity, specificity and correlation rate compared with virus isolation, were 100%, 94% and 95%, respectively. The results showed that the RT-PCR assay using H9 gene specific primers, directly on tissue samples, could be used in rapid detection and subtyping of H9 AIVs as a useful alternative to virus isolation assay

Molecular analysis of three iranian isolates belonged to 793/B serotype of infectious bronchitis viruses

Infectious bronchitis [IB] disease is one of the important respiratory diseases of poultry that causes annually large economic losses in poultry industry of Iran. The aim of this study is molecular characterization of S1 gene of Iranian infectious bronchitis viruse [IBV] belonged to 793/B serotype. The whole S1 gene of three local IBV strains in RT-PCR, showed a band above 1600 base pair [bp] in gel electrophoresis. RFLP analysis using 3 restriction enzymes, Hae3, Hind3 and Ecor1, showed 793/B serotype pattern. The S1 gene of these strains were sequenced and compared with 30 reference IBV strains. Identity Plot [IP] of nucleic acids and amino acids sequences were also designed. Moreover, nucleic acids differences in 1659 bp of S1 gene were calculated by distance method: nucleotide: Kimura 2-parameter and finally, the phylogenetic tree of S1 gene sequence strains with the highest validity in branching were designed. Three Iranian strains belonged to 793/B genotype with nucleotide differences of 5.64-6.07% to UK/793/B as a prototype of 793/B and 26.02-26.16% to H120 as a vaccinal strain. Regarding to low homology and weak cross protection between 793/B serotype and Massachusetts vaccinal strain, it would be a possible cause for failure in vaccination and outbreak of IB disease in vaccinated flock of poultry industry

[Isolation and identification of infectious bronchitis viruses poultry farms of Iran]

Objective: detection of infectious bronchitis viruses by RT- PCR/RFLPs in poultry farms of Iran

Design: longitudinal study from 1997 to 2003

Samples: tracheas, lungs and kidneys of suspected flocks to respiratory diseases

Procedure: from 1997-2003, tissues samples including lung, trachea and kidney, had been prepared from broiler and layer flocks were submitted to avian virology laboratory of poultry diseases section in order to isolate respiratory viral diseases viruses. A total number of 50 infectious bronchitis viruses [IBV] were isolated in embryonated chicken eggs. The infected embryos displayed stunting, urate deposition in the mesonephros or death after three passages. Twelve isolates that had showed typical signs in embryos, were selected for molecular identification. Viral RNA was extracted by RNxTM plus [CinnaGen Co.] using chloroform 1 Isoamyl alcohol, Isopropanol, Ethanol and DEPC water. cDNA was prepared from extracted RNA with RT enzyme, RH primer, RT buffer, dNTP and Rnase inhibitor. For PCR reaction, buffer PCR ]OX, MgC12, S loligo5' and 3' primer, ampli Taq DNA polymerase and dNTP were added to cDNA and then PCR was conducted in thermal cycler. The PCR products were analyzed on a 1% agarous gel and Ethidurn Bromide staining. The S1 glycoprotein genes of IBV strains appeared to be above 1600 bp in size. PCR products were digested by HaeIII, EcorI and HindIII, according to the manufacture's recommendation

Results: base on RFLPs patterns and comparison with RFLP references patterns [793A3, D274, M41, H120], 8 of 12 strains showed 793/B pattern and the rests [4 of 12] showed Mass patterns in RFLPs

Clinical implications: regarding to low homology and weak cross protection between 793B serotype and vaccinal strain [Massachusetts] prevention and a controlled strategy against IB should be altered