Molecular Detection of Avian Infectious Bronchitis Viruses in Live Bird Markets, Gilan Province.

Coronaviruses (AvCoV) which include infectious bronchitis virus (IBV) and other bird coronaviruses belong to the genus gammacoronavirus, subfamily Coronavirinae. One of the most prominent representatives of gammacoronavirus genus is infectious bronchitis virus (IBV) which is a highly contagious viral pathogen of chickens causing considerable economic losses to the poultry industry. IBVs mostly affect the respiratory, urinary, and reproductive tracts leading to a substantial drop in production. Backyard poultry in the villages usually share their food and water with free flight birds which puts them at serious risk of disease transmission. Furthermore, the poor hygienic measurements which are often used in backyard flocks make them a potential reservoir for diseases that can be transferred to commercial poultry flocks. Live bird markets (LBMs) which receive live poultry to be resold or slaughtered and sold onsite play a significant role in spreading infectious diseases among the different bird species. In the present study, a number of 354 cloacal swab samples were collected from different bird species from LBMs of Gilan province. Subsequently, after RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR) technique was carried out using specific primers of S1 gene to detect coronavirus infectious bronchitis virus. Two samples from backyard chickens were reported to be positive to coronavirus which were named Iran/Backyardchicken 96/2017 and Iran/Backyardchicken 94/2017. The results of the phylogenetic analysis demonstrated that these two isolates are placed in QX and IS-1494 strains, respectively. On a final note, the obtained results highlighted the role of live birds offered in LBMs in the epidemiology of IBV and the transmission of the virus to the industrial flock.

Molecular Detection of Gamma Coronaviruses in Bird Parks of Iran.

Gamma Coronaviruses (GCoVs) are distributed worldwide, affecting a wide range of bird species, the beluga whale, and bottlenose dolphins. Because of the limited proofreading capability in the viral encoded polymerase, they emerge genetically diverse. There has been no molecular surveillance data to describe the epidemiology of GCOVs in avian species. The present study was conducted to detect GCOVs in Tehran birds’ parks, 2015. Cloacal swabs (267 samples) from eight different bird species ((Chickens (Gallus gallus), Pheasant (Phasianus colchicus), Turkey (Meleagris gallopavo), Partridge (Perdix perdix), Quail (Coturnix coturnix), Duck (Anas platyrhynchos), Goose (Anserini),and Guinea fowl (Numididae)) were collected, the viral RNA was extracted, the RT-PCR was performed using QIAGEN one step RT-PCR kit and the primers targeting “3'-UTR” and “Nucleocapsid” genes. The detection rate was approximately 8.99%. GCOVs were detected in the chicken, quail, pheasant, turkey, and the partridge with different prevalence rates. Phylogenetic tree based on partial nucleotide sequences of the N gene clustered the samples into two groups. It is the first report of GCOVs in non-commercial birds in Iran. According to our results, GCOVs are circulating in different avian species, and further studies are needed to isolate these viruses and evaluate their pathogenesis.

Virus neutralization study using H120, H52, 793/B antisera against Iranian infectious bronchitis virus genotypes.

Infectious bronchitis virus (IBV), a major pathogen of the domestic fowl, exhibits extensive antigenic variation. IBV is a member of the Coronaviridae family and the genus Gammacoronavirus. A new infectious bronchitis virus serotype can emerge from only very few amino acid changes within the major peplomer glycoprotein, namely in its S1 part forming the virion spike. Principally, the serotypes are identified by virus neutralization (VN) tests. This study is aimed to investigate the neutralizing efficiency of H52, H120, and 4/91 antiserum against IBV genotypes (IS-1494, IS-720, 793/B, IR-1) recently circulating in Iran. For the first time, we have used cross-neutralization tests for the serological classification of these isolates. In this study, all antisera failed to neutralize all IBV strains. According to the results of our research, cross-protection studies are necessary for the design of a proper vaccination program for IBV circulating genotypes in Iran. The data are useful for the development of new vaccine strategies. Keywords: avian infectious bronchitis; Iran; virus neutralization.

Zataria multiflora essential oil reduces replication rate of avian influenza virus (H9N2 subtype) in challenged broiler chicks.

1. The effect of Zataria multiflora essential oil on replication rate of the H9N2 virus in target organs was determined by real-time PCR. One-day-old broiler chicks were randomly divided into six groups and were challenged with H9N2 influenza. Two groups received either 20 or 40 µl/kg body weight/day Zataria multiflora essential oils (ZM) seven days before the challenge while two other groups received the essential oil at the same dosage but after H9N2 challenge. One group received 4 mg/kg body weight/day of the anti-viral compound amantadine after challenge and the last group received no treatment and served as the control. 2. Groups that received the ZM, before or after H9N2 challenge, and the amantadine treated group showed reduced viral replication in the respiratory and gastrointestinal tracts compared to the control. Supplementation with ZM improved weight gain and FCR in broilers in comparison with the control. 3. The results showed that ZM had a positive effect on reducing viral replication in both the intestine and trachea of H9N2 influenza infected broiler chickens, that led to milder clinical symptoms and better performance.

Combination of H120 and 1/96 avian infectious bronchitis virus vaccine strains protect chickens against challenge with IS/1494/06 (variant 2)-like infectious bronchitis virus.

Avian infectious bronchitis (IB) is a worldwide chicken disease, caused by avian infectious bronchitis virus (IBV) which infects all commercial poultry lines. The present study was done to evaluate protection caused by two different serotype vaccines (Massachusetts and 793/B) in order to evaluate protection against challenge with IS/1494/06-like virus (variant 2-like virus), which is prevalent in the Middle East. SPF chickens were divided into four groups (n = 20). First and second group as negative control group and non-vaccinated-challenged group received no vaccine. Groups 3 and 4 received H120-H120 and H120-1/96 IBV vaccine strains at the 1st and 14th day, respectively. Twenty one days after last vaccination, non-vaccinated-challenged group and vaccinated group were challenged using variant 2-like IBV. Serum samples were collected before challenge to measure humoral immune response of chickens. Five days after challenge, the tissue samples from the trachea, lungs and kidneys were taken to evaluate cilliary activity, viral load (quantitative real-time RT-PCR), and histopathological evaluation. Clinical sign scores were also recorded after challenge. Overall, the results showed a protective efficacy of the used vaccination program. Best cross protection (69.2%) was obtained in the H120-1/96 vaccinated group. Virus replication of the challenged virus in H120-1/96 group compared with H120-H120 group showed a significant reduction of viral load in trachea (1.5×103 compared to 503) and kidneys. Clinical sign scores of the challenged groups showed significant effect of the vaccination program to reduce clinical signs. The trachea pathological scores and histopathological findings in the lungs and kidneys also confirmed better protective efficacy of vaccinated groups. In conclusion, using combination of heterologous IBV vaccine serotypes (Massachusetts and 793/B) would be a better strategy to control variant 2-like viruses, but more evaluation is needed using other circulating isolates to find the best combination of vaccines.

Pathogenicity characteristics of an Iranian variant-2 (IS-1494) like infectious bronchitis virus in experimentally infected SPF chickens.

Avian infectious bronchitis (IB) is a major cause of economic loss to the poultry industry. IB virus primarily affects respiratory tract, but strains differ in their tropism for such other target organs as kidneys and alimentary tract. The objective of this study was to estimate the pathogenicity of an Iranian IB virus (IBV) variant (variant-2) which is one of the most prevalent isolates circulating in Iranian poultry farms. SPF chickens were intranasally inoculated with 104 EID50/0.1 ml of the virus. Sera, fecal swabs, and different tissue samples were collected on different days post infection. Clinical signs, gross pathology, and histological changes were recorded. The amount of virus genome was quantified in different tissues and feces using quantitative real-time PCR assay. The highest viral loads were detected in the feces and cecal tonsils. Real-time PCR results demonstrated variant-2 tropism for respiratory tract, digestive system and renal tissue that is due to its epitheliotropic nature. This is the first pathogenicity study of Iranian variant-2 virus. Based on histology observations and clinical signs this isolate was classified as a nephropathogenic IBV. Further knowledge of IBV pathogenesis permits to perform more effective prevention practice.

Molecular identification of Salmonella Infantis isolated from backyard chickens and detection of their resistance genesby 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.

A study of Newcastle disease virus obtained from exotic caged birds in Tehran between 2009 and 2010.

The aim of this study was to investigate the frequency of the Newcastle disease virus (NDV) infection and its virulence in exotic cage birds over a limited area and time period. A set of 335 samples was collected from 24 different species of exotic unvaccinated cage birds kept in the zoological gardens and bird markets of the Tehran province of Iran during 1.5 years. Except for three pigeons, all of the sampled birds were healthy with no clinical signs of Newcastle disease. NDV was detected in three sick pigeons by haemagglutination assay (HA), haemagglutination inhibition (HI) and reverse transcription-polymerase chain reaction (RT-PCR) tests while two of them were identified as virulent types by RT-PCR. Although the remaining samples were negative by Newcastle-disease-specific HA and HI tests, 35 of them (10%) were identified as positive and 25 (72%) were determined as the velogenic type by RT-PCR test. Five PCR products were sequenced and all were confirmed as NDV but sequences were different from each other and from other sequences from Iran. In total, 14 species (58%) were infected and 10 species were uninfected with the velogenic type without showing any signs. Pigeons are very sensitive to NDV infection and play an important role in its epidemiology. In this study, the PCR test was found to be a more sensitive and powerful method than the HA and HI tests for detection of NDV reservoirs and carrier status in exotic birds. Also, the frequency of infection with the virulent type showed that the exotic birds should probably be considered one of the main causes of recurrent annual epidemics of Newcastle disease in endemic regions.

Nutritional value of raw soybeans, extruded soybeans, roasted soybeans and tallow as fat sources in early lactating dairy cows.

Thirty multiparous Holstein cows (29.8 ± 4.01days in milk; 671.6 ± 31.47 kg of body weight) were used in a completely randomized design to compare nutritional value of four fat sources including tallow, raw soybeans, extruded soybeans and roasted soybeans for 8 weeks. Experimental diets were a control containing 27.4 % alfalfa silage, 22.5% corn silage, and 50.1% concentrate, and four diets with either tallow, raw soybean, extruded soybean, or roasted soybean added to provide 1.93% supplemental fat. Dry matter and NEL intakes were similar among treatments, while cows fed fat diets had significantly (P<0.05) high NEL intakes when compared to control with no fat. Supplemental fat, whether tallow or full fat soybeans increased milk production (1.89-2.45 kg/d; P<0.01) and FCM production (1.05-2.79; P<0.01). Milk fat yield and percentage of cows fed fat-supplemented diets were significantly (P<0.01 and P<0.05 respectively) higher than control. Between fat-supplemented diets, roasted soybean caused highest milk fat yield and extruded soybean caused lowest milk fat yield. There was no significant effect of supplemental fat on the milk protein and lactose content and yield. Feed efficiency of fat-supplemented diets was significantly (P<0.01) higher than control. Body weight, body weight change and BCS (body condition score) of cows, as well as energy balance and energy efficiency were similar between treatments. In conclusion, while there was no significant effect of fat sources on production response of cows, fat originating from heat-treated soybean help to minimize imported RUP (rumen undegradable protein) sources level as fish meal in comparison with tallow and raw soybean oil. In the Current study, there was no statistical significance among nutritional values of oil from extruded soybeans and roasted soybeans.

Phylogenetic study on nonstructural (NS) gene of H9N2 isolated from broilers in Iran during 1998-2007.

Since 1998, H9N2 AI outbreaks have been one of the major problems in Iranian poultry industry. The association of high mortality and case report of H5N1 and H9N2 influenza virus in wild birds in recent years raised the specter of a possible new genetic modified AI virus. In this study, we do phylogenetic analysis on Full- length Nonstructural (NS) genes of seven H9N2 Isolates from Broilers in Iran, Tehran province during 1998-2007. Phylogenetic analysis clearly shows that Iranian H9N2 isolates gene pools, corresponding to just NS allele A. Comparison of nucleotide sequences of isolated viruses revealed a substantial number of silent mutations, which results in high degree of homology in amino acid sequences. In addition, the cluster of Iranian H9N2 isolates could be subdivided into two subgroups, which matched their times of isolation especially around 2006 time line. The high degree of similarity between the NS genes of the Iranian H9N2 isolates supports the hypothesis that these genes originated from a single predecessor. Present result provides useful molecular epidemiological data to understand the dynamics of H9N2 evolution during 9 years in Iran and support earlier phylogenetic observations.

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

BACKGROUND AND OBJECTIVES: 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. MATERIALS AND METHODS: 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. RESULTS: Among 58 Salmonella isolates, 72.4% were Salmonella enterica serovar Enteritidis, 8.7% were Salmonella enterica 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 g/ml for four isolates and other four isolates exhibited resistance to ceftriaxone (MIC 64-256 g /ml). CONCLUSION: 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.

Identification, cloning and sequencing of Escherichia coli strain chi1378 (O78:K80) iss gene isolated from poultry colibacillosis in Iran.

AIMS: To identify, clone and sequence the iss (increased serum survival) gene from E. coli strain chi1378 isolated from Iranian poultry and to predict its protein product, Iss. METHODS AND RESULTS: The iss gene from E. coli strain chi1378 was amplified and cloned into the pTZ57R/T vector and sequenced. From the DNA sequence, the Iss predictive protein was evaluated using bioinformatics. Iss from strain chi1378 had 100% identity with other E. coli serotypes and isolates from different origins and also 98% identity with E. coli O157:H7 Iss protein. Phylogenetic analysis showed no significant different phylogenic groups among E. coli strains. CONCLUSIONS: The strong association of predicted Iss protein among different E. coli strains suggests that it could be a good antigen to control and detect avian pathogenic E. coli (APEC). SIGNIFICANCE AND IMPACT OF THE STUDY: Because the exact pathogenesis and the role of virulence factors are unknown, the Iss protein could be used as a target for vaccination in the future, but further research is required.