Complete Genome Sequencing of an Embryonated Chicken Egg-Adapted Duck atadenovirus A.

Egg drop syndrome (EDS) is prevalent in industrial poultry globally. This disease is caused by Duck atadenovirus A or EDS virus (EDSV), a member of the genus Atadenovirus under the family Adenoviridae. The disease is attributed to significant economic losses in the poultry industry worldwide due to a drop in egg production, reduction in egg quality, and failure to reach maximum egg production. Oil-adjuvant inactivated vaccines, which are widely used in the poultry industry, provide good protection for immunized chickens against EDS. This study aimed to genetically and phylogenetically analyze the full-length genome of an embryonated chicken egg-adapted EDSV strain 127. After extraction of viral DNA from the allantoic fluid, overlapping fragments of the viral genome sequence were generated by polymerase chain reaction (PCR) using 25 pairs of primers. Purified PCR products were subjected to complete genome sequencing by the next-generation sequencing (NGS) approach. The nucleotide homology observed between genomes of the studied strain and that of the original strain 127 (NC_001813) of laying chickens was 99.9%. Its genome was 33,213 bp in length, with a G + C content of 43.01%. A comparison of the genome sequence of the egg-adapted virus with strain 127 revealed only three non-synonymous single-nucleotide polymorphisms (SNPs) between these viral genome sequences. Two mutations of S320G and I62K out of these SNPs were found within the coding regions of fiber and hypothetical proteins which may play a role in the adaptation of EDSV in the embryonated chicken eggs. The full genome sequencing of EDSV using NGS techniques provides insights into the discovery of genetic variants. Moreover, the genome sequence information of the EDSV provides valuable data for vaccine development in near future.

Isolation, Identification and Antimicrobial Susceptibility of AvibacteriumParagallinarum from Backyard Chicken in Retail Markets of Karaj and Tehran Cities, Iran.

Avibacterium (Haemophilus) Paragallinarum (Av. Paragallinarum) is the causative agent of Infectious Coryza (IC) in chickens. Despite the worldwide distribution of IC, no systematic study, to the best of our knowledge, was conducted on isolation and characterization of Av. Paragallinarum in Iran. The present study aimed to isolate and perform antibacterial susceptibility testing (AST) of IC agents from suspected backyard chickens with typical symptoms of IC in avian markets. From 18 collected choanal swab samples, four (22%) isolates of Av. Paragallinarum were detected by culture methods based on satellite growth on blood agar, which was confirmed by the biochemical reaction of Catalase and Oxidase tests and species-specific PCR (HPG-2). The hypervariable region of the hemagglutinin genes of 4 isolates was amplified and obtained sequences were deposited at a gene bank for more characterization. Meanwhile, 12 (66%) positive reactions were detected by observing expected 500 bpb and using PCR (HPG-2) on swab samples. Antibiotic susceptibility testing (AST) of obtained isolates were analyzed using the Kirby-Bauer disk diffusion method on Columbia agar with horse blood. Isolates were found to be resistant to amoxicillin, oxytetracycline, streptomycin, trimethoprim/sulfamethoxazole (up to 75%) and sensitive to cefalexin, ceftriaxone, enrofloxacin, florfenicol, gentamycin, linco-spectin, neomycin, doxycycline (50%), danofloxacin (75%), flumequine (50%), ofloxacin (75%). An intermediate growth inhibitionzone has been observed around antibiotic discs for ampicillin, colistin, erythromycin, penicillin, tiamulin (75%), tylosin (75%). In summary, to the best of our knowledge, this study is the first report of isolation and identification of AvibacteriumParagallinarum from backyard chickens which may be a source of IC for commercial chicken flocks. Moreover, the prevalence of resistance to some antibacterial drugs of IC agents may impose an additional threat to the poultry industry. A more in-depth study is recommended to develop a low-cost autogenous IC vaccine for small-scale flocks of poultry to prevent and manage the disease and establish antimicrobial resistance.

Characterization and full genome sequencing of a velogenic Newcastle disease virus (NDV) strain Ck/IR/Beh/2011 belonging to subgenotype VII(L).

Avian avulavirus 1, better known as Newcastle disease virus (NDV), causes substantial loss to the poultry industry in many developing countries. In this study we have characterized and fully sequenced the genome of a velogenic NDV strain named Beh (Ck/IR/Beh/2011) that has been used in our lab for a number of challenge and immunological studies over the last few years. This strain was isolated from poultry in the city of Behshahr, Mazandaran Province, Iran after an outbreak reported in the region in 2011. The intracerebral pathogenicity index (ICPI) was 1.8 in one-day-old chicks, characteristic of a velogenic NDV strain. Later, the virus was purified using a sucrose gradient centrifugation and used for next-generation sequencing (NGS). The results showed that the genome length was 15192 bp, similar to those of class II velogenic strains. In addition, the phylogenetic analysis based on the complete F gene showed that the NDV strain Beh has an F protein cleavage site 112RRQKR↓ F117 and belongs to the newly identified subgenotype VII(L). Based on the biological and genetic characterization, NDV strain Beh is now the best documented reference isolate representing the novel subgenotype VII(L) in Iran. Keywords: NDV; NGS; velogenic strain, subgenotype VII(L); phylogenetic analysis.

Comparison of the first Iranian native Ornithobacterium rhinotracheale vaccine with conventional vaccine: A challenge study.

BACKGROUND AND AIM: The best strategy to prevent or control an Ornithobacterium rhinotracheale (ORT) infection is vaccination. The present study aimed to compare the efficacy of the first Iranian inactivated ORT vaccine (Razi, Iran), which had been prepared from a native strain, with the Nobilis ORT Inac (Intervet, The Netherlands) through a challenge trial. MATERIALS AND METHODS: Seventy-two 1-day-old specific pathogen-free White Leghorn chickens were used in this study. The birds were divided randomly into four groups. Following the vaccination and challenge of the birds, the efficacy of the Razi and the Intervet ORT vaccines was evaluated by serological, bacteriological, and molecular methods. RESULTS: The antibody titer in vaccinated groups was determined to be significantly higher than unvaccinated birds. In addition, the difference in postmortem lesion scores between the vaccinated and unvaccinated birds was significant. The differences in the means of the antibody titers and postmortem lesion scores in birds that were vaccinated by the Razi and Intervet ORT vaccines were not significant. CONCLUSION: Considering the results of this study, it can be concluded that the Iranian native ORT vaccine was comparable to the Intervet vaccine. The Razi ORT vaccine has effectively decreased the duration of the ORT infection and can effectively protect the chickens against an ORT infection.

Isolation and identification of Ornithobacterium rhinotracheale in broiler breeder flocks of Guilan province, north of Iran.

The aims of the present study were to isolate and serotype, determine the Seroprevalence, Drug susceptibility and diagnosis of infection by Polymerase Chain Reaction (PCR). In this study 460 serum samples and 220 tracheal swabs, 90 ovaries and oviduct swabs, 90 misshapen egg shells swabs were collected from 22 broiler breeder flocks of 5 companies. Serological results showed that all of the 22 flocks (100%) were positive for ORT infection. Ornithobacterium rhinotracheale (ORT) antibodies were detected in 289 (62/83%) out of the 460 serum samples. ORT was detected from tracheal swabs of seven flocks (31/81% or 3/18% out of 220 tracheal swabs). There was significant correlation between flock different ages and ORT titers (p<0.05), but correlation of flock ages and ORT isolates was not significantly different (p>0.05). Seven flocks infected with ORT were detected positive in PCR but bacteria were Isolated from only five culture. No ovaries and oviducts, misshapen egg shell swabs yielded ORT. A 784 bp fragment of the 16S rRNA gene was amplified using ORT specific primers in the PCR. All the isolates were identified as serotype A by Rapid Agglutination Test. Drug sensitivity test using standard disk diffusion technique was performed with 27 antibiotics. Antibiotic susceptibility for Quinolons family was seen more than the others and Cephalosporins family except to Cephalexin. The isolates were 80-100% susceptible to Tetracycline family and the most antibiotic resistant were seen for Aminopenicillins, Polypeptides, Sulfanamides and 80-100% resistant to Aminoglycoside family. Eighty percent of the isolates were resistant to Licomycin and 60% were moderate sensitive to Lincomycin. This study is the first report of prevalence of ORT, bacterial isolation, biochemical characteristics, serotyping and molecular method (PCR) in broiler breeder flocks in Guilan province of Iran.