Effect of Vaccination on Distribution and Immune Response of Avian Influenza Virus H9N2 in Coturnix coturnix.

Influenza viruses can multiply in quails and be transmitted to other animal species. As vaccination reduces virus shedding in chickens, the effect of the killed H9N2 avian influenza virus (AIV) on tissue distribution and virus shedding was evaluated in quails. One hundred 20-day-old quails were divided into six equal groups, kept in separate pens, and fed ad libitum. Before vaccination, blood samples were randomly collected from the wing veins. Four groups were vaccinated with the inactivated H9N2 Razi Institute vaccine at 21 days subcutaneously at the back of neck. Three weeks later, two groups were re-vaccinated. Two weeks later, at the age of 56 days, three groups were challenged with 100 µL of allantoic fluid containing 105 EID50 H9N2 through the oculonasal route. Blood samples were collected from quails at 42, 56, 63, and 70 days from each group to determine AIV antibodies by the hemagglutination inhibition test. Three quails were randomly selected and euthanized from each group on days 1, 3, and 6 post-inoculation (PI). Tissue samples were collected, and the RT-PCR test was performed. No clinical signs or gross lesions existed in any of the groups during the experiment. However, the virus was detected in different tissues on the first, third, and sixth days after the challenge in unvaccinated challenged birds. Virus detection was significantly more frequent in the quails vaccinated once and challenged than in the twice-vaccinated challenged group (P≤0.05). On the third day of PI, the virus was detected in some organs of the challenged groups. On the sixth day of PI, the virus was detected only in the lungs of two unvaccinated and once-vaccinated challenged birds. It was concluded that the vaccination of quails against AIV H9 is necessary to protect them from clinical signs, as well as respiratory tract and intestine replication. Two-time vaccination significantly protects the respiratory and intestine tracts, compared to one-time vaccination (P≤0.05).

Avian Influenza-Killed Vaccine on Tissue Distribution and Shedding of Avian Influenza Virus H9N2 in Ducklings.

Ducks play an important role in the transmission of avian influenza to poultry farms. Because of the importance of vaccination in reducing virus shedding, this study evaluated avian influenza-killed vaccine H9N2 on tissue distribution and shedding of avian influenza virus H9N2 in ducklings. One hundred-day-old ducklings were purchased and, after bleeding from 20 birds, were kept in four separate rooms under standard conditions. Groups 1 and 2 were vaccinated at 9 days, and groups 2 and 3 were challenged with 0.1 ml of allantoic fluid containing 105 EID50 (A/chicken/Iran/Aid/2013(H9)) virus intranasally at 30 days. Group 4 chicks were kept as the control group. Chicks were observed two times daily. On days 1, 3, 5, and 8 after inoculation, 3 chicks were randomly selected from each group and cloaca and trachea swabs samples were collected from each bird. Then the ducklings were euthanized and trachea, lung, spleen, intestine, liver, and brain tissue samples were collected for molecular detection. The virus was detected in the tissues and tracheal and cloacal swabs by polymerase chain reaction (PCR), and anti-AIV titres were measured by HI test. The results showed no clinical signs in the challenged groups. In the vaccinated challenged group, virus was detected only in cloacal swabs, but in the unvaccinated challenged group, virus was detected more in tracheal swabs than in cloacal swabs. In challenged-unvaccinated chicks, virus was detected in the trachea and lungs, and in challenged-vaccinated birds, virus was detected in the intestines. In conclusion, vaccinating ducks against the AI H9N2 virus reduced shedding and tissue distribution of AI viruses in challenged ducks.

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.

Phylogenetic analysis of the hemagglutinin genes of 12 H9N2 influenza viruses isolated from chickens in Iran from 2003 to 2005.

In the present study, the hemagglutinin genes from 12 influenza viruses of the H9N2 subtype were isolated from chicken flocks in different provinces of Iran from 2003 to 2005, amplified and sequenced. All of the 12 isolates showed similar sequences at the cleavage site, RSSF/GLF, bearing eight potential glycosylation sites and sharing the characteristic deduced amino acid residues alanine-190, glutamine-226, and glutamine-227 at the receptor-binding site. Ten out of these 12 isolates possessed leucine at position 226, which prevails in the sequences found in human H2 and H3 strains. Overall, the presence in these Iranian poultry H9N2 viruses of the sequence known to bind to human-type receptors and the presence of antibodies in the human population of Iran to H9N2 showed that it is possible for circulating H9N2 avian influenza viruses in Iran to infect humans. Hence, extensive surveillance of H9N2 in this country is highly recommended.