Molecular characterization of circulating avian metapneumovirus, subgroup B, in broiler chickens, Iran, 2016-2018.

BACKGROUND: Avian metapneumovirus (aMPV) infection has significant economic impacts on the poultry industry all around the world. AIMS: The aim of this study is molecular investigations of different types of aMPV in broiler farms in different provinces of Iran from 2016 to 2018. METHODS: Tracheal and oropharyngeal swabs were collected from two hundred broiler chickens with respiratory signs in ten provinces of Iran, including Kurdistan, West Azerbaijan, Semnan, Esfahan, Sistan and Baluchistan, Qazvin, Khuzestan, Fars, Gilan, and Khorasan Razavi from February 2016 to December 2018. After RNA extraction, the presence of aMPV was confirmed using N gene special primers. Then, subtype-specific primers were utilized to differentiate the specific subtype. All positive samples were sequenced. RESULTS: As a general trend, the percentage of aMPV positive chickens increased gradually over time. All samples were clustered together and placed in the subtype B aMPV group. Although 2 samples from 2016 and 2 samples from 2018 were placed in a separate branch, most of the current study samples of 2016, 2017, and 2018 revealed six segregated sub-branches, and they were placed close to other isolates of 2011 and 2013 from Iran. CONCLUSION: The current field study indicated the presence of aMPV in a considerable number of areas in Iran. Thus, the role of this virus in broiler respiratory complex should not be neglected.

Outbreak Investigation of Officially Reported and Highly Pathogenic Avian Influenza (H5N8 Subtype) in Iran During 2016.

On 14 November 2016, an outbreak of highly pathogenic avian influenza (HPA) was reported from a commercial layer farm located in Malard, Tehran Province, Iran. This study aimed to investigate the HPAI H5N8 outbreaks in Iran. The questionnaire was prepared and completed through interviews with farm owners or field observations at the time of disease onset from November 2016 to February 2017. The HPAI H5N8 infection was confirmed in 30 different locations including 10 villages (33.3%), nine-layer farms (33%), two broiler breeder farms (6.67%), one layer breeder farm (3.3%), one turkey farm (3.3%), one partridge farm (3.3%), five national parks (16.7%), and one wetland (3.3%) in 12 provinces of Iran. The cumulative incidence rates of disease in villages, layer farms, broiler breeder farms, layer breeder farms, partridge farms, and turkey farms were 0.02%, 0.87%, 0.55%, 6.25%, 7.14%, and 0.69%, respectively. The findings reflect that among the investigated variables at infected locations, new birds entering the home in villages, live bird markets, inappropriate biosecurity conditions, transporting manure during the breeding period, close proximity of a common road to infected farms, and poultry movement inside (pullet) and outside were the most frequently observed possible risk factors for these outbreaks. In conclusion, attention should be focused on the study of the dynamics and movements of domestic poultry, investigation and modification of the structure of industrial poultry farms, training for all related people, enhancement of passive surveillance, an increase in biosecurity, raising the awareness of the authorities on the importance of the infection, and provision of the required credits and facilities.

Comparison of autogenous and commercial H9N2 avian influenza vaccines in a challenge with recent dominant virus.

BACKGROUND: Avian influenza (AI) caused by AI virus subtype H9N2 is a prevalent viral disease with enormous economic losses in poultry farms through significant respiratory and gastrointestinal manifestations. The degree of protection obtained from a vaccine strongly depends on the level of antigenic similarity between challenge and vaccine virus. AIMS: The study aimed at investigating the possible effects of continuous antigenic changes occurring in circulating Iranian viruses since 1998 on the commercial vaccines outcome by using vaccine seeds from earlier outbreaks for inhibiting viral replication in target organs of broilers challenged with the recent isolate. METHODS: Ninety broilers at one day of age were randomly allocated into 5 groups and vaccinated with autogenous or commercial vaccines (A or B). Two remaining groups consisted of challenged without vaccination and intact birds. Quantitative real time-polymerase chain reaction (qRT-PCR) was performed on the trachea and faecal samples of challenged chickens with recent H9N2 virus to determine viral load. Moreover, humoral antibodies titers were evaluated by hemagglutination inhibition (HI) assay. RESULTS: There was no significant difference in H9N2 viral load in the trachea among vaccinated groups on 5 days post challenge (DPC), but on 15 DPC, the autogenous vaccine significantly lowered viral load compared to commercial vaccines (P≤0.05). No significant differences in faecal swab's viral load was observed between autogenous and commercial vaccine A, and both of them significantly inhibited viral load compared to unvaccinated group (P≤0.05). In addition, the autogenous vaccine elicited the highest HI titer. CONCLUSION: Inactivated vaccines that use isolates from previous outbreaks are no longer able to induce proper immunity against recent H9N2 viruses. It seems the time to change vaccine strains to more recent isolates that have better antigenic similarity with current circulating H9N2 viruses in the region has come.

Molecular Monitoring of D1466 Genotype of Avian Infectious Bronchitis Virus in Iran: A Retrospective Study in 2013-2017.

Infectious bronchitis is one of the most common diseases in the poultry industry in many countries, especially in the regions with a dense poultry farming industry. Gammacoronavirus is the etiologic agent of this disease, with the chickens and poultries as the natural reservoirs of the virus. Various strains of infectious bronchitis virus have been reported in poultry around the world. In terms of pathogenicity, this virus can induce a spectrum of diseases ranging from the moderate respiratory tract to kidney and reproductive diseases. The serotypes of this virus do not cause cross-immunity against each other. This issue makes it difficult to control the disease. Based on the analysis of the highly variable region of the glycoprotein S1 gene, the isolated strains in Iran were classified into seven different phylogenetic groups, including Massachusetts, QX, IS-720, IS-1494, 793/B, IR-1, and IR-2. The D1466 genotype has not been reported in the country; however, the killed vaccine is used in broiler breeder farms. In this study, tissue specimens were collected from 700 farms (i.e., broiler, egg-laying, and broiler breeder farms) suspected of infectious bronchitis within 2013-2017. The samples were examined using real-time reverse transcription-polymerase chain reaction. The D1466 genotype was not detected in any of the studied specimens. Due to the lack of immunity of the D1466 serotype against the dominant types in the country, one has to be careful in choosing the right vaccine. It is necessary to perform continuous monitoring of the circulation status of the various serotypes of viruses in the country to identify the dominant and possible new serotypes for the utilization of the appropriate vaccine.

Effectiveness of Thermostable Vaccine for Newcastle Disease Produced by the Razi Institute on Backyard Poultry in Iran during 2015.

Newcastle disease causes many economic losses to the poultry industry in most countries. This disease is endemic in Iran. Backyard poultry is considered the reservoir of Newcastle virus; however, there is either no vaccination program against Newcastle, or it is performed in a restricted manner. Commercial live vaccines are inactive and sensitive to temperature; moreover, vaccine delivery to villages and remote areas requires special equipment and high cost to maintain the cold chain. This study evaluated the effectiveness of a thermostable Newcastle vaccine produced by the Razi Institute (ND.TR.IR) on the backyard poultry. In four provinces, at least 4 villages were selected as the treatment group, and the same number was selected as the control group. At least, 30 birds were sampled in each village. In each group, blood samples were collected before vaccination and 2 weeks later, and the serum titer of the samples was examined with the haemagglutination inhibition test. The arithmetic mean and standard deviation of the sample titers at the rural level were compared using paired t-test before and after vaccination in each group. Moreover, Repeated Measures ANOVA was utilized to compare the vaccinated and control groups in terms of the titer changes before and after vaccination. In this study, 584 and 389 samples were taken from the treatment (53 households in 20 villages) and control groups (33 households in 14 villages). The mean serum titer values of Newcastle were 4.51±3.03 and 6.64±2.48 in the treatment group before and after vaccination, respectively (P<0.001). The increase in mean titer of the treatment group (2.31 log) was statistically higher than that in the control group (0.66 log) (P<0.001). Out of 584 birds, 517 (88.5%) ones had titer above 3 in the second turn in the treatment group. The thermostable vaccine (ND.TR.IR) produced by the Razi institute is suitable for backyard poultry, which immunizes them against Newcastle disease. Appropriate vaccination programs for backyard poultry should be made; moreover, vaccination of backyard poultry can be effective in preventing the circulation of the field viruses.

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.

Serological Survey of Avian Influenza (H9N2) in Commercial Ostrich Farms in Iran, 2015.

The aim of this study was to determine the seroprevalence of avian influenzaH9N2 subtype in the industrial ostrich farms and its geographical distribution. This cross-sectional study was conducted from January to June 2015. A total of 40 farms were selected from different provinces of Iran, from each of which 11 ostriches (n=440) were sampled. The sera samples were examined using 4 hemagglutination units of H9N2 antigens. A frequency distribution was used to describe the responses to the survey questions. The mean titers between provinces were compared using one-way analysis of variance. According to the results, 21 (47.5%) out of 40 farms and 108 (24.5%) out of 440 ostriches tested positive in the HI-H9N2 test. There were statistically significant differences between the mean titers of samples in different provinces (P<0.001). The current study was conducted on unvaccinated ostriches. The results showed that H9N2 had a high seroprevalence at both farm and bird levels. The findings of this study can be for the further investigation of infection in ostrich farms in order to consider this species in the surveillance programs of the Iranian Veterinary Organization. The detection and isolation of viruses and epidemiological investigation are necessary for the persistent use of H9N2 vaccines in some ostrich farms.

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.