Two Novel Avian Influenza Virus Subtypes Isolated from Domestic Ducks in North of Iran.

Avian Influenza Viruses (AIV) are the causative agents of Avian Influenza (AI), which is a contagious and zoonotic disease in birds. Among birds, wild waterfowls and ducks are the primary and natural reservoirs of low pathogenic avian influenza viruses (LPAI). This study aimed to identify and differentiate between two AIV subtypes (i.e., hemagglutinin and neuraminidase from domestic ducks by hemagglutinin inhibition (HI) and neuraminidase inhibition (NI) assays. To this end, 962 cloacal swabs were collected from domestic ducks being sold at different Iranian Live Bird Markets in Gilan, Mazandaran, and Golestan provinces, located at the southern coast of the Caspian Sea. The samples were inoculated in 10-day-old embryonated specific pathogen-free chicken eggs, and subsequently, harvested allantoic fluids were subjected to agar gel immunodiffusion, HI, and NI assays. In total, five positive samples, including two H4N2 and three H3N2 AIV subtypes were identified. Isolation of H4N2 and H3N2 viruses has never been reported from Iranian domestic ducks previously. This finding further suggests the diversity of LPAI viruses in Iranian ducks and also shows that the HI and NI assays are highly efficient in determining AIV subtypes.

Evaluation of viral load and expression level of apoptotic genes in selected tissues of two hybrids of commercial broiler chickens challenged with infectious bronchitis virus: a comparative study.

Background: Infectious bronchitis virus (IBV) causes severe economic losses worldwide. IBV has a broad tissue distribution with different viral loads in different tissues. Additionally, IBV can induce apoptosis in infected cells. Aims: The present study aimed to evaluate the role of the genetic background of chickens in viral load and the expression level of apoptotic genes in different tissues of two hybrids of commercial broiler chickens (Ross 308 and Cobb 500) challenged with IBV. Methods: Chickens at 21 days of age were nasally challenged with 200 µL of allantoic fluid containing 104 EID50/ml of Iranian variant-2-like IBV (IS/1494). The expression level of apoptotic genes (Fas, FasL, Bax, and Bcl-2) in the tracheal and renal tissues and the amount of viral load in the tracheal, renal, and cloacal swab samples were investigated two, five, and seven days after IBV infection by RT-qPCR assay. Results: The amount of viral load and apoptotic the expression level of apoptotic genes in the tracheal (two and five days after infection) and renal samples (seven days after infection) were significantly higher in the Ross challenged group than in the Cobb challenged group.Furthermore, no difference was observed in the cloaca viral load on sampling days. Conclusion: To our knowledge, this is the first report that evaluated the role of the chickens' genetic background in the amount of viral load and the expression level of apoptotic genes against IBV. Further studies are needed to investigate the pathogenic characteristics of IBV in Ross 308 and Cobb 500 chickens.

Report of a new meq gene size: The first study on genetic characterisation of Marek's disease viruses circulating in Iranian commercial layer and backyard chicken.

1. In recent months, several outbreaks with clinical signs of MDV-1 were reported in Iranian parent and laying hen farms, in addition to backyard chickens. Several meq gene sequences from these outbreaks were amplified and molecularly characterised.2. The meq protein sequences revealed three different sizes, namely the standard 339 aa, a shorter form of 338 aa lacking a proline residue at position 191, and a very short (vs) size of 265 aa. Based on sequence and size, the 265 aa meq has never been reported from international research groups before. The protein has only one PPPP repeat motif suggesting it belongs to a highly virulent strain.3. The standard meq sequences showed 100% BLAST identity to the vv+ isolate Polen5. However, the 338 aa form clustered to the clade usually reported from North America.4. This is the first report on genetic analysis of MDV-1 from Iran, but further study is required to obtain a better picture of the diversity and prevalence of different MDV-1 strains circulating in the country's farms, backyard poultry and other bird species.

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.

Molecular detection of fowl adenovirus 7 from slaughtered broiler chickens in Iran: the first report.

BACKGROUND: Fowl adenoviruses (FAdVs) are responsible for a variety of clinical symptoms, with an increasing significance in the poultry industry throughout the world. Typical diseases caused by FAdVs include inclusion body hepatitis (IBH), hepatitis-hydropericardium syndrome (HHS), gizzard erosion (GE), respiratory disease, and hemorrhage in muscles and organs. AIMS: During 2020, broiler chickens from the north of Iran showed ecchymotic and petechial hemorrhages in thigh and breast muscles at the slaughterhouse. Hemorrhages were observed in 10% to 60% (with an average of 20-30%) of chicks per flock. To find out the etiology of these lesions, the present study was conducted. METHODS: Different environmental factors were investigated, and FAdV, infectious bursal disease virus (IBDV), and chicken infectious anemia virus (CIAV) were detected using molecular assays. RESULTS: Among the viruses tested, FAdV was detected by polymerase chain reaction (PCR), and sequence analysis clustered the virus into species E, serotype 7. CONCLUSION: This is the first report on FAdV-7 existence among poultry in Iran. Effective screening of the chicks at slaughtering age should be performed from the whole country.

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.

Prevalence Determination of m. Hominis and m. Genitalium in the Semen Samples in the Northeast of Iran Using Culture and Multiplex Polymerase Chain Reaction.

Infertility has recently become a growing social and economic world problem. Genital mycoplasmas, such as Mycoplasma hominis and M. genitalium, are most frequently associated with several adverse effects on men’s fertility. The present study aimed to determine the prevalence of M. hominis and M. genitalium in the semen samples in thenortheast of Iran. During thiscross-sectional study from February to May, 2018, 100 semen samples were collected from 100 infertile men in Mashhad, Khorasan Razavi province, northeast of Iran. The presence of M. hominis and M. genitalium was detected by cultivation, polymerase chain reaction (PCR), and Multiplex PCR assays. The colony of mycoplasma was confirmed by Diene’s stain; moreover, arginine hydrolysis, glucose, and urea utilization were evaluated. The following semen indices were analyzed according to World Health Organization guidelines for semen analysis: color, volume, appearance, liquefaction, viscosity, concentration, pH, leukocyte concentration, progressive motility, morphological normality, motile sperm concentration, functional sperm concentration, sperm motility index, and functional sperm. The gene of 16SrRNA (GPO1& MGSO primers) was used as the target gene of the Mycoplasma genus in PCR assay. Multiplex-PCR was performed with a specific primer for conserved regions in the 16SrRNA gene for M. hominis (RNAH1& RNAH2 primers) and the 140-kDa Adhesion Protein Gene for M. genitalium (MG1 & MG2 primers).According to the results,9 (9%) samples were PCR-positive for Mycoplasma spp , while there were 7 (7%) cases isolated by cultivation. M. hominis was detected in 8 (8%) samples by Multiplex PCR, while there was no evidence for M. genitalium. The mean age scores of all infertile and infected men were obtained at 31 and 30 years, respectively. The study could not show any statistical correlation between mycoplasma infection and abnormal semen parameters. The heterogeneity of mycoplasma prevalence in the reports can be ascribed to differences in geographic areas, the sensitivity of the identification method, condition of the group (fertile/infertile), sample size, and operator proficiency. Various results have been reported in numerous studies conducted on the relationship between mycoplasma infection and abnormal semen parameters.

Detection and Phylogenetic Study of Peste des Petits Ruminants in Iran, 2019: Updated Data.

Peste des Petits Ruminants (PPR) is caused by a morbillivirus from the Paramyxoviridae family and the infected animals, especially goats, that show clinical signs of necrotic stomatitis, enteritis, and pneumonia. The PPR virus has four lineages closely related to the geographical regions. Sufficient awareness of the lineage of the virus helps monitor the disease in different regions of a country. Phylogenetic studies have led to implementing strategies against new lineages that may enter a given country from the neighboring countries. The present research aimed to study the PPR virus (PPRV) detected phylogenetically by PCR in a small ruminant flock with PPR clinical signs. The goats in a flock in Alborz province showed clinical signs of PPR, and 10% died. Oral swabs and blood samples were taken from two affected goat flocks. The RT-PCR was conducted to detect PPRV RNA, and the sequence of the obtained RNA was analyzed phylogenetically. Moreover, all the samples were positive for the presence of PPRV and belonged to lineage IV. The isolates had high homology with each other and with the isolates from different countries. To inhibit the entrance of new isolates to Iran and reduce the incidence of outbreaks in Iran, it is essential to control the animals’ movement across the borders and increase the vaccination coverage throughout the country. To eradicate PPR, an extensive vaccination program should cover small ruminant populations throughout the country.

laying Farm: Up to Date Data on a Fowlpox Outbreak in Phylogenetic Analysis in Iran, 2018.

Fowlpox is an economically significant viral disease in poultry, characterized by two forms of clinical signs, including cutaneous and diphtheritic lesions. This infection can have several adverse effects on flock performance, such as a reduction in egg production and growth and an increase in mortality. In winter 2018, an infection suspected to fowlpox was reported from a Hy-line W-36 laying farm in Isfahan province, Iran. The birds were 38 weeks of age and showed obvious diphtheritic signs in mucous membranes with increased mortality and reduced egg production. In total, 20 samples were collected from diphtheritic lesions (Trachea and Esophagus) of infected birds. The Polymerase Chain Reaction method was used to amplify a 578 bp fragment of the poxvirus 4b core protein gene. Phylogenetic relationships of avian poxviruses are usually analyzed using the 4b core protein-coding gene sequences with molecular weights of 75.2 kDa. The major elements had the fowlpox genome, and sequencing was performed for one isolate as representative. The nucleotide sequence result showed that this isolate (FP\UT-POX-2018) had a similarity rate of 99.53% with the previous Iranian fowlpox isolate (FP\GHPCRLAB.3) sequenced in the GenBank.Moreover, there was a 100% similarity among the current isolate nucleotide sequence, FP/NobilisVarioleW, and FP/FPV-VR250. The derived phylogenetic tree showed that these isolates were clustered in A1 subclades. Therefore, Iranian isolates of fowlpox virus have remained in the same subclade of phylogenetic classification (subclade A1), and they show high genomic similarity with previous isolates of Iran. Veterinarians and farmers must not underestimate fowlpox. However, they should consider the importance of vaccination against this disease like any other disease care.

The emergence of Q1 genotype of avian infectious bronchitis virus in Iran, 2019: the first report.

BACKGROUND: Avian infectious bronchitis (IB) is an infectious viral disease of chickens. The effective protection of chickens against many different infectious bronchitis virus (IBV) variants is not achieved unless the circulating genotypes in the region are identified and the cross-protection of the potential of vaccines in use is assessed. AIMS: In a monitoring program of IBVs, a new genotype was identified in the north of Iran, 2019. This work was conducted to isolate and characterize this new IBV genotype. METHODS: Tracheal tissues were collected from chickens showing signs of respiratory involvement. Specimens were homogenized and inoculated to the allantoic fluid of embryonated specific pathogen-free (SPF) eggs. Infectious bronchitis virus was detected using real time-polymerase chain reaction (RT-PCR). The hypervariable region of the IBV S1 gene was amplified for sequencing. RESULTS: Positive samples were phylogenetically analyzed, and both positive isolates were clustered with Q1 IBV strains. CONCLUSION: This is the first report of the Q1 outbreak in Iran. More investigations are needed to find the role of Q1 IBV in the respiratory disease complex of chickens.

Avian Influenza (H9N2 Subtype) in Iranian Broiler Farms: A Cross-sectional Study.

The present study aimed to determine the seroprevalence of H9N2 influenza in broiler farms at the time of slaughter in Iran. A total of 747 birds were sampled from 74 Farms in 13 provinces within 2013-2016. The obtained sera were investigated using the hemagglutination inhibition (HI) test. Out of 74 sampled farms and 747 birds, 57 farms (77%) and 445 (59.57%) birds were reported to be seropositive. In 2013, 10 farms and 110 birds were sampled out of which three farms (29.6%) and 29 birds (30%) were seropositive. In 2014, 24 farms and 220 birds were sampled out of which 22 farms (91.6%) and 220 birds (86.6%) were positive in six provinces. In 2015, 30 farms and 278 birds were sampled out of which 5 farms (16%) and134 birds (48.2%) were positive in four provinces. Finally, in 2016, 7 farms (70%) out of 10 sampled farms and 62 birds (59%) out of 105 sampled birds were positive for H9N2 in eight provinces. The mean titer of units in 2013 was statistically lower, as compared to that in 2014 (p <0.01). In addition, the proportion of positive serum units in 2013 was statistically lower, as compared to that in 2014 (p <0.001). In general, the prevalence of H9N2 was high indicating the continuous circulation of the virus in Iran. Given the importance and impact of this virus on the poultry industry, people’s livelihood, and public health, more epidemiological studies are needed to evaluate the effectiveness of the adopted measures and methods in controlling the H9N2 virus.

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 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 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.

Investigation of Avian Influenza Viruses (H9N2-H5nx) in Pigeons during Highly Pathogenic Avian Influenza Outbreaks in Iran, in 2016.

Avian influenza (AI) virus (H9N2 and H5 subtypes) infections in birds cause major concerns around the world. The majority of the avian species, such as domestic, pet, and wild birds, are natural and experimental hosts of avian influenza viruses. There are global concerns about members of the Columbidae family, namely pigeons or doves, for their role as the potential interspecies bridge in influenza A viruses ecology. The acquired scientific data in this regard is still not clear since there are doubts about whether or not they transmit viruses between susceptible populations, and spread viruses among farms during outbreaks. To monitor H5 and H9 influenza virus infection status in the rural, backyard, and domestic birds, an annual active surveillance program was performed from September to October 2016. In December 2016, an outbreak of highly pathogenic avian influenza (HPAI) virus subtype H5N8 was detected in a layer farm in Tehran province, Iran. The present research was conducted to study H9N2 or H5 infections in pigeons within HPAI H5N8 2016 outbreaks and annual national AI surveillance in Iran. For this purpose, cloacal swabs and tissue samples (trachea, lung, brain, liver, heart, pancreas, and cecal tonsil) were collected and examined by real-time reverse transcription-polymerase chain reaction (RT-PCR) method and virus isolation. Results of the tests performed on the swab and tissue samples were negative for H5 nor H9N2 viruses. The samples in real-time RT-PCR that after three passages still showed negative results in HA and molecular tests were considered negative. Moreover, the Newcastle disease virus was isolated in most of the samples taken from dead pigeons, after inoculation in embryonated chicken eggs.

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.

Trend of Changes in the Titer of Antibody against Avian Influenza Virus H9n2 during Raising Period in Vaccinated and Unvaccinated Broiler Farms in Qazvin Province, Iran: A Cohort Study.

Avian influenza virus (AIV) H9N2 is endemic in Iran and its large-scale circulation in the poultry industry of the country is devastating. This virus was first reported in the industrial poultry populations of Iran in July 1998. Some of the published studies showed that inactivated avian influenza (AI) vaccines are capable of inducing an immune response and providing protection against morbidity and mortality in different countries (Vasfi et al., 2002; Tavakkoli et al., 2011). Low pathogenicity avian influenza subtype H9N2 virus has been reported to have a zoonotic potential and widespread distribution in Iran. Therefore, water-in-oil emulsion vaccines are employed to control the disease in chickens (Nili and Asasi, 2003). This cohort study was conducted during July 2016-November 2017 in broiler chicken farms of Qazvin province, Iran to investigate the serological change trends in broiler chickens in this region. Level of immunity against the H9N2 virus was evaluated by hemagglutination inhibition assay. Fifteen farms out of thirty enrolled units used AI H9N2 killed vaccines. The minimum of mean antibody titers (MATs) was 4.54-2.42 and the maximum of MATs was 4.54+2.42 on day 3. In addition, the minimum and maximum MATs on day 50 were 0.4-0.64 and 0.4+0.064, respectively. The transfer rate of H9N2 AIV antibodies from the serum of breeders to the serum of chickens was calculated as 60.35% in our study. A significant difference was revealed between the maternal mean antibody titers (MMATs) and the MATs on day 3 (P<0.001). In addition, the difference between the MATs on day 3 and the MATs on day 10 was found to be significant (P<0.01). Moreover, MATs were significantly different between the vaccinated and unvaccinated herds on day 40 (P<0.05), while no significant difference was observed on days 3, 10, 20, and 30 (P>0.05). According to the results of this study, antibody titers in the vaccinated farms did not reach the protective level until the end of the rearing period. Most of the unvaccinated herds experienced a spurt in antibody titers due to exposure to the virus. Consequently, biosecurity measures must be implemented more seriously and strictly in broiler farms.

Prevalence of Avian Influenza in Live Bird Markets, Bird Gardens, and Zoos in Iran in 2015: A Cross-sectional Study.

Avian Influenza (AI) H9N2 is endemic in Iran; therefore, it is necessary to estimate the disease prevalence among birds in live bird markets (LBMs) and assess the risk spread across the country. Accordingly, this study aimed to estimate the prevalence of AI subtypes in LBMs, bird gardens, and zoos during October and November 2015 in Iran and investigate the associated risk factors. Data related to independent variables for birds and premises were collected using a prepared questionnaire which included items about previously known potential risk factors associated with avian influenza infection. Serological testing was carried out to detect the antibodies against H5, H7, and H9. Regarding H5 and H7, the antigens H5N2 and H7N1 were used in this study. Positive samples on the first test were examined with the second antigens, namely H5N1 and H7N7. Moreover, sera with titers ≥4 (i.e. log2) were considered positive and premises with at least one positive bird were considered as positive units. In total, 87 premises were included in this cross-sectional study. Serum samples were examined utilizing hemagglutination inhibition, and RT-PCR was conducted on swab samples. Regarding the molecular test, the RNA was extracted using the High Pure Viral RNA Kit (Roche, Germany). In addition, real-time RT-PCR was conducted based on the described method. The seroprevalence rates of H9N2 were 83.9% and 31.8% at the premises and bird levels, respectively. Totally, 9.2% of pooled swab samples were positive for H9N2. However, all sera and swab samples were negative for H5 and H7. Hot and humid weather (OR=0.13, 95% CI 0.02 – 0.78) as well as bird-keeping condition (i.e., enclosed area) (OR=0.11, 95% CI 0.012 – 1.02) were protective factors for H9N2. High seroprevalence rate of H9 indicates that the disease is endemic in Iranian LBMs. Active surveillance must be carried out in LBMs, especially in the northern provinces of Iran. In addition, cleanliness and improved hygiene would be useful to prevent the spread of disease in LBMs.

Genotyping of avian infectious bronchitis virus in Afghanistan (2016-2017): the first report.

BACKGROUND: Avian infectious bronchitis (IB) is a highly contagious viral disease which affects the poultry industry. The virus exists in a wide variety of genotypes, and phylogenetic analysis has been used to classify infectious bronchitis virus (IBV) strains. AIMS: The object of the study is a molecular characterization of circulating IBV in Afghanistan as a first study. METHODS: The tracheal tissue specimens from 100 different commercial broiler flocks with respiratory distress in Afghanistan were collected during 2016-2017. After real-time reverse transcriptase-polymerase chain reaction (RRT-PCR), IBV-positive samples were further characterized. A 390 bp hypervariable spike glycoprotein gene segment was amplified using Nested PCR, sequenced, and analyzed. RESULTS: The results of real-time RT-PCR showed that 45/100 of the mentioned flocks were IBV positive. Phylogenetic analysis of all positive samples revealed that IBV strains were clustered into two distinct genotypes: LX4 (GI-19) (9/45) and IS-1494 like (GI-23) (34/45). Also, 2 of the 45 samples remained uncharacterized. CONCLUSION: It is the first study focusing on the molecular epidemiology of IBV in Afghanistan, extending our understanding of IB in the region. These results showed the high rate of IB infection in Afghanistan broiler farms and confirm the continuing monitoring of IBVs to modify the vaccination program.

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.