Investigation of Newcastle Disease Virus Infection in Pet Birds in the Southwest of Iran.

Newcastle disease virus (NDV) is a serious threat to the international poultry industry. Therefore, to determine the role of pet birds (Psittaciformes and Passeriformes) in its spread and epidemiology, the presence of this virus in these birds was investigated. In this study, fecal and cloaca swabs from 63 Psittaciformes and 37 Passeriformes, along with tissue samples of dead birds, including proventriculus, trachea, lungs, and intestine, were collected from breeding and sales markets as well as the birds referred to Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran. Isolation of the virus was performed by injecting the suspension of the samples into the allantoic fluid of fertilized eggs, and NDV was detected in the achieved allantoic fluids by reverse transcription polymerase chain reaction. The NDV was detected in 13 allantoic samples. The partial F gene sequences of 10 positive samples were investigated, and their genetic relationship with each other as well as with other isolates in the gene bank was marked. Consequently, subgenotype VII.1.1 (VIId) was in the locus of all 10 viruses. By the amino acid cleavage site sequences of F protein, 10 isolates were determined as velogenic NDV. Moreover, all sequences were similar to each other and other Iranian isolates. Furthermore, the 112RRQKR/F117 pattern was the main amino acid (aa) sequence in the F-protein Cleavage site for VIId genotype isolates.

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.

Effects of Selected Adjuvants on Immunogenicity and Protectivity of Pasteurella multocida Bacterin Vaccine in Chickens.

Avian pasteurellosis (fowl cholera) is an important disease affecting domestic and wild birds all over the world. Although the capsular type A of Pasteurella multocida is mostly involved, other capsular types are occasionally incriminated. The present study aimed at investigating the effect of some adjuvants on immunogenicity and protectivity of P. multocida bacterin in chickens, compared to an Iranian commercial vaccine. Eight-week-old chicken pullets were double vaccinated with an interval of three weeks. Vaccine immunogenicity testing was conducted using an in-house indirect enzyme-linked immunosorbent assay and assessing serum antibody titers at 7, 14, and 21 days post-primary and 14 days post-secondary immunization. The possible adverse effects were recorded by a poultry-disease expert. For evaluating the vaccine protection rate, chickens were subjected to 2×Lethal Dose 50%of a virulent P. multocida strain two weeks post-secondary immunization. The rate of live and normal animals was regarded as protection rate 7days after the exposure. The findings showed that oil adjuvants Montanide ISA 70-and Montanide ISA 71-containingvaccines (with or without saponin) caused a powerful immune reaction than the aluminum adjuvanted vaccine and commercial vaccine (P<0.05). Significant protection against challenge was merely induced by the oil adjuvanted vaccines (P<0.05). The majority of the studied chickens showed inflammation at the injection site (yellow) throughout the trial. Vaccines made by Montanide ISA 70 and Montanide ISA 71 are novel and effective inactivated vaccines that are able to cause significant protection to fowl cholera disease.

Molecular identification of aviadenoviruses in broiler chickens suspected to inclusion body hepatitis in Golestan province, Iran.

BACKGROUND: Fowl adenoviruses (FAdVs) are distributed widely throughout the world, and domestic avian species of all ages are susceptible. Fowl aviadenoviruses (FAdVs) can be separated into 5 different species (A-E) with various genotypes and 12 serotypes. Some geno- or serotypes induce hepatitis-hydropericardium syndrome (HPS), inclusion body hepatitis (IBH), and adenoviral gizzard erosion (AGE). AIMS: Detect FAdVs serologically and molecularly and sequencing of FAdVs in broiler flocks in Golestan province. METHODS: From December 2017 to June 2018 liver tissues and blood samples were collected from 31 broiler flocks suspected of IBH. Polymerase chain reaction (PCR) was applied on liver samples and the positive samples were sequenced and antibody against FAdVs was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Out of 31 flocks, the titers of 29 flocks (93.5%) were high in ELISA test for FAdVs and 22 flocks (70.96%) were positive in PCR test. Sequence analysis indicated the isolates belonged to D and E genotype of adenovirus. CONCLUSION: Inclusion body hepatitis caused by FAdVs, are spreading increasingly in broiler flocks of Golestan province and more attention and surveillance programs of breeder and broiler farms are needed to develop preventive measures. Moreover, vaccination of poultry farms in Iran should be considered by more complement studies.

Molecular identification of reovirus in broiler type flocks in Golestan province, Iran.

BACKGROUND: Avian reovirus (ARV) has a global distribution in nature and most clinical signs are found in broiler type chickens. AIMS: This study was conducted to detect and identify reovirus infections from vaccinated breeder chickens and their progenies. METHODS: A total of 20 tissue and blood samples were collected from vaccinated broiler breeders and their progenies with gastrointestinal or performance problems during peak production. Antibody titers were measured by indirect enzyme-linked immunosorbent assay (ELISA) tests. RNA extraction from tissue samples was performed and cDNA was prepared and directly used in the polymerase chain reaction (PCR). Nucleotide sequences were bilaterally determined using internal primers. The analysis of the nucleotide sequences and their related amino acids was performed by the specialized Molecular Evolutionary Genetics Analysis software (6th version). RESULTS: The virus variant was detected in two vaccinated broiler breeders and five broiler flocks. The vaccine strains in breeder flocks included S1133, SS412, 1733, 2408 belonging to genotype 1 from the reovirus phylogenetic tree. Sequence 7 from the isolated reovirus based on the σC revealed that they were different from the reovirus vaccine, and that 6 isolates belonged to genotype 1 of the phylogenetic tree while 1 isolate belonged to branch 4 of the phylogenetic tree. CONCLUSION: The study showed that the new reovirus strain isolated from vaccinated birds differs from common strains used in the vaccines. It is therefore essential to prevent the effects of the field reovirus on the performance of industrial poultry, by updating and inventing new commercial vaccines, live and killed, against the reovirus.

Experimental Infection of Turkeys with A Virulent Newcastle Disease Virus Isolated from Broiler Chickens.

Newcastle disease (ND) is a highly contagious infection of many avian species, mainly chickens and turkeys, with a devastating impact on worldwide poultry production. The ND accounts for heavy losses in Iranian poultry flocks. There are some reports regarding the epidemiology of this infection in Iran. This study was performed to investigate the infection of turkeys with a Newcastle disease virus (NDV) isolated from a broiler chicken flock in southwestern Iran during 2013. For the purpose of the study, 70 day-old Wishard bronze poults were allocated into two groups of control (n=25) and infected (n=45). At 32 days of age, each bird in the infected group was inoculated with 0.1 mL (50 &mu;L per eye) of NDV-infected allantoic fluid through an ocular route and received 105 EID50 of viral inoculum. On the other hand, the birds in the control group were inoculated with phosphate buffered saline by the same route. Swab samples were taken from both groups at different time points, namely from 1 to 21 days postinoculation, and verified for NDV infection by using reverse transcription-polymerase chain reaction (RT-PCR). Both groups were also examined serologically by haemagglutination inhibition test. Clinically, the infected turkeys exhibited anorexia, severe depression, sitting on the hock joint, white to greenish (sometimes bloody) diarrhea, neurological disorders, and mild respiratory problems. Out of 45 inoculated birds, 9 (20%) cases died. Based on RT-PCR, virus shedding was observed in the challenged birds 3-8 days postinoculation. The NDV was detected more in tracheal swabs (50%) than in cloacal swabs (12.5%). The infected birds showed a high seroconversion. Therefore, the NDV circulating in Iranian chicken flocks has the potential to cause a serious illness in commercial turkeys. The vaccination of turkeys, as well as biosecurity, should be considered carefully to prevent the ND outbreaks in the future.

Detection and Isolation of H9N2 Subtype of Avian Influenza Virus in House Sparrows (Passer domesticus) of Ahvaz, Iran.

Avian influenza (AI) is an acute infectious disease with worldwide significance causing extensive economic losses in the poultry industry. Avian influenza viruses (AIVs) belong to the family Orthomyxoviridae and categorized in the genus influenza virus A. These viruses have been isolated from more than 100 species of free-living birds. Migratory birds are considered as reservoirs for AIVs and are the major agents responsible for global outbreaks. The Passeriformes are found in most parts of the world and cover a variety of habitats from rural to urban areas. House sparrows are members of the family Passeridae and due to their free flying, are strongly associated with seabirds, indigenous, and industrial poultry. The aim of this study was to determine the role of house sparrows in AIV (H9N2) circulation in the Ahvaz region. The intestinal and tracheal samples were taken from 200 sparrows around Ahvaz during 2017. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using specific primers in order to detect M and H9 genes of AIVs. The positive specimens in the PCR for the M gene were inoculated into 9-11-day-old embryonated chicken eggs via the allantoic fluid. The results showed that 11 out of 200 samples were positive for the two genes of M and H9. According to the findings of the present study, house sparrows are infected with H9N2 and pose a threat to commercial poultry. These birds may play a significant role in the transmission of AIV between wildlife and domestic animals. Therefore, this issue is important to be considered in preventive measurements.

Detection of Mycoplasma gallisepticum and Mycoplasma synoviae among Commercial Poultry in Khouzestan Province, Iran.

Mycoplasmas are important avian pathogens, which can cause both respiratory disease and synovitis in poultry that result in considerable economic losses to the poultry industry all over the world. The aim of this study was to determine the prevalence of Mycoplasma gallisepticum and Mycoplasma synoviae infections among commercial poultry flocks in Khouzestan province, Iran, using the polymerase chain reaction (PCR) technique. Totally, 290 tracheal swab samples were collected from 19 broiler flocks and 4 layer-breeder flocks, with or without respiratory signs, in different areas of Khouzestan province within six months. The PCR tests were applied for the specific amplification of 16S rRNA (185 bp) and vlhA (392 bp) genes. Out of 100 swab samples obtained from the layer-breeder flocks, 1 and 72 specimens were positive for M. gallisepticum and M. synoviae, respectively. In this regard, out of the 4 layer-breeder flocks, 1 (25%) and 4 (100%) flocks were positive for M. gallisepticum and M. synoviae, respectively. However, none of the studied broiler flocks were M. gallisepticum- or M. synoviae-positive. According to the results, the PCR technique could be concluded as a rapid method for the accurate identification of M. gallisepticum and M. synoviae infections in commercial poultry flocks. The results were indicative of the low prevalence of M. gallisepticum in the studied flocks in Khouzestan province. On the other hand, M. synoviae was widely distributed among layer-breeder flocks in this province.

Detection of Newcastle Disease, H9N2 Avian Influenza, and Infectious Bronchitis Viruses in Respiratory Diseases in Backyard Chickens in Ahvaz, Iran, in 2014-2015.

Newcastle disease virus (NDV), avian influenza virus (AIV), and infectious bronchitis virus (IBV) are the most prevalent viral pathogens in the Iranian poultry industry. This study aimed to reveal the presence of these viruses in the backyard chickens in Ahvaz, located in the Southwest of Iran. A total of 100 chickens with respiratory signs and mortality were examined by taking the blood samples as well as tracheal and cloacal swabs. Most of the chickens had not received any vaccine. The blood samples were assessed for the antibodies against NDV and AIV by haemagglutination inhibition test, and against IBV by enzyme-linked immunosorbent assay. The swab samples were utilized for molecular detection using reverse transcription polymerase chain reaction (RT-PCR). Based on the results of the serologic test, 77%, 45%, and 38.4% of the birds were positive for NDV, AIV, and IBV, respectively. In the RT-PCR, 95% of the birds were positive for one of the three viruses. The detection rates of NDV, AIV, and IBV were 60%, 34%, and 55%, respectively. The coinfections of AIV/NDV, AIV/IBV, NDV/IBV, and AIV/NDV/ IBV were observed in 13%, 4%, 23%, and 7% of the sampled chickens, respectively. The results demonstrated that the Iranian backyard chickens were infected with NDV, AIV, and IBV. This could pose a threat to the commercial poultry; therefore, preventive measures need to be implemented in this regard.

Comparison of three diagnostic methods for the detection of Toxoplasma gondii in free range chickens.

Detection of Toxoplasma gondii in free range chickens is an indicator of the prevalence and distribution pattern of T. gondii in the environment. For this purpose, serologic assays especially modified agglutination test (MAT) is the main approach in the literature. The main goal of this study was to compare the polymerase chain reaction (PCR) based on amplification of first internal transcribed spacer (ITS-1) of ribosomal DNA gene, ELISA, and MAT to demonstrate T. gondii infection in free range chicken. A total of 106 adult free - range chickens were killed. Blood, whole heart and brain samples were taken. Sera were examined for the presence of T. gondii antibodies by ELISA and MAT as well. Selected tissues were used for PCR and bioassay in mice. The results revealed that 48.11%, 51.89%, 46.23% and 27.36% of chickens were positive in ELISA, MAT, PCR and bioassay in mice respectively. Good correlation between the results of PCR, ELISA and MAT were detected, but not with bioassay in mice. Compared with PCR, the sensitivity and specificity of ELISA were 92.16% and 96.36% respectively and also for MAT, the sensitivity was 81.81% and the specificity was 92.15%. The specific diagnosis of T. gondii infection in chickens is central to a better understanding of the epidemiology and dynamics of transmission among the various host population and is particularly important for planning effective optimal prevention and control programs. Our data in the present study demonstrated that PCR, ELISA and the MAT are helpful and precise methods to detect T. gondii in naturally infected free-range chickens.

Serum protein profiles in domestic pigeons naturally infected with Mycobacterium avium subsp. avium.

BACKGROUND: Avian tuberculosis is an important disease affecting all species of birds and is most often caused by Mycobacterium avium or Mycobacterium genavense. Blood proteins are important diagnostic constituents in gastrointestinal, hepatic, renal, and infectious diseases. OBJECTIVE: The aim of the study was to compare serum protein profiles of domestic pigeons (Columba livia var. domestica) infected with Mycobacterium avium subsp. avium (MAA), with healthy pigeons. METHODS: Serum samples were collected from 80 pigeons with clinical signs of tuberculosis, all kept in the same loft. All birds were necropsied and cultured for mycobacteriosis; positive cultures were typed for MAA by PCR reactions targeting 16S rRNA, IS901 and IS1245. The concentration of total serum proteins was determined by the biuret method and spectrophotometry. Individual protein fractions were analyzed by cellulose acetate electrophoresis and extrapolated based on total protein concentration. For statistical analysis, the infected birds were compared with healthy pigeons. RESULTS: A total of 37 pigeons with culture results positive for MAA were selected and allocated to 2 groups, a culture-positive group with macroscopic lesions of tuberculosis and another without macroscopic lesions. Six protein fractions were identified: prealbumin, albumin, alpha-1, alpha-2, and beta globulins and gamma globulins. Concentrations of total protein, beta globulins and gamma globulins were statistically significantly higher in the infected pigeons when compared with the control group. There were no significant differences between the groups of birds with or without macroscopic lesions. CONCLUSIONS: Statistically significant differences in total protein, and beta and gamma globulin concentrations in all infected pigeons suggest that serum protein electrophoresis represents a nonspecific but valuable indicator for tuberculosis in pigeons.

Characterization of Eimeria Species in Commercial Broilers by PCR Based on ITS1 Regions of rDNA.

BACKGROUND: Coccidiosis is an intestinal disease of chickens caused by various species of protozoan parasites within the genus Eimeria. Diagnosis and genetic characterization of different species of Eimeria are central to the prevention, surveillance, and control of coccidiosis. The aim of this study was to detect different chicken Eimeria species from several areas in Khuzestan, southwest Iran. METHODS: From February to September 2008, PCR assay as well as parasitological examinations was applied for the identification of field isolates of Eimeria parasites around Ahvaz, center of Khuzestan, southwest Iran. Data were analyzed by the Kappa statistic test. RESULTS: Eimeria maxima, E. necatrix, E. tenella, E. acervulina and E. mitis were detected in this study. The prevalence of Eimeria spp. was 31.5% (126 of 400) and E. tenella was the most prevalent species in Khuzestan. Based on the Kappa statistical test, a good correlation between the results of PCR and traditional biometrical methods was only observed for E. maxima. CONCLUSION: The present study is the first on the prevalence of Eimeria species in Khuzestan, based on the molecular findings. We believe that traditional methods are not sufficiently reliable for specific diagnosis of Eimeria species in chickens and PCR based amplification of DNA sequence of parasite, could resolve this problem.

The role of outer membrane proteins of Ornithobacterium rhinotracheale in attachment to chicken tracheal epithelium.

Ornithobacterium rhinotracheale (ORT) infections cause major losses to the poultry industry. In search for factors implicated in the pathogenesis of ORT infections, the role of outer membrane proteins (OMPs) in the interaction of ORT with chicken tracheal epithelium was investigated. For this purpose, immune sera were prepared against total extracted OMPs, whole cell bacteria and three major OMPs of 45, 53 and 70 kDa and used in bacterial adherence inhibition assay. The results showed antibodies against a 53 kDa OMP significantly (p < or = 0.05) inhibited the bacterial adherence to chicken tracheal epithelium up to 78%.

A survey of the prevalence of infectious bronchitis virus type 4/91 in Iran.

To evaluate the prevalence of infectious bronchitis virus (IBV) type 4/91 in Iran, tracheal swabs from 77 broiler flocks in 16 provinces were collected at the slaughterhouse. Swabs were subjected to RNA extraction and tested by RT-PCR, followed by a type-specific nested PCR. The viral RNA was detected in 33 samples (42.8%) from different provinces. The results indicate a relatively high prevalence of IBV type 4/91 in Iran and necessitate revising the vaccination programme against this disease.

Spontaneous lesions in the thyroid of sheep and goats in Iran.

A slaughterhouse survey conducted in Ahwaz (Iran) revealed higher incidence of pathological lesions in the thyroid glands of goats (6.54%) than in sheep (2.8%). Goitre was found in 4.99% and 1.96% of slaughtered goats and sheep, respectively. However, ultimobranchial body cysts were present three times more in sheep than in goats.