Characterization of Newcastle disease virus in broiler flocks with respiratory symptoms in some provinces of Iran.

BACKGROUND: Newcastle disease, is one of the most important diseases of the poultry industry, has many economic losses. The aim of this study was to isolate and determine the molecular identity of Newcastle disease virus in 40 broiler flocks with respiratory symptoms in four provinces of Iran. METHODS AND RESULTS: Samples of farms with respiratory symptoms were collected from different regions of Isfahan, East Azerbaijan, Golestan, and Khuzestan provinces and inoculated into 9-day-old embryonated chicken eggs. The Reverse-transcription polymerase chain reaction (RT-PCR) was performed to detect the Newcastle disease virus in allantoic fluid. Of the 40 flocks, the virus was isolated and identified in 16 flocks. The PCR products of 16 isolates were sequenced, and a phylogenetic tree was drawn. Accordingly, six isolates were in genotype II and ten isolates were in subgenotype VII.1.1 (VIId) of class II. CONCLUSION: Both genotypes were present in all four provinces. The isolates of Khuzestan province showed the greatest diversity compared to the other three provinces. The similarity of isolates belonging to genotype II in this study was observed with Pakistan, China, and Nigeria, and other isolates were similar to previous isolates in Iran. Also, the highest amino acid sequence in the F-protein cleavage site was 112RRQKR/F117 for VII.1.1 (VIId) genotype isolates and 112GRQGR/L117 for II genotype isolates.

A histopathological and immunohistochemical study of experimental infected turkeys with a virulent Newcastle disease virus.

Newcastle disease (ND) is a highly contagious infection of many avian species, mainly chickens and turkeys, with a devastating impact on worldwide poultry production. This study was designed to examine the effect of virulent ND infection in turkey's tissues and the tissue tropism of the virus. During the previous study period, poults were inoculated at 32 days of age with 105 EID50 virulent Newcastle disease virus. Three poults on days 0, 1, 2, 3, 4, 6, 7, and 14 postinoculations (PI) were selected from each group. They were euthanized by intravenous sodium pentobarbital injection. After macroscopic observation, to histopathological and immunohistochemical studies, the spleen, bursa, cecal tonsils, intestine, proventriculus, lung, kidney, and brain were sampled. Clinically, the infected turkeys exhibited loss of appetite, severe depression, down on hock joint, white to greenish (sometimes bloody) diarrhea, nervous signs, and mild respiratory problems. Out of 45 birds inoculated, 9 (20%) died. Histopathological effects in lymphoid tissues included necrosis and penetration of mononuclear cells on day 4 PI, and subsequent follicular lymphoid depletion on days 6 and 8 PI was observed. Based on the immunohistochemical test, on day 3 in cecal tonsils and spleen, and on day 8 PI, all of them were positive for virus antigen. In conclusion, the NDV circulating in Iranian chicken flocks has the potential to cause severe illness in commercial turkeys.

Molecular identification of infectious bronchitis virus isolated from respiratory diseases in some Iranian broiler flocks.

Infectious Bronchitis (IB) is an acute, highly contagious disease associated with respiratory signs in young chickens and reduced egg production and quality in layers. The purpose of this study was to isolate and identify the infectious bronchitis virus in broiler flocks with respiratory diseases in four provinces of Iran. The specimens from forty IB suspected flocks from different regions of Isfahan, East Azerbaijan, Golestan, and Khuzestan provinces were collected, and the trachea, lung, and cecal tonsils were sampled. The samples were inoculated into 9- to 11-day-old embryonated chicken eggs. After collecting the allantoic fluid, RT-PCR was carried out to detect IB viruses. The results showed that IBVs were isolated from 30% of the flocks in these four provinces. The positive samples, according to a partial S1 gene sequence, were more investigated. Comparing nucleotide and amino acid sequences showed that the four isolates had the most similarity to the Pakistani 793/B strain (GI-13 lineage). The three isolates had the most considerable similarity in amino acid and nucleotide sequences to Iraqi and Iranian QX-like viruses (GI-19 lineage). Two isolates had 96 to 98% resemblance to Iranian variant-2 (GI-23 lineage) isolates. One isolate was found to belong to the Massachusetts serotype (GI-1 lineage) having 100% similarity in its amino acid sequence to the Massachusetts serotypes in GenBank. The phylogenetic relationship of the isolates shows complexity and diversity concerning different sequences and geographical regions.

Molecular detection and phylogenetic properties of isolated infectious bronchitis viruses from broilers in Ahvaz, southwest Iran, based on partial sequences of spike gene.

Infectious bronchitis (IB) is a highly contagious disease involving mostly upper respiratory tract in chickens, leading to significant economic losses in the poultry industry worldwide. One of the major concerns regarding to IB is the emergence of new types of infectious bronchitis viruses (IBVs). The purpose of this study was to identify the IBVs isolated from Iranian broiler chickens with respiratory symptoms. Twenty-five broiler flocks around Ahwaz (southwest of Iran) were examined for IBV. The specimens including trachea, lung, liver, kidney, and ceacal tonsil, were collected from diseased birds and inoculated into chicken embryonated eggs. Harvested allantoic fluids were subjected to reverse transcription polymerase chain reaction (RT-PCR) using primers in order to amplify spike 1 (S1) gene of IBV. The RT-PCR products of four IBV isolates were sequenced. The results showed that from 25 examined flocks with respiratory disease, 12 flocks (48.00%) were positive for IBV. In phylogenetic analysis, our isolates were closely related to the QX-like viruses such as PCRLab/06/2012 (Iran), QX, HC9, HC10, CK/CH/GX/NN11-1, CK/CH/JS/YC11-1, CK/CH/JS/2010/13, CK/CH/JS/2011/2 (China), QX/SGK-21, QX/SGK-11 (Iraq) with nucleotide homology up to 99.00%. This study indicates the role of IBVs in the respiratory disorders of broiler flocks located in southwest Iran, and also the existence of a variant of IBV, which is distinguishable from the other Iranian variants.

Embryonated pigeon eggs as a model to investigate Neospora caninum infection.

It has been shown that embryonated chicken eggs can be used as animal models for experimental infections. The aim of the present study was to investigate pigeon embryonated eggs as animal models for experimental neosporosis. An infection with Neospora caninum Nc1 isolate was conducted in chicken and pigeon embryonated eggs to evaluate LD50. After calculation of LD50, 2LD50 of tachyzoites were injected into the eggs. Macroscopic changes of each embryo were observed, and immunohistochemistry (IHC) and molecular methods were used to investigate the parasitic distribution in the tissues. In the present study, histopathological changes were considered, and sections of those used for histopathological examination including the heart, liver, brain and chorioallantoic (CA) membrane were also subjected to IHC. Pigeon embryos showed more macroscopic changes than chicken embryos. A hemorrhage of the CA membrane was the main gross lesion. Microscopic examination of tissues revealed acute neosporosis due to hemorrhage, necrosis and infiltration of mononuclear inflammatory cells. Based on IHC and molecular results, the parasite DNA was detected in the liver, heart and CA membrane. As with chicken embryonated eggs, these results reinforce the susceptibility of pigeon embryonated eggs to N. caninum, and provide new insights into using an inexpensive and available animal model for N. caninum research. The results of the present study suggest that pigeon embryos may be a good choice for studying the biology of N. caninum in living organisms.

Molecular characterization and phylogenetic study of the fusion genes of Newcastle disease virus from the recent outbreaks in Ahvaz, Iran.

Newcastle disease (ND) is an acute and highly contagious disease affecting many domestic and wild species of birds. Its effects are most notable in domestic poultry due to their high susceptibility and the potential for severe impacts of an epizootic on the poultry industry. In this study, partial sequences of fusion genes of three Newcastle disease virus (NDV) isolates collected during 2013-14 outbreaks from the vaccinated commercial broiler chicken farms with high mortality around Ahvaz city (Southwest of Iran) were characterized. All three isolates showed the amino acid sequence 112RRQKRF117 at the C-terminus of the F2 protein and phenylalanine at the N-terminus of the F1 protein residue 117. These amino acid sequences were identical to a known virulent motif. The phylogenetic analysis revealed that the Iranian ND isolates in this study are closely related to the genotype VIId of class II NDV strains. Our results specified that there are velogenic NDV circulating in Ahvaz commercial broiler flocks and causing outbreaks in poultry industry.

Viral quantity and pathological changes in broilers experimentally infected by IRFIBV32 isolate of infectious bronchitis virus.

An Iranian isolate of avian infectious bronchitis virus IRFIBV32 was quantified in experimentally infected broilers using real-time reverse transcriptase polymerase chain reaction and histopathological changes was investigated. Thirty-six 3-week-old commercial broilers were inoculated by 10(5) ELD50/0.1 ml of the virus. On the various days post inoculation (dpi) different tissues were collected. The virus strongly started the replication in trachea at 1 dpi and reached to the maximum titer at 3 dpi. The highest IBV RNA level was shown in this organ. In lung, the virus was replicated with the titer lower than that of the trachea, but it rose up more at 5 dpi. The kidneys were the tissues with the least viral genome copy number, although the duration of the virus presence was considerable. The virus replicated in testes sooner than ovaries also disappeared sooner but the maximum viral yield in the ovaries was more. The virus titer in the studied tissues had an interesting fluctuation especially in caecal tonsils. Testes and ovaries were the organs that the virus could reactivate without using any chemical. The most severe lesions were observed in tracheae but they appeared in the lungs later. Lymphocyte infiltration in the kidneys was noted from 5 dpi even sooner than the lungs. There were no lesions in the caecal tonsils, testes and ovaries in spite of the virus replication in a high titer.

Changes of several acute phase factors in broiler chickens in response to infectious bronchitis virus infection.

This study was performed to clarify the acute phase response following infectious bronchitis virus inoculation. Ninety clinically healthy 1-d-old Ross chicks were randomly assigned into 2 groups: control (n = 20) and infected group (n = 70). At the age of 20 d, all birds in the infected group were challenged intranasally with allantoic fluid containing 10(5) embryo lethal dose (ELD50)/0.1 mL of the infectious bronchitis virus. Blood samples were collected from 20 clinically healthy and 70 infected chicks at prior and 1, 2, 3, 5, 7, 11, 13, 15, and 20 d postinoculation. On d 1, 7, and 11 postinoculation 4 chickens from the experimental group and 2 chickens from the control group were randomly selected. Their trachea, lungs, and cecal tonsil were collected for virus detection and quantitation by real-time reverse-transcription PCR assay. In the serum the acute phase proteins (haptoglobin and serum amyloid A), pro-inflammatory cytokines (interferon-γ and tumor necrosis factor-α), and serum sialic acid (total, TSA; lipid-bound, LBSA; and protein-bound, PBSA) concentrations were measured using validated standard procedures. All variables were significantly higher in the infected birds after virus inoculation compared with the healthy group (P < 0.05). There were positive correlations between all variables in the infected group. Correlation coefficients were significantly positive between haptoglobin and interferon-γ, LBSA and TSA, and TSA and LBSA (P < 0.05). There were positive correlations among viral RNA and all studied variables; however, these correlations were not statistically significant (P > 0.05).

Pathogenesis and tissue distribution of avian infectious bronchitis virus isolate IRFIBV32 (793/B serotype) in experimentally infected broiler chickens.

Infectious bronchitis (IB) is one of the most important viral diseases of poultry. The aim of this study was to investigate the distribution of avian infectious bronchitis virus isolate IRFIBV32 (793/B serotype) in experimentally infected chicken. Ninety-one-day-old commercial broilers were divided randomly into two groups (seventy in the experimental and twenty in the control group). Chicks in the experimental group were inoculated intranasally with 10(5) ELD50/0.1 mL of the virus at three weeks of age. The samples from various tissues were collected at1, 2, 3, 5, 7, 11, 13, 15, and 20 days postinoculation. Chickens exhibited mild respiratory signs and depression. Viral RNA was detected in the kidney, lung and tracheas on days 1 to 13 PI, in the oviduct between, days 3 and 13, in testes between days 1 and 11 PI, and in the caecal tonsil consistently up to day 20 PI. The most remarkable clinical signs and virus detection appeared on day 1 PI. Data indicated that the number of infected chickens and viral RNA detection from tissues was reduced with increasing antibody titer on day 20 PI. The results demonstrated that the IRFIBV32 virus has wide tissue distribution for respiratory, urogenital, and digestive systems.