Isolation, identification, antibiotic resistance profile and molecular analysis of Ornithobacterium rhinotracheal isolates from turkeys.

BACKGROUND: Ornithobacterium rhinotracheal (ORT) infects numerous birds, particularly chickens and turkeys. ORT is an emerging bacterial pathogen of global concern in the poultry industry. As ORT is rapidly spreading throughout commercial poultry, it requires intensive studies of its epidemiology, diagnostic procedures, molecular typing, virulence genes and antimicrobial resistance. OBJECTIVES: The present study was conducted in isolation and identification of ORT from slaughtered turkeys. METHODS: Cleft palate swabs of 200 were collected from slaughtered turkeys and cultured on blood agar. ORT was characterized using biochemical tests and PCR targeting the ORT 16S rRNA gene. Virulence genes of isolates were determined targeting adenylate kinase (adk), copA and virulence-associated protein D (vapD) genes. Additionally, diversity of ORT isolates was performed by enterobacterial repetitive intergenic consensus (ERIC) and RAPD PCR. Disk diffusion was used to determine the antibiotic sensitivity of the isolates. RESULTS: ORT was identified in 23 (11.5%) samples using both the biochemical tests and PCR. The result of detecting virulence genes showed that all the isolates (23: 100%) had the adk gene, whereas two (8.7%) isolates had the copA gene, and seven (30.43%) isolates had the vapD gene. Molecular typing of isolates revealed 21 different patterns by RAPD PCR assay using M13 primer and 20 distinct patterns by ERIC PCR test. Both ERIC and RAPD PCR were distinctive methods for investigating the genetic diversity of ORT isolates. The antibiotic resistance test showed that 18 (78.26%) isolates were resistant to gentamicin, amikacin, cefazolin, streptomycin and penicillin. All isolates (100%) were resistant to cloxacillin and fosfomycin. CONCLUSIONS: This study showed the prevalence of ORT in turkey and high resistance of this bacterium to many common veterinary antibiotics. Moreover, both ERIC and RAPD PCR are distinctive methods for investigating the genetic diversity of ORT isolates. These data may help monitor antibiotic resistance and typing of ORT in epidemiological studies and serve as the foundation for designing region-specific vaccines for future use.

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.

Hematologic values in domestic pigeons naturally infected with Mycobacterium avium subsp. avium.

Avian mycobacteriosis is an important disease of birds and is most often caused by Mycobacterium avium or Mycobacterium genavense. However, little information on the hematologic changes associated with this infectious disease in pigeons has been published. The aim of this investigation was to compare the hematologic parameters of domestic pigeons (Columba livia var. domestica) naturally infected with M avium subsp. avium (MAA) with clinically healthy pigeons. Blood samples were collected from 12 pigeons with suspected mycobacteriosis and 12 clinically healthy pigeons. All the birds with suspect infections were necropsied, and affected organs were cultured and examined on histopathology for mycobacteriosis. Total leukocyte and erythrocyte counts were performed on each blood sample with the Natt and Herrick method using a Neubauer hemocytometer. White blood cell (WBC) differential counts were performed on Giemsa-stained blood smears. Packed cell volumes (PCVs) were measured using the microhematocrit technique. Hemoglobin concentrations were measured with a spectrophotometer using the cyanomethemoglubin method. Mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentrations (MCHCs), and mean cell volumes (MCVs) were calculated manually. All of the infected birds had typical histopathologic findings of avian mycobacteriosis, which were confirmed using microbiologic and molecular methods to detect MAA. The hematologic data from the two groups were compared. The total WBC, heterophil, lymphocyte, and monocyte counts were significantly higher, and the PCV, HGB, MCH, and MCHC values were significantly lower in the infected birds compared with the clinically healthy pigeons.

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.

Effect of experimental infectious bursal disease virus on clinical signs and pathogenesis of avian influenza virus H9N2 in turkey by real time PCR.

Infectious bursal disease virus (IBDV) in turkeys may result in immunosuppression, and inability of turkeys to resist nonpathogenic or less pathogenic organisms. A total number of 120 day-old commercial male turkeys were purchased and blood samples were collected from 20 day-old turkeys, remaining 100 were divided into four equal groups and kept in separated rooms. Groups 1 and 2 were infected with 104 CID50 of IBDV via intra-bursal route on day 1; Groups 1 and 3 were each infected with 106 EID50 of AIV (H9N2) via the oculo-nasal routes on day 30. All groups were vaccinated against Newcastle disease vaccine (NDV). Detection of avian influenza virus H9N2 in trachea and cloaca swabs and in the tissues, was confirmed by Real-time polymerase chain reaction. Anti- NDV-AIV and anti-IBD titers were measured using HI and ELISA tests, respectively. The present study showed that infectious bursal disease changed the pathogenesis of (AIV) H9N2 by affecting AI virus replication and resulted in an increase shedding due to prolonged duration of sever clinical signs. The extent of shedding and virus replication need further study.

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.

Detection and subtyping avian metapneumovirus from turkeys in Iran.

Avian metapneumovirus (aMPV) causes diseases like rhinotracheitis in turkeys, swollen head syndrome in chickens and avian rhinotracheitis in other birds. Causing respiratory problems, aMPV adversely affects production and inflicts immense economic losses and mortalities, especially in turkey flocks. In recent years, several serological and molecular studies have been conducted on this virus, especially in poultry in Asia and Iran. The purpose of the present study was detecting and subtyping aMPV by reverse transcriptase polymerase chain reaction (RT-PCR) from non-vaccinated, commercial turkey flocks in Iran for the first time. Sixty three meat-type unvaccinated turkey flocks from several provinces of Iran were sampled in major turkey abattoirs. Samples were tested by RT-PCR for detecting and subtyping aMPV. The results showed that 26 samples from three flocks (4.10%) were positive for viral RNA and all of the viruses were found to be subtype B of aMPV. As a result, vaccination especially against subtype B of aMPV should be considered in turkey flocks in Iran to control aMPV infections.

Characterization of isolated pigeon paramyxovirus-1 (PMV-1) and its pathogenicity in broiler chickens.

Characterization of isolated pigeon paramyxovirus-1 (PMV-1) and its pathogenicity in broiler chickens were studied. Two hundred and thirty-two samples collected from 50 unvaccinated pigeons lofts suspected to Newcastle disease from private houses and bird markets from Ahvaz, Iran. Swab samples from cloaca and oropharynx of live pigeons and from trachea, lung, liver, spleen, kidney, brain, proventriculus and cecal tonsil of dead pigeons suspected to ND were collected. Isolation of the PPMV-1 was performed through intra-allantoic inoculation of 9- to 11- day-old embryonated chicken eggs. The RNA extraction and cDNA synthesis were conducted. With PCR, multiplication of cleavage site of F gene was carreid out and PCR products were sequenced and phylogenetic comparison on isolates was performed. For pathogenecity study of isolated PPMV-1, one hundred sixty day-old broiler chicks were divided into four equal groups. Groups 1 and 2 chicks vaccinated against ND by B1 vaccine at nine days. Groups 3 and 4 were kept as unvaccinated control groups. Groups 1 and 4 chicks were challenged with 105EID50 of highest virulent isolated PPMV-1 by ocular route at day 29. The results indicated PPMV-1 is enzootic in Ahvaz pigeons and all isolates were virulent Newcastle disease virus with 112KRQKR*F117 motif. For study pathogenicity of pigeon isolate in chickens, they challenged with most virulent isolate, showed respiratory signs, conjunctivitis and in some cases depression and lethargy. In conclusion, isolated PPMV-1 is a virulent NDV and can infect chickens and produce mild ND in unvaccinated chickens.

The Effects of Isolated Fractions of Mesobuthus eupeus Scorpion Venom on Humoral Immune Response.

BACKGROUND: Many elements such as immunosuppressive, chemotactic and anti-inflammatory peptide that could effect on human and animals physiologic system were determined in venom. This study evaluated the use of Mesobuthus eupeus scorpion venom fractions as an immunomodulator. METHODS: The venom fractions collected from Khuzestan Province in South West of Iran were purified by ion exchange chromatography. Elution of the bounded elements was done by using a linear gradient of sodium chloride (0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5 and 2 molar). The fractions were analyzed by Bradford spectrophotometric and SDS-PAGE method. After treatments of chicken with venom fractions and sheep red blood cell (SRBC), direct haemagglutination test in microtiter plate was used for the determination of the chicken SRBC antibody titer. RESULTS: The fraction released by NaCl 1.25M had the highest protein concentration. The highest and lowest antibody titer was determined at the fifth (NaCl 0.75 molar) and seventh fraction (NaCl 1.25 molar), respectively. CONCLUSION: Different protein profile of isolated fractions, were associated with various effect on immune response. Both enhancing and suppressing of the chicken humoral immune response to SRBC were observed after M. eupeus faction's venom treatment. It is due to biological functions of venom components. Purification of these elements would provide the new agents for immune responses manipulation.

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.

Isolation, identification and antimicrobial sensitivity of Ornithobacterium rhinotracheale in broilers chicken flocks of Khuzestan, Iran.

Ornithobacterium rhinotracheale (ORT) is a bacterium associated with respiratory disease, growth retardation, decreased egg production and mortality in chickens and turkeys. The objective of this study was isolation, identification and evaluation of antimicrobial susceptibility of ORT bacterium in slaughtered broilers chicken flocks based on cultural and molecular tests in Khuzestan province, south-west of Iran. A total of 210 tracheal swab samples were collected from 21 broiler flocks slaughtered in abattoirs of the province. The results of cultural and biochemical tests showed that 23 (10.95%) isolates from tracheal swabs of 4 flocks (19.04%) were identified as ORT, but according to molecular characterization, 18 (8.57%) ORT isolates were positive in PCR assay and produced the predicted 784 bp amplification product. Finally, using the disk diffusion method, the drug resistance patterns of ORT isolates were determined against a panel of commonly used antimicrobial agents. Antimicrobial susceptibility test revealed that all isolates (100%) were sensitive to tetracycline, florfenicol and cephalexin. The highest antimicrobial resistance (89.00%) was seen for fosfomycin, sultrim and gentamicin. The results of present research showed that there was significant difference between the isolation rates of ORT from various areas of the province. As well, our findings indicated that the simultaneous use of both cultural and molecular techniques results in more comprehensive outcomes in the isolation and identification of the organismfrom understudy hosts.

Drug susceptibility testing of Mycobacterium Avium subsp. Avium isolates from naturally infected domestic pigeons to avian tuberculosis.

OBJECTIVE: Avian tuberculosis is one of the most important infections affecting most species of birds. Several mycobacterial species have been identified causing avian tuberculosis, and the organisms confirmed most frequently are Mycobacterium avium and Mycobacterium genavense. Any species of birds can be infected with M. avium. Generally, domesticated fowl or captive wild birds are affected more frequently than those living in the wild. M. avium can not only infect all species of birds, but can also infect some domesticated mammals to cause disease, usually with localized lesion. In immunocompetent individuals, M. avium complex isolates produce localized soft tissue infections, including chronic pulmonary infections in the elderly and cervical lymphadenitis in children, but rarely any disseminated disease. In patients infected with HIV and AIDS or in other immunocompromised individuals, M. avium complex isolates frequently cause severe systemic infections. The importance of avian tuberculosis and the risk of its zoonotic spread motivated our interest to determine the drug susceptibility testing of M. avium subsp. avium isolates from naturally infected domestic pigeons to avian tuberculosis. METHODS: Based on their clinical signs, 80 pigeons suspected with avian tuberculosis were subjected to the study. Out of the 51 identified isolates, 20 M. avium subsp. avium were subjected to the test. Drug susceptibly testing was performed according to the guidelines by Centers for Disease Control and Prevention and using proportional method. RESULTS: In the drug susceptibility testing, all isolates were resistant to streptomycin, kanamycin, ethionamide, and thiophene carboxylic acid hydrazide. Additionally, 3, 2, and 1 isolates were susceptible to isoniazid, rifampin, and ethambutol, respectively. To date, no study has documented the drug susceptibility testing of M. avium isolates from infected birds to avian tuberculosis. Pigeons are extensively kept in urban and rural areas for homing and racing purposes; thus, they can infect people and farm animals exposed to their droppings containing pathogenic M. avium, and the severity of drug resistance of these isolates indicate lethality in immunocompromised individuals and incurable lymphadenitis in immunocompetent individuals. CONCLUSION: We suggest drug susceptibility testing for more nontuberculous mycobateria, particularly M. avium complex isolated from infected birds and humans, as well as molecular basics of drug sensitivity in order to detect resistance genes of pathogenic M. avium subsp. avium.

Effects of dietary vitamin E on mucosal maltase and alkaline phosphatase enzyme activities and on the amount of mucosal malonyldialdehyde in broiler chickens.

The effects of dietary vitamin E levels on mucosal maltase and alkaline phosphatase (ALP) enzyme activities and on the amount of mucosal malonyldialdehyde (MDA) in broiler chickens were studied in the present study. One hundred and eighty of male day old broiler chicks (Ross 308 strain) were randomly assigned into five groups, each with three replicates and 12 chicks in each replicate. Chickens in group A were fed corn-soy- based diet, while those in groups B, C, D and E were fed the same diet with 20, 60, 180, and 540 mg kg(-1) vitamin E supplement (d-alpha tocopherol), respectively. Six birds were randomly chosen from each group, and were euthanized on days 10, 21, 32, and 42 of age. One segment of small intestine outset was homogenized and mucosal ALP and maltase activity were measured. Moreover, mucosal lipid peroxidate amount was measured to reveal the impact of vitamin E on oxidative stress. Maltase activity was increased with the increase of vitamin E up to 60 mg kg(-1) of diet while with further levels, it was decreased. Addition of 60 mg kg(-1) of vitamin E to the diet significantly increased ALP enzyme activity (p ≤ 0.001). Addition of 540 mg kg(-1) of vitamin E supplement to the diet led to the minimum amount of MDA at 32 days of age. It may be concluded that supplementation of broiler's diet with 60 mg kg(-1) of vitamin E can increase mucosal maltase and ALP enzyme activity.

Comparison of four different culture media for growth of Mycobacterium avium subsp. avium isolated from naturally infected lofts of domestic pigeons.

BACKGROUND AND OBJECTIVES: Diagnosis of avian tuberculosis by conventional culture method is still considered as the "gold standard" technique. The main objective of this study was to compare growth of Mycobacterium avium subsp. avium on four specific Mycobacterial cultures such as glycerinated Lowenstein-Jensen medium, pyruvate-enriched Lowenstein-Jensen medium, mycobactin J-supplemented Herrold-egg yolk medium and plain Herrold-egg yolk medium. MATERIALS AND METHODS: Eighty out of more than 600 pigeons were selected based on their clinical signs and poor health conditions. The birds were numbered and their clinical signs were registered in the working sheets, and under standard condi-tion, euthanized, subjecting to necropsy examinations, followed by bacterial culture on four specific media for Mycobacterium avium subsp. avium, including glycerinated Lowenstein-Jensen (LJG) medium, pyruvate-enriched Lowenstein-Jensen medium (LJP), mycobactin J-supplemented Herrold-egg yolk medium and plain Herrold-egg yolk medium. RESULTS: Fifty one Mycobacterium avium subsp. avium were isolated from pigeons. Mycobactin J-supplemented Herrold-egg yolk media yielded greater number of colonies in shorter incubation time in compare with other media. CONCLUSION: It was concluded that most of the isolates need mycobactin as a growth factor.