Detection of cytolethal distending toxin [cdt] Genes in Campylo-bacterjejuni and Campylobacter coli isolated from the intestinal of commercial broiler chickens, turkey and quail of Iran

Campylobacter jejuni and Campylobacter coli are zoonotic bacteria which are frequently associated with human diarrhea. Sharing of the cytolethal distending toxin [cdt] genes in Campylobacter is common and is considered species specific. In this study we focused on detecting the presence of cdt gene in C. jejuni and C. coli isolated from broilers, turkeys and quails of Iran. Cecal samples were randomly collected from 240 broiler chickens, 100 meat type turkeys and 100 quails after slaughtering. We used PCR as a method for detecting cdt genes. In broilers, 93% of 58 C jejuni positive samples possessed cdt gene and in all cases the three different subunits of cdt genes were present. However, only 56% of 14 C. coli isolates in broilers had contained cdt genes, while one fourth having all three subunits present. In turkeys, around 65% of 34 C. jejuni positive samples had cdt gene present with 38% possessing all three subunits of cdt genes. But all 5 C. coli isolates had all three subunits cdt gene. In quails, 67% of 30 C. jejuni positive samples were identified by cdt gene, 20% of those possessed all three gene subunits. On the other hand, all 28 C. coli isolates of quails had cdt gene present while 36% of those held all three gene subunits. Our data is indicating the isolation, culture and cdt PCR amplification approaches in this study seemed to be efficient. However, the presence of different variation of Campylobacter cdt gene types in our sample isolates signifies the necessity of further functional gene studies to elucidate which gene type combinations result in encoding effective toxins

[Pathogenicity indices of Newcastle disease viruses isolated from Iranian poultry flocks in Iran]

Newcastle disease [ND] is caused by serotype I of avian paramyxoviruses. The ND virus [NDV] strains are conveniently grouped as velogenic, mesogenic, lentogenic, and nonpathogenic-intestinal pathotypes. The purpose of this study was to determine the pathogenicity indices of the isolated NDVs from poultry flocks in Iran. Samples were provided from poultry flocks in different provinces of Iran and prepared for NDV isolation. From many isolated NDVs, 12 isolates belonged to 10 provinces with highly populated poultry farms which were selected for this study. A clone for each of these virus isolates was generated using limiting dilution procedure. Then, the mean death time [MDT], intracerebral pathogenicity index [ICPD, and intravenous pathogenicity index [IVPI] were determined for each virus clone and compared with those of standard virulent strains such as Hertz 33.56 and Texas GB. The results showed that the pathogenicity indices of the NDVs in the present study ranged from 41.6 - 60 hr for MDT, 1.76 - 1.91 for ICPI, and 2.68- 2.88 for IVpI indicate which the velogenic type of our viruses. The findings of this study suggested that the very virulent NDVs currently circulating in Iranian poultry flocks are close to and even more virulent than standard virulent NDVs. Isolation, identification, pathotype determination, and molecular characterization of Iranian NDVs may help authorities to make right decisions to reduce the risks posing the Iranian poultry industry

[Molecular identification of mycoplasma agents in suspected contagious caprine pleuropneumonia]

Contagious Caprine Pleuropneumonia [CCPP] is one of the common infections in the middle east regions. So far, there has not been received any report about isolation and identification of these agents in Iran. The aim of this study is to diagnose and isolate mycoplasma agents in suspected goat flocks. Total of 100 pneumonic lung specimen from 20 CCPPsuspected flocks were collected from abbatoirs close to Kermanshah during 1384-1386 and had been sent to Microbiology Lab. Gross lesions showed hepatization with grey and white lesions [consolidation] and motley appearance with or without fibrin. The minced tissue were inoculated to PPLO broth agar. After multiple passages, typical mycoplasma colony was isolated from 4 flocks [22/2%]. Mycoplasma DNA was also extracted based on phenolchloroform method and subjected to generic PCR with specific primers. In addition to the perivious positive samples from tissue culture, 5other flocks also showed contamination with Mycoplasma organisms in PCR tests[45%]. Then, the samples were determined for Mycoplasma mycoides cluster infection, M. capricolum capripneumonia and M. mycoides mycoides [L.C], using M. agalactia as negative control, with specific primers in PCR, there has showed no contamination to these strains. However, to declare "free status " from CCPP in goat flocks requires more developed researches and much more samples in further investigation

[Diagnosis ofornithobacterium rhinotracheale using polymerase chain reaction [PCR]]

Ornithobacterium rhinotracheale [ORT] has been identified as one of the emerging respiratory bacterial pathogens in turkey and chicken flocks. Diagnosis of ORT infection has been faced some difficulties in isolation and identification of the bacterium based on the biochemical properties. A reliable identification method that can be used in routine laboratory investigations is of importance. The Purpose of this study was diagnosis of ORT using polymerase chain reaction [PCR] in comparison with culture. The PCR was optimized using the primer combination OR16S-F1 and OR16S-R1, positive control of ORT bacteria, and 7 other bacteria such as Haemophilus paragallinarum and Pasteurella multocida as negative control. All samples were prepared for DNA extraction by use of phenol- chloroform method. Sixty pooled tracheal and infraorbital sinus samples from 30 broiler flocks slaughtered at an abattoir in Gazvin province were examined for presence of ORTusing culture and PCR assay. Afragment of 784-bp was amplified in 5 positive known ORTsamples, but not in other 7 bacteria as negative controls. 19 out of 60 primary samples including 11 homogenized tracheal samples and 8 infraorbital sinus swabs, and also all 17 suspected and purified colonies identified microbiologically as ORTwere positive in PCR assay. One out of 41 negative primary samples in PCR test became positive through the culture. Then the propagated bacterium was confirmed in PCR. Fifteen out of 30 flocks [50%] were positive in PCR test and only one of them was negative in culture. Upon the results the PCR method of this study can be used as a reliable and rapid identification of ORT in samples suspected to ORT infection. However, it is better to use a combination of the PCR and culture in order to maximize the diagnosis of ORT infections

Pathogenicity and haemagglutinin gene sequence analysis of Iranian avian influenza H9N2 viruses isolated during [1998-2001]

Sixteen avian influenza [AI] H9N2 viruses were isolated from disease outbreaks in different parts of Iran during [1998-2001]. These AI isolates were used for pathogenicity, haemagglutinin [HA] gene variation and phylogenetic analysis. Results in both pathogenicity tests and HA gene cleavage site sequence detection represented a non-highly pathogenic feature for all Iranian AI isolates studied. The cleavage site motif [R-S-S-R] of all AI isolates however, indicated that they had capability of becoming highly pathogenic viruses following 2 nucleotide substitutions at this region. Based on 450 nucleotides region obtained for local isolates and those for referenced viruses available in Gene Bank database used in phylogenetic analysis, all viruses placed on 3 distinct groups, 2 for Iranian and 1 for reference viruses. Among the reference AI viruses, isolates from Pakistan, Saudi Arabia and 1 from Germany showed less differences with Iranian AI isolates. Results also revealed that the circulating viruses in neighbouring provinces have been remained with less mutation for about 2 years

Isolation and detection of Mycoplasma gallisepticum by polymerase chain reaction and restriction fragment length polymorphism

Mycoplasmas were isolated from tracheal and air sac samples of the suspected flocks to have mycoplasmosis and cultured on Frey's medium. Forward and reverse primers were selected on the basis of known sequences of Mycoplasma gallisepticum [MG] and Mycoplasma synoviae [MS] 16S rRNA genes. These primers successfully amplified a 780 bp fragment of the target DNA in MG. Restriction fragment length polymorphism [RFLP] by three restriction enzymes [REs], Hpal, Hpa II, and Mbol was performed for each PCR product. According to the RFLP results, 55 samples were detected as MG; no MS was detected. The PCR results were confirmed by sequencing of a selected amplicons. Results showed that PCR and RFLP are rapid and useful for diagnosis of both cultured as well as field samples of suspected flocks to have infection with MG. In addition, PCR could be performed with at least 100 CFU of MG per each PCR reaction