Detection of Mycoplasma gallisepticum by 16S rRNA PCR with specific primers in clinical samples

Mycoplasma gallisepticum (MG) as one the major pathogens of birds, causes significant economic losses in poultry industry. The main purpose of the present study was to detect Mycoplasma gallisepticum in clinical samples using the 16S rRNA PCR method. For serological screaming test, 18 commercial laying farms and 8 broiler breeder farms were selected and rapid serum agglutination test (RSAT) was performed. For polymerase chain reaction sampling, 10 of the 17 farms that were positive in RSAT were selected and 109 sterile swab samples were collected from the palatine cleft, trachea, air sacs and lungs in each farm. Three swabs from three birds were placed in test tube containing 1 ml of phosphate buffered saline and transferred to laboratory form PCR testing. In this study, specific primers for 16S rRNA gene were used. The aforementioned primers are totally specific for MG and can be differentiated from other Mycoplasmaand bacteria present in the trachea of poultry of the 26 farms examined, 17 farms were positive in RSAT serologic test. The 530 bp PCR product produced by specific primers of all field strains appeared on electrophoresis gel in 46 samples from 10 farms accounting to 42.2%.The 16S rRNA PCR with very high sensitivity can be employed in definitive diagnosis of Mycoplasma gallisepticum infection in vitro

Detection of Mycoplasma synoviae infection in broiler breeder farms of Tehran province using PCR and culture methods

Mycoplasma synoviae (MS) is an important avian pathogen that can cause both respiratory disease and joint inflammation synovitis in poultry, inducing economic losses to the Iranian chicken industry especially breeder farms. The aim of this study was to use the MS specific PCR and culture methods in order to detect of M. synoviae from breeder farms where located in Tehran province. A total of 475 samples including choanal cleft, trachea, ovary and /or joint cavities from 23 broiler breeder farms of Tehran area were collected. Samples were cultured in PPLO broth media supplemented for MS isolation. The bacteria DNAs were extracted by phenol/chloroform method. Specific published primers amplify a 207 bp region of the 16S rRNA gene of MS were used for PCR method. Out of 475 samples, 146 cultures were shown positive and typical Mycoplasma colonies, 85 samples were also identified MS based on agglutination test with specific MS antiserum and the PCR method. A total of 122 samples, a band with 207 bp was shown as MS specific PCR product in electrophoresis. In addition to these 85 samples that were positives in both culture and PCR, 37 samples that had not grown in Mycoplasma media were positive in MS specific PCR. A total of 292 samples were negatives in both culture and PCR methods. 122 positive samples out of 475 samples (25.7%) were belonged to 7 breeder farms (30.4%). On conclusions, the MS infection of broiler breeder farms of Tehran area was confirmed truly. From the results, as the PCR method reduces the time consuming, an effectiveness and efficient for detection of M. synoviae infection of chicken breeder. It is then suggested that the PCR method could be an alternative method for culturing

Detection of Mycoplasma synoviae in clinical samples by VlhA-PCR method

As one of the major pathogens of avian species, Mycoplasma Synoviae causes significant economic losses to the poultry industry. The main purpose of this study was to detect Mycoplasma Synoviae in clinical samples using the VlhA-PCR method. For serological screening test, 373 serum samples were collected from 25 breeder farms and rapid serum agglutination test conducted which revealed that 143 samples equivalent to 19 breeder farms were positive. For VlhA-PCR assay, 20 of the previously mentioned breeder farms were selected and sterile swab were collected from the palatine cleft, trachea, air sacs and lungs. Three swabs from 3 birds were placed in a test tube containing 1 ml of PBS and transferred to the laboratory for PCR test. Specific primers for VIhA gene were employed in this study. The PCR product from specific primers showed 350-400 bp for all field isolated on electrophoresis gel in 8 farms. VlhA-PCR with high sensitivity could be employed in definitive diagnosis of Mycoplasma Synoviae infection in the laboratory

Detection of Mycoplasma capricolum capricolum from goats of Qom province, Iran

Mycoplasma capricolum subsp capricolum (Mcc) is one of the etiological agents of contagious agalactia(C.A) in goats which can cause significant economic losses. The aim of this study was to detect Mcc from goats of Qom province in Iran. A total of 111 samples were collected from suspected goats to C.A and cultured in PPLO broth supplemented for Mcc isolation. The bacteria DNAs were extracted from clinical samples and the PCR assay was performed to detect Mycoplasma genus, cluster Mycoides and Mcc from culture. Out of the 111 samples, 33(29.7%) sample cultures were shown positive and typical Mycoplasma colonies in PPLO agar culture method, 53 (47.7%) samples were scored positive by Mycoplasma genus PCR, 8 (7%) of the samples were scored positive by using myciodes cluster PCR and finally 2(1.8%) of the samples were scored positive by using Mcc PCR method. The result of this study showed that Mcc was detected for the first time from Qom province in Iran. Therefore, Qom could be one of the geographical distribution and efficient factors of Mcc in Iranian goats. The lung samples and lymph nodes also could be significant samples for detection of Mcc

Prevalence and molecular characterization of verotoxin-producing Escherichia coli O157:H7 in cattle and sheep in Shiraz-Iran

Shiga toxin producing Escherichia coli have been associated with HUS, HC and TTP in human. We found recto-anal mucosal sample in sheep as well in cattle is the main site for E. coli O157 localization. 1246 E. coli isolates from 872 both healthy and diarrheic animals were analyzed, by screening for the presence of Shiga toxin-producing (VT 1 and VT 2) and intimin (eae) genes used Multiplex PCR. 87(9.75%) VTEC from 52 cattle and 28(7.90%) from 28 sheep were isolated. VT2 gene was found to be more frequent than VT1 in cattle (54.02% vs 26.43%), in contrast the same genes in sheep (21.42%vs25%). There was observed significant difference in the origin of VT positive sheep in close contact with farms of cattle origin. Having cattle and sheep with each other was a possible risk factor. The animal was kept in pen was more localized than tethered. Young cattle were documented strongly significant high prevalence rate in E. coli O157:H7 than older, but no effect of age was observed on the occurrence of E. coli O157 in sheep. Both diarrheic and healthy animals were shed E. coli O157:H7 in their feces. Sheep and dairy cow were not illustrated any significance differences geographical region and seasonal variation with E. coli O157:H7 prevalence rate

Isolation and identification of Mycoplasma agalactiae by culture and polymerase chain reaction (PCR) from sheep of Qom province, Iran

Contagious agalactia (C.A) is an infectious syndrome of sheep that is characterized by mastitis andsubsequent failure of milk production, arthritis, abortion and keratoconjunctivitis. Mycoplasma agalactiae(M. agalactiae) is the main cause of the disease in sheep. The aim of this study was isolation andidentification of M. agalactiae with culture and polymerase chain reaction (PCR) assay from sheep of Qomprovince in Iran. A total of 102 samples were collected from milk secretion, eye, ear and joint exudates ofsheep. All samples were cultured in PPLO broth supplemented for M. agalaciae isolation. The bacteriaDNAs were extracted by phenol/chloroform method and the PCR assay was applied for detecting ofMycoplasma genus in 163bp fragment of 16S rRNA gene and M. agalactiae in 375bp fragment oflipoprotein gene from culture as same as in clinical samples. Out of the 102 samples, 19(18.63%) cultureswere shown positive and typical Mycoplasma colonies in PPLO agar culture diagnostic method and59(57.8%) were scored positive by Mycoplasma genus PCR, 19(18.62%) of the samples were scoredpositive by using M. agalactiae PCR as diagnostic method. Out of the 102 samples, 19 samples wereshown both positive in the culture and PCR, 42 samples were shown both negative in the culture and PCR.40 samples were negative in the culture and positive in PCR whereas only one sample was positive inculture and negative in PCR. The results showed that the more isolations of M. agalactiae were taken from milk and less in joint samples. M. agalactiae was one of the main factors of contagious agalactia that was detected for the first time from sheep in Qom province