Isolation and identification of Mycoplasma agalactiae by culture and Polymerase Chain Reaction in Sheep and Goat Milk Samples in Kordestan province, Iran

Contagious agalactiae (C.A.) is one of the most common disease affecting small ruminants which is caused by Mycoplasma agalactiae. This disease is particularly widespread around the world and Iran is one of the countries that C.A. is present. The aim of this study was isolation and identification of M. agalactiae (MG) with culture and PCR technique in milk samples in Kordestan province, Iran. A total of 367 milk samples were collected from sheep and goat. Specific published primers amplify a 375 bp gene of MG were used for PCR. Twenty (5.5%) out of 367 were positive in PPLO agar and 5 (25%) out of these isolates were positive with Mycoplasma agalactiae primers. Four (75%) out of 5 isolates was from sheep and 1(25%) from goat. Result of PCR with 367 milk samples showed that 11(3%) of them were positive with these primers. The isolation of M. agalactiae showed that C.A is present in Kordestan province and our results suggested that PCR method because of reduces the time consuming could be an alternative method beside culture

Large scale production of Blackleg vaccine by fermenter and enriched culture medium in Iran

In all biological systems growth is defined as increase of chemical compounds. Bacteria can achieve to balanced growth if they are growing in a medium, which are completely adapted to it. Clostridium chauvoei, (Clostridium feseri) is an anaerobic, spore forming, motile, and polymorph bacteria, which its size varies from 0.5-1 to 3-8 micron and could be observed as individual bacterium, diplo, and rarely streptococcus. Blackleg is a fatal disease of young cattle. It produces an acute local infection, and the resulting blood poisoning leads to rapid death. Clostridium chauvoei and, less frequently, Clostridium septicum are the most commonly responsible organisms. Vaccination is the only effective means for controlling of blackleg disease. Several kinds of vaccine are available commercially. It is 4 decades that blackleg vaccine is produced in Razi institute and because of enhanced demand of country, decision was made to improve the production procedure of this vaccine using large-scale fermenter, so the aim of this study was adaptation of Clostridium chauvoei to growth and proliferation in fermenter for preparation of a potent vaccine. Accordingly attempts were made to prepare and formulate the ingredients in order to obtain high yield of Clostridium chauvoei in culture medium by fermenter. All experiments were done in two sets: A-growth in glass bottles using conventional culture medium and B-growth in fermenter using conventional culture medium similar to A and also enriched culture medium. Results showed high yield of Clostridium chauvoei suspension in fermenter after 10 hours, using enriched culture medium (more than 1,480,000,000 organisms/ml), but no significant changes was obtained in glass bottles conditions comparing to the fermenter conditions. The safety and potency of the prepared vaccine was determined in sheep and guinea pigs according to British pharmacopoeia (veterinary) with satisfactory results. Since this research has been successfully done in Razi research institute, so the mono valent inactivated blackleg vaccine, using the enriched culture medium is currently producing by fermenter and is used for immunization of cattles in Iran

Isolation and identification of Mycoplasma agalactiae by culture and Polymerase Chain Reaction in sheep and goat milk samples in Kordestan province, Iran. Archives of Razi Institute

ABSTRACT Contagious agalactiae (C.A.) is one of the most common disease affecting small ruminants which is caused by Mycoplasma agalactiae. This disease is particularly widespread around the world and Iran is one of the countries that C.A. is present. The aim of this study was isolation and identification of M. agalactiae (MG) with culture and PCR technique in milk samples in Kordestan province, Iran. A total of 367 milk samples were collected from sheep and goat. Specific published primers amplify a 375 bp gene of MG were used for PCR. Twenty (5.5%) out of 367 were positive in PPLO agar and 5 (25%) out of these isolates were positive with Mycoplasma agalactiae primers. Four (75%) out of 5 isolates was from sheep and 1(25%) from goat. Result of PCR with 367 milk samples showed that 11(3%) of them were positive with these primers. The isolation of M. agalactiae showed that C.A is present in Kordestan province and our results suggested that PCR method because of reduces the time consuming could be an alternative method beside culture

Occurrence of Beta2 toxigenic Clostridium perfringens isolates with different toxin types in Iran

Clostridium perfringens is an important cause of enteric diseases in both human and animals. The bacteria produce several toxins which play key roles in the pathogenesis of diseases and are classified into five toxin types, on the basis of the differential production of Alpha, Beta, Epsilon and Iota toxins. In this study a single PCR assay was developed and used for detection of cpb2 gene to identify the Beta2 harboring isolates among different types of C. perfringens isolated from animal enteric diseases in Iran. It was found that cpb2 presents among C. perfringens isolates types A, B, C and D with 54.5% (6/11), 62% (13/21), 42.8% (6/14), 69.25% (9/13), respectively. Totally 34 of 59 (56.7%) isolates screened by PCR were cpb2-positive. This is the first report of cpb2 positive isolates of C. perfringens causing enteric diseases of animals in Iran. Further studies to demonstrate the exact role of Beta2 toxin in pathogenesis of the bacterium is suggested

Fusion of Clostridium perfringens type D and B epsilon and beta toxin genes and it’s cloning in E. coli

Designing and producing a proper fusion construction is the most important problem of producing large quantities of a properly folded functional protein. This construction should have all necessary components of a real gene. A good designed fusion gene construction could be cloned into a good and suitable host. Clostridium perfringens is an important pathogen of humans and livestock and produces numerous toxins including epsilon and beta which are responsible for severe diseases. In the present study a new construction containing Clostridium perfringens type D epsilon toxin gene and type B beta toxin gene was designed. At the first step two pairs of primers were used for these genes amplification. At the next step epsilon forward and beta reveres primers were used to produce a chimeric gene containing amplified partial cds of etxD and partial cds of cpbB which are linked together by the AEAAAKEAAAKA fragment as a small linker. The method was based on fusion PCR and using of Pfu DNA polymerase, which has a proofreading activity. The fusion gene inserted into pJET1.2blunt and cloned into E.coli strain TOP10. Based on the latest information, this is the first design and cloning of epsilon-beta fusion gene and also this is the first time that PCR fusion strategy is used for Clostriadial gene fusion, which could be used for development of a recombinant epsilon-beta fusion protein vaccine. This construction also could serve as a model for development and production of novel fusion protein for other potential proteins and toxins