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ThesisItem Open Access STANDARDIZATION OF PCR FOR DETECTION AND SEROGROUPING OF Dichelobacter nodosus(Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2010-08) ANUPAMA, R; SREENIVASULU, D (Major); SRILATHA, Ch; SREEDEVI, BABSTRACT : Traditional methods used for diagnosis of footrot were based on smear examination, isolation and characterization of D. nodosus from clinical samples. This is time consuming and difficult because the D. nodosus organisms are fastidious in nature and require anaerobic conditions in addition to enrichment media. To overcome these problems it is proposed to standardize PCR for detection and serogrouping of D. nodosus from the clinical samples. Dichelobacter nodosus JKS – 05 B cultures obtained from Srinagar was revived and maintained in the laboratory. The cultures were used for standardization of PCR techniques. The colony of D. nodosus JKS – 05 B appeared as flat concentric zones with finely granulated surface texture. Morphology of D. nodosus was found to be Gram negative, large or slightly curved rods with terminal swollen ends. Gelatin gel test showed hydrolysis of gelatin around the wells containing broth heated to 68˚C for 16 minutes, which indicated the presence of thermostable enzyme which is a characteristic feature of virulent strain. The PCR for targeting 16SrRNA gene was standardized for direct detection of D. nodosus. A 30 cycles PCR reaction with annealing temperature of 60˚C (for 5 cycles) and 58˚C (for 25 cycles) with 25 mM MgCl2 were found optimum for amplification of 783 bp product of 16SrRNA gene. Multiplex PCR targeting fimA gene was also standardized for identification of D. nodosus serogroups using the similar cyclic conditions. Analysis of epidemiological data revealed that footrot is a seasonal disease noticed mostly during North-East monsoon period in Andhra Pradesh. The disease occurrence was related to the rainfall in the area. A total of 778 outbreaks of footrot were recorded during the period of 10 years from 1998 to 2008. The maximum number of outbreaks was recorded in Mahaboobnagar district. A total of 15 outbreaks were attended in selected villages of Chittoor and Nellore districts of Andhra Pradesh during the period of November 2009 to July 2010 and collected the materials for isolation and serogrouping of D. nodosus. Of the 371 samples 76 were found positive for the presence of D.nodosus using 16SrRNA gene targeting PCR. When the samples were further subjected to multiplex PCR targeting fimA gene revealed the presence of D.nodosus serogroups A (19), B (24), C (6). Serogroups both A and B (21) were found infecting the sheep. The study revealed that multiple serogroups were found infecting the sheep and causing footrot in Chittoor and Nellore districts of Andhra