Hazari Ragini, Kumar Aman, Maan Sushila, Kumar Arvind

Hemorrhagic septicemia (HS) is a major disease of cattle and buffaloes characterized by an acute, highly fatal septicemia with high morbidity and mortality. Sporadic cases are more difficult to diagnose clinically and hence diagnosis at an early stage is required for control of the disease. The present study was conducted to compare the temporal sensitivity of multiplex PCR and bacterial isolation to diagnose hemorrhagic septicemia due to Pasteurella multocida at an early stage i.e. before the appearance of clinical signs in mice. Multiplex-PCR (mPCR)was evaluated for simultaneous as well as temporal detection and identification of P. multocida at type level for early and accurate diagnosis of hemorrhagic septicemia (HS). Swiss albino mice were experimentally infected with 100 colony forming units (CFU) of the bacteria P. Multocida TypeB:2 Strain P52. Heart blood samples were collected, 2, 4, 8, 12 and 24 h post infection for bacterial isolation as well as detection and identification by mPCR. The P. multocida was isolated from the heart blood sample collected as early as 8 h post infection while mPCR detected the bacterial DNA as early as 4 h post infection before the appearance of clinical signs hence mPCR for P. multocida Type B:2 was found to be more sensitive than the bacterial isolation for diagnosis of HS.

Restriction enzyme analysis of VP7 gene of Indian isolates of bluetongue virus.

The genome segment 7 of two Indian isolates of bluetongue virus (BTV) from Avikanagar (BTV-1-western India) and Hyderabad (BTV-Untyped Hyderabad-southern India) was amplified by RT-PCR using two sets of VP7 specific primers. A sequence of 1137 bp comprising the complete coding sequence of the VP7 gene from Avikanagar isolate and a 1154 bp full-length sequence from BTV-UT Hyderabad isolate were amplified. Further, restriction enzyme digestion of these full-length amplicons, using EcoRI, PstI and TaqI revealed that genome segment 7 from both isolates were different from each other by absence of any EcoRI site in the BTV-UT Hyderabad isolate. There were also variations in the number and position of restriction sites for TaqI enzyme in these two isolates. Taql has two sites in the Avikanagar isolate whereas four sites in BTV-UT Hyderabad. The restriction digestion fragments obtained after PstI digestion were differentiated on the basis of their distinct sizes in both isolates. Comparison of their in silico restriction profiles with that of other isolates from different countries revealed that the two Indian isolates belonging to different parts of India had variations in their VP7 gene which was also distinguishable from at least some isolates from Australia and South Africa. Hence the restriction enzyme (RE) based analysis might be a useful tool in determining the genetic diversity in genome segment 7 and also for tracing their evolutionary relationships.