Nearly 32.7 million population of buffaloes present in Pakistan which makes about 15% of the world buffalo population. For water buffaloes Brucellosis is a pricey disease (Bubalus bubalis). In Pakistan 5 known breeds of buffalos are; Ravi, Nili, Kundi Nili-Ravi and Aza Kheli. The study was conducted on 200 animals of two breeds including Nilli-Ravi and Kundi around farms of Punjab. 200 serum samples from two breeds were screened for brucellosis. Dormant infections and lengthy incubation of the pathogens bounded the effectiveness of programs based upon the elimination of infected animals. As comparative genomics is playing a pivotal role in the genetic dissection of complex traits such as infectious diseases resistance using this information we can oppressed for disease resistance with genetic choice as a method to the regulator of brucellosis in our local breeds such as Nili-Ravi buffalo. Blood sample (5mL) was collected in EDTA vacutainers from each animal. Serum from blood samples were collected in 3 mL eppendorf tubes with the help of sterile pipettes. Serum samples were screened out for brucellosis by Rose Bengal Plate Tetst (RBPT). Rose Bengal Antigen was purchased from Veterinary Research Institute (VRI), Lahore. The antigen was stored at 40C in refrigeration during the study according to manufactures recommendation. DNA quantification was performed on “Thermo Scientific NanoDrop spectrophotometer ND-2000” and by running extracted DNA on 0.8% agarose gel. Nanodrop spectrophotometer provides direct and easy measurements within a 5 second only by using just a pipette and wipe. PCR for amplification was done with a total volume of 20μl by adding primer pair and extracted DNA to the PCR master mix in following concentrations. 2μl of forward and reverse primer was taken respectively. 4 μl of PCR grade water was added and DNA was taken in 2 μl
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Quantity. The total volume of master mix obtained was 10 μl. The thermocycler (Bio-Rad, CFX ™, Singapore) was set with the conditions for the total of 35 cycles of PCR. Initial denaturation of DNA samples were performed at 940 C for 5minutes. Then in 2nd cycle denaturation was performed at 94 0C for 30 seconds. Annealing step was performed at 63 0C for 30 second. Then, initial extension of DNA samples was conducted at 720 C for 30 second. Finally, extension was performed at 720 C for 5 minutes to get results. The PCR product was confirmed by running the PCR product on 1.5 % Agarose gel electrophoresis was performed at 110 Volts for 30 minutes. In Nilli-Ravi, among 12 positive samples by PCR, 08 were found resistance to brucellosis through sequencing study of Nramp 1 BB, only one sample was found to be susceptible to brucellosis through genotypic study of Nramp1 AA and 3 samples were found to be neutral may be susceptible or resistance to brucellosis. In Kundi, among 38 positive samples by PCR, 03 were found resistance to brucellosis through sequencing study of Nramp 1 BB, and 6 sample were found to be susceptible to brucellosis through genotypic study of Nramp1 AA and 29 samples were found to be neutral may be susceptible or resistance to brucellosis. This research clearly demonstrates that Kundi is more susceptible to brucellosis then Nilli-Ravi. Conclusion: We concluded that Nilli-Ravi is more resistant to brucellosis as compare to Kundi breed Natural resistance against brucellosis in cattle is linked to the Nramp 1 gene. Polymorphisms at the bovine Nramp 1 3/ untranslated region (3 UTR) are associated with natural resistance against brucellosis.