A total of 200 cattle were divided into five groups including: Group A Sahiwal cattle, Group B Jersy cattle, Group C Frisian cattle, group D Sahiwal cross Jersy and group E Sahiwal cross Frisian. Out of total of 200 serum samples from suspected cattle we found 155 samples positive by RBPT and 109 were positive for Brucella abortus by PCR. Comparison of presence of Brucella abortus was statically made in all five groups using chi square.
The study was conducted on 200 animals of five breeds including Sahiwal, Jersey Cross Sahiwal, Frisian Cross Sahiwal, Fresian and Jersey around farms of Punjab. Blood sample (3mL) was collected in EDTA vaccutainers 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 Test (RBPT). RBPT positive samples were stored at 40C for further processing. 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. PCR for amplification was done with a total volume of 20 μL by adding primer pair and extracted DNA to the PCR master mix in following concentrations.
2ml of forward and reverse primer was taken respectively. 4ul of PCR grade water was added and DNA was taken in 2 ul quantity. The total volume of master mix obtained was 10 ul. The thermocycler (Bio-Rad, CFX ™, Singapore) was set with the conditions for the total of 35 cycles of PCR. For optimization process of primers different options for PCR reaction mixture
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and PCR cyclic conditions were tried for two objectives. To get maximum amplification, by using minimum volume of chemicals. Changing the volume of magnesium chloride, deoxynucleotide triphosphate (d NTPs) and Taq polymerase, amplification can be increased. Primers annealing temperature is considered critical for optimization.
Denaturation of DNA samples were performed at 94 0C for 5minutes. Annealing step was performed at 63 0C for 30 second. Then, initial extension of DNA samples was conducted at 72 0C for 30 second. Finally, extension was performed at 72 0C for 5 minutes to get results. The PCR product was confirmed by running the PCR product on 0.8 % agarose gel electrophoresis was performed at 100 Volts for 30 minutes. The NRAMP1 gene encodes a divalent cation transporter, located in the phagolysosomal membrane of macrophages, which has been associated with resistance to intracellular pathogens. In cattle, natural resistance against brucellosis has been associated with polymorphisms at the 3′ untranslated region (3′UTR) of the NRAMP1 gene, which are detectable by single-strand conformational analysis (SSCA).
Genetic selection of domestic animals resistant to pathogens has been applied mostly to farm
animals, particularly cattle. Identification of genes linked to natural resistance may allow for a
better understanding of natural resistance with obvious practical implications. These genes may
also function as markers for prediction of genetic resistance against specific diseases.
Recommendations:
From this study we concluded that Nramp1BB gene is resistant to brucellosis, while Nramp1 AA is susceptible to brucellosis.
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 By gene knock out technique breeds resistant to brucellosis can be produced.
 Criss Crasper technique can be used for gene knock out process.