Bovine Tuberculosis (bovine TB) is a chronic disease of animals and has been known for the significant zoonotic impact. Immune mechanisms necessary for protection against Bovine TB are poorly understood. Interferon-γ cytokine has been reported critically and it is important to study its role in immunity against Bovine TB.
Blood samples were collected from 100 Animals from Peri-urban areas of Lahore, Gujranwala and Okara, Pakistan. Genomic DNA was extracted from the samples. Specific primers were designed to amplify specific portion of IFN-γ gene. Amplified products were sequenced and analyzed by bioinformatics tools. Interferon-γ assay was performed from blood collected in heparin coated vacutainers for the quantification of interferon-γ cytokine in different groups of animals. Blood samples from mycobacterium infected symptomatic and symptomatic animals were processed in Haematology analyzer for complete blood count.
Genetic sequencing of bovine Interferon gamma gene (IFN- γ) help in finding out the Genetic Variations to characterize its role in resistance against Mycobacterium bovis infection. This study help in finding out the confirmed markers for natural resistance against bovine TB that can be used in future selection and breeding programmes. The comparison of hematological values and Interferon gamma level of different groups of animals help us for the detailed diagnosis and prognosis of the disease.
The collected data from hematological analysis of Mycobacterium infected symptomatic animals (Group A), Mycobacterium infected asymptomatic animals (Group B) and non-infected animals/control Group (Group C) was analyzed using the statistical technique of comparing more than two groups i.e. Analysis of variance (ANOVA), One way ANOVA through SPSS 16.0.
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Mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration were found non-significant (p>0.05). White blood cells, Lymphocytes, Platelets, Mean platelet volume and Mean corpuscular volume were found significant (p<0.05). Granulocytes, Red blood cells and Red cell distribution width values were found highly significant.
Interferon gamma assay provided confirmation about the presence of disease in the animals by indicating interferon gamma level to insight the undergoing pathogenesis which was helpful in the detailed diagnosis of the disease. Later on it helped us in the confirmation of false positive results by Tuberculin test.
Final results revealed four intronic variations in different groups of animals. Three of them were found in Group A and B and one was found in Group C (non-infected animals) by Primer 1 (P1). Intronic variations don’t have significant effect but they may have an impact on the regulation of the gene.
We found Transversions (T > A), (A > T), (T > G) were found in mycobacterium infected symptomatic group of animals (Table: 4.7). Transversion (C > G) at and deletion (G >_) was found in this group and exclusive presence of these SNP’s in this group can be considered significant and responsible for the infection. Transversion (A > C) and addition (_ > G) were found in mycobacterium infected asymptomatic group of animals. These two SNP’s are significant as they have been found only in this group. We can infer that the presence of these two SNP's is responsible for the infection along with making them asymptomatic towards the disease.
It was noted that Transition (G > A), (T > C) has been found common in mycobacterium infected symptomatic and asymptomatic group of animals. This common mutation at same position in both groups is quite significant and could be attributed to the occurrence of disease.
Summary