PCR BASED IDENTIFICATION OF BRUCELLA MELITENSIS FROM BLOOD AND MILK SAMPLES OF GOAT AND SHEEP SURVEYED AT NORTH GONDAR, ETHIOPIA M.Sc. THESIS

Abstract

Brucellosis is contagious and communicable zoonotic disease caused by bacteria of genus Brucella. Within the genus, the species Brucella melitensis is the pathogenic and causative agent of sheep and goat. The aim of this study was to identification of Brucella melitensis from blood and milk of goat and sheep by using PCR. A cross sectional prevalence survey was conducted from Maksegnit (tsion), Dinzaz, and Enfiranze kebele in Gondar Ethiopia. These study areas were selected purposively following community based breed improvement program in the study area. A total of 125 blood and milk samples (Goat, n=62 and sheep, n=63) were collected from middle of March to the beginning of September 2016. The DNA from each sample was isolated and quality of DNA was determined by OD 260nm/ 280nm ratio by using spectrophotometer and agarose gel electrophoresis. PCR was performed by targeting OMP-31(720bp) gene which is immunogenic gene in B. melitensis. PCR products were analalyzed using 1.5% agarose. 16.66% of blood and 12.31% of milk samples taken from goat and sheep were found positive in PCR. Out of total 125 samples 62 and 63 samples belongs to goat and sheep respectively. Out of 62 samples collected from goat 30 and 32 samples were blood and milk respectively. The prevalence of B. melitensis infection in goat blood and milk was found 23.3% and 9.4% respectively. Out of 63 samples collected from sheep 30 and 33 samples were blood and milk respectively. The prevalence of B. melitensis in sheep blood and milk was found 10 % and 15.15 % respectively. The overall prevalence of B. melitensis infection in goat and sheep in the study areas was found 14.4 %. The sensitivity and specificity of PCR targeting OMP-31 gene in blood and milk samples of goat were found (100%); (66.6%) and 100%, (100% respectively and in blood and milk of sheep were found (100%), (60%) and (100%), (100% respectively. The prevalence of B. melitensis infection in Dinzaz, Maksegnit and Enfiranze study areas were found 17.02 %, 11.63 % and 14.4 % respectively. The prevalence of B. melitensis at flock level in small (1-5), medium (5-15) and in large (>15) study areas were found 26.3 %, 8.5 % and 0.8 % respectively. By using multivariable logistic regression fit for detection of PCR putative risk factors for small ruminant B. melitensis in the study area. Age of small ruminant (OR=4.792, CI95%:1.349-17.021) and origin of animal (OR=5.43, CI95%:1.6890-17.454) were significant risk associated B. melitensis infection of small ruminant p=0.015 and 0.005 were recorded respectively. The prevalence of B. melitenesis from the study area in Dinzaz getting higher chance of the other study area. In this study the prevalence of B. melitensis infection in blood samples was found higher than the milk samples. The prevalence of B. melitensis infection was found higher in goat than the sheep. In the individual owner of small ruminant were interviewed there was lack of awareness and poor management of reproductive health problem which contributed the expansion of B. melitensis infection in the improvement system. This research in an important milestone in Ethiopia to diagnose caprine and ovine brucellosis at molecular level. It is strongly believed that accurate diagnosis will lead to prevention and control of this disease by good management of animals.