Development of National Diagnostic Standard Operating Procedures (SOPs) for Isolation, Identification and Typing of Brucella Isolates

Abstract

Brucellosis is primarily a disease of animals which is transmitted to man either directly or indirectly, and it continues to be a zoonosis of worldwide public health and economic importance. It is caused by 10 known species of the genus Brucella. The causative bacterium was named in honor of Sir David Bruce the discoverer of Brucella melitensis. This research done at Department of Brucella in Central Veterinary Research Laboratories (CVRL). The main objective of this research was to develop a national Standard Operating Procedures (SOPs) for isolation and characterization in the Brucella Laboratory. SOPs are sets of step-by-step instructions compiled by the laboratory scientists to help workers carry out routine operations. SOPs aim to achieve efficiency, quality output and uniformity of performance and regulations which should be in alignment with the international standards. This research focused on studying the methods of diagnosis of Brucella in the laboratory by isolation, classification and characterization using different culture media. Typing of Brucella isolates was carried out by both classical biochemical identification and advanced Polymerase Chain Reaction (PCR) test. In this study two isolates were used successfully isolated from suspected samples which were knee joint hygromal fluid from a bovine and blood sample of a febrile patient who did not respond to antibiotic or anti malarial therapy. In colony counts, cultured the isolates in the enriched liquid media, which were Brain-Heart Infusionbroth, potato infusion broth, nutrient and Laurel Treptose under anaerobic incubation condition, the minimal number of the bacterial colonies was calculated as one colony per one ml of media. The ecometric tested Six solid nutrient culture media, which were potato agar, Tryptose, Tryptose soya agar, Thyr Martin agar, Serum dextrose agar and blood agar for growth. The Biochemical methods were used including, anaeropic incubation, production of H2S, growth in media which contain Thionine 2%, SafraninO 5% and Basic fuchsin 2% , testing the colonies with mono specific anti sera A and M (for B. abortus and Brucella melitensis). Finally the growth of the two isolates was tested in the presence of five brucella phages. The two isolates were typed by both conventional methods and PCR were Brucella abortus biovar 6 from the knee hygromal and Brucella melitensis biovar 1 from the human blood sample.. In Ecometric technique the isolates were 100% growth in all nutrient media under anaerobic incubator of 37oC for 3-5 days. In aerobic incubation the growth of Brucella abortus was 55%-85%, the best media was blood agar and Tryptose soya agar followed by Tryptose agar. The growth of Brucella melitensis in agar medium at 37oC for 3-5 days was lower and to varying degrees from 25%-30%. . In colonies count, the best medium for growth of Brucella abortus was potato broth followed by Brain heart infusion, the best medium for growth of Brucella melitensis was Brain heart infusion followed by followed by potato broth The results showed that all media supported the propagation of the brucella cells . In biochemical tests the anaeropic incubation helped the rapid growth of two Brucella isolates sulfur dioxide was found to be produced from B.abortus and not from Brucella melitensis. In dyes showed that Brucella abortus did not grew in the solid media containing the three dyes where as brucella melitensis grew in all media containing the three dyes. In mono specific anti sera A and M gave the classical picture of the two isolates. B. abortus positive result with mono specificanti sera(A) and negative result with anti-sera (M), B. melitensis was positive result with mono specific (M) anti sera and negative result with (A) anti sera. In bacterio phages lysed the colonies of the B. abortus isolate except the phage rough canis which gave negative result.All five phages fail to lyse the Brucella melitensis colonies. In DNAs extracted from the isolates were amplified using a species specific AMOS cocktail. each isolate was amplified alone, the use of the multiplex primer PCR as a rapid and differentiating screening test was illustrated. When PCR technique was used using AMOS primers (Abortus, Melitensis, Ovis, Suis), the result of isolated samples was Brucella abortus from animal isolate and brucella melitensis from human isolate. On conclusion the type of solid in ecometric test and broth media support and improved the best growth of two isolates proved to be of value in isolation and identification and typing of brucella species.