Isolation and characterization of streptococcus bovis from rumencontent of awassi sheep in iraq

This is the first study in Iraq aimed to isolate and characterize Streptococcus bovis from rumen of Awassi sheep . Ten sheep with different ages fed on grain base diet for three days were used to collect 20 ruminal fluid samples twice at fourth and fifth days by rumenocentesis method , samples cultured on selective media (Modified membrane-bovis agar(M-BA) ,broth of basal medium and modified blood brain heart infusion) ,the isolates were identified according to their morphological, physiological ,biochemical tests and serological by Lancefield group.Cultural characteristic on the selective medium M-BA showed two types of streptococci :first type (23) comprised the majority of isolates ,this type characterized by high acid producing streptococci formed mucoid ,creamy ,orange –centered colonies and second type (4) characterize by low acid producing formed small white colonies . Morphologically the isolates were identified as gram positive ,the cells were oval or spherical , singles, pairs & short chains of 4 to 8 cells. The organisms were found to full under the Lancefield group D.All isolated bacteria grew on broth of basal medium post incubation at 45C ̊ ,but not grew at 10C ̊ and 50C ̊. Adding of 2% NaCl permit the growth, while in 6.5% NaCl didn’t grow. And did not grew on adding 0.04 % Potassium tellurite . All isolates produced lactic acid but ammonia production from arginine was negative, no hemolysis on blood agar . All isolates ferment starch, lactose, fructose, glucose, raffinose and cellobiose.The results of culturing and Physiological and biochemical tests showed that 27 isolates has the characterictices of Streptococcus bovis in 3 types (S1,S2 &S3) ,first type of colony divided into two strain (S115)(S28)according to difference In arabinose. And (S3) called on the second type white pigmented isolates which did not ferment inulin Introduction Streptococcus bovis was recognized as an important ruminal bacterium in terms of carbohydrate fermentation in early 1950s (1) but was not classified as an active proteolytic bacterium until 30 years later. In different animals the population of this bacterium remains lower under normal feeding ( forage diet ) ,but its numbers can increases significantly following dietary change when large of starch or sugars were added to the diet ( 2,3,4). Streptococcus bovis is a grampositive bacteria, it was a ruminal bacterium that can produce large amounts of lactate and its involved in the onset of ruminal acidosis ( 1,5 ) . During period of rapid growth , lactic acid production by S.bovis can cause decline in rumen pH to the point where growth rates of other rumen bacteria are inhibited ( 6,7,8). Kamra ( 9) showed that the anaerobic conditions in the rumen are maintained by gases generated during fermentation e.g. Carbone dioxide , methane and traces hydrogen ,and some of the oxygen with feed consumed by the animal is utilized by the facultative anaerobes present in the rumen and thus a perfect anaerobic condition is generated and maintained.Hungate (2) was developed technique “roll tube or hungate method“for cultivation of strict anaerobes in rumen, and this technique is very convenient for isolation of S. bovis.This study aimed to determine the presence of S. bovis in rumen of sheep fed with grain base diet and to characterize the isolates by different bacteriological, biochemical and serological methods. AL-Qadisiya Journal of Vet.Med.Sci. Vol./12 No./1 2013 ___________________________________________________________________________ 45 Material and methods 1Animals:Ten Awassi sheep in different ages were run as a single flock on alfa alfa and green berseem grass pasture ,sheep were housed indoors in individual pens and fed on high grain-based diet with access to fresh water at all times for three days were used for isolation of Streptococcus bovis at fourth (10 samples ) and fifth days(10 samples) from ruminal fluids of these animals. 2Local modified anaerobic apparatus A local anaerobic apparatus was designed by As'ad jassim and Afaf Abdulrahman similar to hungate apparatus(2) with some modification and used for purification of commercial carbon dioxide gas by passage on heated copper column to forming copper oxide (Picture No1). The apparatus consist from:1-CO2 tube 5Kg (Aeroticiv ,Italy)2-CO2 regulator3-Rubber conduit hose4-the pyrex glass column consist of an a-inner glass (65 x1.5cm) inserted with heater are used to attach the electrical supply b-outer glass(75x 3cm) was provided by glass stopper was contain two opens for enter CO2gas and electrical wire to apparatus.5-Copper wire 5 meter is wrapped around an inner glass6Holder 7-filter 8electeric supply 9-Conduit hose was divived into two leads(10,11) provided with needles gauge 20 at its end for flushing the flask and roll tubes at same time with O2 free CO2. Picture No. (1) local anaerobe apparatus 3-Solution and media:a-Reducing solution prepared a freshly:each 1 ml containing 0.026 g cysteine – HCl and 0.026 g Na2S.9H2o after boiling then pre –reduced by bubbling with CO2 gas on ice until cold . t This solution was used for preparation of media b-Carbonate solution: (8%Na2CO3 solution) , used for preparation of media . c-Resazurin solution:-0.1g resazurin in 100