ISOLATION OF BACTERIAL ORGANISMS FROM BILE AND INTESTINAL CONTENT OF APPARENTLY HEALTHY SLAUGHTERED CHICKENS IN JOS AND ENVIRONS BOT C.J 1* ; WOMA T. Y 2 ; ABIAYI E 3 ; MANGUT P. E 1 ; and ISHAYA D 4 1 Federal College of Animal Health and Production Technology, N.V.R.I Vom. 2 Viral Research Department, N.V.R.I Vom. 3 Central Diagnostic Laboratory, N.V.R.I Vom. 4 Taraba State College of Agriculture, Jalingo. * Correspondence: 08035142064. Summary The aim of this study was to isolate and identify microbial organisms associated with bile and intestinal content of slaughtered chickens in Jos and environs. The gall bladder and swab of intestinal contents of fifty chickens presented for slaughter were collected for culture, isolation and identification. Six (6) similar bacteria isolates based on Gram s reaction and biochemical tests were identified from both organs: Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enteroccoci faecalis and Bacillus subtilis. In bile, forty-four (44) organisms were isolated on Blood Agar (BA) out of which Bacillus (27; 61.36%) and Staphylococcus aureus (9; 20.45%) occurred most. Other organisms found in the bile on BA include Enteroccoci faecalis (7; 15.90 %) and one (2.27%) yeast cell. On MacConkey Agars (MCA), 19 Isolates consisting of Klebsiella pneumonia (12; 63.16%) and Escherichia coli (7; 36.84%) were identified from the bile. A total of seventy-nine (79) organisms were identified from the intestine, forty-seven (47) on BA consisting of Staphylococcus aureus (24; 51.06%), Bacillus subtilis (21; 44.68%) and Yeast (2; 4.26%). While Escherichia coli (23; 71.87%) and Klebsiella pneumonia (9; 28.12%) were identified on MCA. It is important to control the incidence of these bacterial infections in poultry to save the farmer from economic losses and the consumer from public health risk. Keywords: Bacteria, Isolation, Chickens, Jos, Public health. INTRODUCTION Bacteria are prokaryotic microscopic organisms, varying in size from 0.1-1.0 micrometer (4). They are parasitic, saprophytic, and free living-organisms which can be ovoid or spherical, rod-like or cylindrical bacillus (bacilli), curved, spiral-shaped and coil shaped spirochaete organisms. They are classified according to their morphological features, staining reactions, sizes and antigenic structures (5). Bacteria can be primary pathogens, opportunist or co-pathogens (6). A large group of microorganisms of the family Enterobacteriacea colonize the intestinal tract of animals including chickens. Enterococcus is found in the intestinal tract of chickens and can be considered as normal microflora of the intestinal tract (2). Poultry keeping provides a means by which rapid transformation towards provision of consumable protein can be achieved. Poultry diseases caused by these organisms and parasites are a major concern to the industry and result to economic loses to the farmer and the nation (7). 61
The most frequently reported causes of food borne illness are due to major enteric and systemic organisms (8, 9). In poultry production, bacterial organisms have cause diseases which lead to retarded growth, reduced egg production, high economic losses and consequently serious impact on human health. Bacteria diseases of poultry minimises the supply of protein for the increasing human population. The poultry industry is of great significance in the socio-economic development of Nigeria, it provides the most readily available protein in the form of eggs and meat, however the sudden onset of diseases that result in high mortality of chickens caused by disease agents have become the undermining factor. Therefore, prophylactic measures against such agents will help most farmers to control infections before it lead to the outbreak of disease in their flock. The aim of this work was to isolate and identify bacterial organisms found in bile and intestinal tract of apparently healthy slaughtered chickens in Jos and environs and to proffer relevant recommendations and create awareness based on the findings to the farmer. MATERIALS AND METHODS Sample collection and processing Samples were collected from a total of fifty (50) slaughtered chickens at random in 3 poultry slaughter slab; behind railway station Jos (15 samples), Yankaji market Jos (15 samples) and Kugiya market Bukuru (20 samples). Bile and intestinal samples were collected from each of the fifty birds, and tied in sterile polythene bags, given a total of 100 specimens. The samples were labelled, preserved on ice pack in a cooler, and then transported to the Laboratory for analysis. The bile and intestinal contents were streaked on blood and MacConkey agar and incubated at 37 o C for 24 hours and then examined for evidence of bacteria growth. Bacteria identification Colonies were first identified macroscopically for their sizes, consistency, elevation, nature of edge, lactose fermentation on MacConkey agar and haemolysis on Blood agar. These colonies were recorded as mixed, pure or scanty growth. Mixed colonies were then sub-cultured for purification on fresh agar plates. Colonies were Grams stained and the resulting organism subjected to biochemical tests for their identification as described by (3). Gram staining Gram staining was done as described by (5). Briefly, Crystal violet was added to the smear fixed on a microscope slide. All the bacteria that were able to absorb this dye were stained, and a diluted solution of iodine was added to decrease the solubility of the purple dye within the cell by combining with the dye to form dye - iodine complex. Then an organic solvent, acetone was added. This solvent readily removes the purple dye-iodine complex from some, but not all genera of bacteria. A red dye such as carbol fuschin was then applied which stains all bacteria. Bacteria that were decolorized by the acetone appeared red and are called gram negative bacteria. Those that retain the purple dye mask of crystal violet are called gram positive bacteria. Catalase and Oxidase tests Isolates were emulsified in a drop of 3% hydrogen peroxide (H2O2) solution on a clean glass slide. The production of abundant gas bubbles indicated the culture to be catalase positive. Absence of bubbles indicated that the culture 62
was catalase negative (5). Samples were also subjected to the oxidase test as described by (4). The colonies were picked from the agar using a sterile wire loop into a clean filter paper, two or three drops of the standard oxidase test solution was placed on the filter paper so as to cover the surface of the organisms. Oxidase positive organisms rapidly developed a purple colour. Triple sugar ion (TSI) and Dulcitol tests Some colonies of suspected organisms were stabbed on TSI slant as described by (4). The tubes were observed for hydrogen sulphide (H2S) production and gas production evidenced by air space below the bottle or air bubbles along the stab line. Suspected colonies were also inoculated into dulcitol and incubated at 37 C for 24 hours. TABLE 1: SIM (hydrogen sulphide, indole and motility) test The SIM (hydrogen sulphide, indole and motility) medium was used to test for hydrogen sulphide (H2S) production, indole and motility according to (5). Each SIM tube was inoculated by stabbing to a depth of about 5mm with straight wire followed by incubation over night at 37 0 c. For the indole test, a few drops of Kovac s reagent was added into the SIM tube which was then shaken, a red colour in the reagent layer indicated indole production. RESULTS AND DISCUSSION A total number of fifty (50) samples each of bile and intestine were cultured and the result for both samples on MacConkey agar and Blood agar are shown in Table 1. Six (6) different species of bacteria were isolated. Two (2) were enteric while four (4) were non- enteric. TOTAL FREQUENCIES OF BACTERIAL ORGANISMS FROM BILE AND INTESTINE ORGANISMS BILE INTESTINE TOTAL PERCENTAGE BA MCA BA MCA (%) Staphylococcus aureus 9 _ 24 _ 33 16.5 Klebsiella pneumoniae _ 12 _ 9 21 10.5 Escherichia coli _ 7 _ 23 30 15 Bacillus Subtilis 27 21 48 24 Enteroccocus faecalis 7 _ 7 3.5 Yeast cell 1 _ 2 _ 3 1.5 No growth 6 31 3 18 58 29 Total No. of organisms 44 19 47 32 142 100 Key: BA-Blood Agar, MCA-MacConkey Agar 63
Out of the 200 plates, colonies on Blood agar and Macconkey agar were Gram stained and subjected to biochemical tests. The results of gram staining are shown in Table 2. Table 2: Organisms Frequencies and Percentages Base on Grams Reaction. Gram staining Frequency % Positive cocci 40 20 Positive rods 48 24 Negative rods 51 25.5 Yeast cell 3 1.5 No growth 58 29 TOTAL 200 100 Table 3 below shows the occurrence of Haemolysis on Blood Agar (BA) and Lactose Fermentation on MacConkey Agar (MCA) from the different bacteria isolates. TABLE 3: Biochemical Frequencies Base on Haemolysis and Lactose Fermentation Organisms Biochemical test Total B-Haemolysis Lactose Fermentation Escherichia coli 30 30 Staphylococcus aureus 34 34 Klebsiella pneumoniae 21 21 Enteroccocus faecalis 7 7 Total 41 51 92 It is important to control the incidence of bacterial infection in poultry farms to safeguard economic losses due to mortality of birds and the consequent public health risk associated with the outbreaks of zoonotic diseases. In poultry, enteric bacteria are associated with severe mortality and predominant amongst these agents are the Escherichia genera (2). However, Staphylococcus spp, Streptococcus spp and Enterococcus spp are known to show yellowish diarrhoea and rapid loss of weight as clinical manifestation (1, 8). In this study, six (6) species of organisms were isolated from both Bile and intestinal contents. These organisms were further subjected to biochemical tests. Amongst the organisms isolated were Bacillus subtilis, Staphylococcus aereus, Escherichia coli and Klebsiella Pneumoniae. This is in agreement with previous studies (2, 6, 8). However, some yeasts were also observed. A total of 41 bacteria colonies were B- haemolytic on Blood agar (Staphylococcus aureus 34 and Entercoccus 7) while 51 isolates were Lactose Fermenters on MCA (Escherichia coli 30, and Klebsiella pneumoniae 21). 64
This work confirms other research done particularly where Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Enteroccoci faecalis and Bacillus subtilis have been isolated. These organisms constitute a problem and responsible for the losses in the poultry industry. Clinical manifestations are usually diarrhoea, emaciation and mucoid faecal discharges (8). Infections of the bile and intestine caused by Bacteria lead to mortality and retarded growth in poultry farms. Thus to avert such losses, birds should be purchased from reliable hatcheries or farms because certain bacteria organisms can penetrate eggs through the shell and can spread to other chickens during hatching. It is important to control the incidence of these bacteria infections in poultry to save the farmer from losses and the human community from outbreaks of zoonotic diseases. The isolated bacteria organisms can be controlled by acidifying the water for poultry consumption; by regularly cleaning of the poultry environment; changing of litters; by providing clean drinking water ad libitum; and by adhering to strict bio-security measures. Addition of antimicrobial feed additives in the feed of poultry is being discouraged to curb the emergence of drug resistance. These preventive measures will help reduce the enteric bacteria load which disrupts poultry production and trade and pose public health concerns for poultry products consumers. 3. Cowan, S.T and Steel, K.G (1974). Manual for identification of medical bacteria. 2 nd Edition Cambrigde University press. 4. Cruikshank, R; Dugaid, J.P; Mannoin, B.P and Swain, R.H.A (1975). Essentials of Veterinary Microbiology. 5 th Edition Academic press London. Pp166-170 5. Delong, E and Pace, N (2001). Environmental diversity of bacteria and archaea syst biol 50 (4) 470-8 doi 10 6. Evans, A.S (1978). Causation and disease. Henle-Koch s postulates revisited. Yale Journal biol med 49;175-195. 7. Oluyemi,J.A;Roberts,F.A (2007).Poultry Production in Warm Wet Climates.Published by spectrum Books Limited,Ibadan Nig.Pp 2and 16 8. Rajesh, C; Tao, V.D.P; Gomez, V.J.C; Shukia, S.K. and Banerjee, P.S (2001). Diseases of poultry and their control. 1 st edition. Pp 123-124 9. Roger,Y.S; John,L.I; Malden,C;Shem,N.C (2007). General microbiology 5 th edition,published by Macmillian press Ltd Pp45-4 References 1. Begley,M.C, Grahams, G.M and Hill, C (2005).The interaction between Bacteria and Bile. FEMS Microbiol Rev 29;625-651. 2. Collins, C.J and Patricia, T.V (1970).The family Enterobacteriacea. Microbiology Method. Pp 552-591. 65