Occurrence of Escherichia coli and Salmonella species in some livestock (Poultry) Feeds in Mando, Kaduna, Nigeria

The study aimed at assessing the proximate composition, isolation, characterization of some Enterobacteriaceae from two (2) brands of poultry feeds marketed in Mando, Kaduna, Nigeria. A total of sixteen (16) samples of two (2) different poultry feeds (starter and finisher) from four (4) poultry farms in mando were collected and subjected to proximate and microbiological analysis. The proximate analysis was carried out using standard techniques and procedures. All the feed samples were cultured on separate media which include Eosin methylene blue (EMB), nutrient Agar (NA) and SalmonellaShigella Agar (SSA) media using standard procedures. The antibiogram of the selected antibiotics were evaluated against the test isolates. The result of proximate analysis of the starter and finisher feeds indicated that the Dry matter of starter feed had the highest percentage composition of 95.02% and crude fiber of the finisher feed had the lowest composition of 3.78%. The highest number of bacterial load was recorded to be 10.0×104 CFU/g for the feed sample A (starter feed) and 12.0x104CFU/g was recorded for the feed sample B (finisher feed) which had the highest number of bacterial load recorded among the two (2) different poultry feeds analyzed. The bacteria isolates were identified as Salmonella species and Escherichia coli. Total viable count (TVC) of Salmonella species and E. coli in the feed samples (starter and finisher) ranges from 3.0×104CFU/g to 12.0×104CFU/g. Both organisms (Salmonella species, E. coli) were found as 37.5% and 25% of the analyzed feeds (Broiler starter and broiler finisher) samples, respectively. There was no level of significant (p>0.05) difference between the level of contamination of Salmonella species and E. coli in the two different feeds analyzed, as p=0.06 and p=0.13 for Salmonella species, and E. coli respectively. Sample A and B (Starter and Finisher) feeds had the highest number of Salmonella species occurrence with six ( 6) positive samples while E. coli was recorded in four (4) samples of A and B (Starter and Finisher) feeds. The result of the antibiogram indicated that ciprofloxacin (30 μg), Gentamycin (30μg), Perfloxacin (30μg) and Tarvid (30μg) was effective against Salmonella species and Escherichia coli. The significant of spread of the species of the Enterobacteriaceae in livestock feeds requires the need for effective quality assurance and control, good hygiene practices in production and proper handling of the poultry feeds.


iNtRODUCtiON
All domesticated birds by man are referred to as poultry. These birds include domestic duck, fowl, geese, guinea fowl, turkey, ostriches and pigeons as reported by 54 . Livestock feeds can serve as a medium for a range of microbial contaminants such as bacteria (Salmonella sp., Shigella sp. E.coli etc) moulds (Fusarium sp. Aspergillus flavus, Aspergillus paraciticus etc) and their mycotoxins 32 . Many bacteria from the family enterobacteriaceae are mostly associated with environmental contaminations of feed ingredients. This poultry feeds contamination family include many genera and species of bacteria namely; Salmonella, Enterobacter and Escherichia coli 26 . The usual and common feeds compositions include soya beans, complete (whole) cereals, vitamins and vegetables such as tridax, Amaranth (Amaranthus sp.) and water leaf (Talinum fruticosum). Feeds in general has been implicated to be a major sources for transmission of bacteria and other microorganisms to the processing plant of the farm. Most animals harbour pathogens which are of food borne which serves as a good source of contamination, which is of significant in the spread of Escherichia coli and Salmonella species in humans 7 . These bacteria can survive for prolonged periods of time without multiplication on materials with low moisture contents therefore providing for the possibilities of the bacteria to be mechanically transmitted from one site to another through fomites, including contaminated feeds 10 . Feeds are formulated from different ingredients with different possible levels of Salmonella and Escherichia coli contamination. A research on cereal ingredients in the UK showed that animal feeds were contaminated with Salmonella and Escherichia coli at the farm level, whereas 92% of the meat and bone meal samples tested in the United States were contaminated with Salmonella and Escherichia coli and in the Netherlands 31% Salmonella contamination was recorded in fishmeal samples 50. Feed manufacturing facilities are therefore considered as critical contamination points for Salmonella and Escherichia coli entry into the food chain. The feeds and their ingredients are also a significant sources of extensive contaminations by antibiotic resistant bacteria along side multidrug resistant strains of Salmonella sp. 5. Other bacteria such as Escherichia coli Streptococcus sp. and Entercoccus sp. have been isolated in feeds. The constant control of bacteria contaminations in feeds has shown to improve performances in production from poultry and it also reduces the occurrences of salmonella in farm environment breeding animals, and their products. The increase in chicken production has resulted in high demand of feeds, and consequently, proliferation of feed mills, some of which operates under substandard conditions. This may result into packaging of feed contaminated with pathogens and thereby spreading diseases to both humans and farms. Despite advances in medical science, infections due to Salmonella and E. coli strains remain the most important food borne diseases (FBDs) of human. Moreover, a majority of FBDs are implicated with the consumption of contaminated poultry eggs and meats. Though many approaches have been employed to counter these infections both in human and poultry, application of effective antibiotics (therapy) is the main control strategy 40 . The research aimed at assessing the occurrence of Escherichia coli and Salmonella species in some livestock feeds in Mando, Kaduna.

Sample Collections
Sixteen (16) samples in total of two ( 2) different chicken feeds: (Broiler starter and finisher) were collected. Eight (8) samples each starter and finisher were collected from four (4) different poultry farms: four (4) per farm in Mando, Kaduna State. The feed samples were aseptically collected separately into sterile universal containers and were well labeled with regards to the feeds after seeking an informed consent from the poultry/livestock farmers. The feed samples were transferred to the Microbiology laboratory, Kaduna State University, Kaduna, Nigeria for proximate and bacteriological analysis.

Proximate Analysis of Chicken Feeds
Proximate analysis of the some chicken feed samples were carried out for percentage total dry matter, crude protein, moisture, crude fat, ash, crude fibre and carbohydrate content using the methods described by Bukar and Saeed MD 9.

Media Preparation
All culture media used in this research were prepared with regards to manufacturer's instructions. The media used include: NA, SSA, EMB and MHA.

Isolation of Bacteria from Chicken Feeds total Viable Count (tVC)
Twenty five (25) grams of each feed sample were homogenized into 225ml of peptone water. One (1) ml of the homogenized sample was suspended in 9.0 ml distil water, then serial dilutions from10 -1 to 10 -5 was carried out, One (1) ml of each two dilutions (10 -3 and 10 -4 ) was inoculated into petri dishes containing nutrient agar for each respectively. The petri dishes was incubated at 37 o C for 24 hours. Colonies that appeared on the plate were counted and recorded in CFU/g. The Total Viable Bacterial Count (TVC) was carried out as described by Atere et al 6 . Viable colonies from nutrient agar were inoculated into EMB and SSA agar for each bacterial isolates respectively. The plates were incubated at 37 o C for 24 hours 39.

Characterization and Identification of Bacteria from Chicken Feeds
The characterization and identification of bacteria isolates from samples of chicken feeds were based on Grams staining and selected biochemical tests which include Catalase , Indole production, Voges-Proskauer (VP), Methyl red, Citrate, Coagulase and Triple Sugar Iron Agar (TSI) test described by Grant et al 22 and Dougnon et al 43 .

Antimicrobial Susceptibility Testing of Selected Antibiotics against the Bacteria Isolates
Mueller Hinton Agar was prepared according to the manufacturer's instruction. The bacteria suspension for each respectively was prepared and compared with the turbidity of 0.5 McFarland standard which is approx. cell density (1,5X10 8 CFU/mL) ( standardization of inoculum) , then 5 μl of the prepared bacteria suspension was placed on each of the series of plates with already different concentrations of the antimicrobial agent using a replicator device. The plate were incubated at 37 o C for 24 hours as described by Mathew et al 33 .

Statistical Analysis of Data
Data generated were statistically analyzed using IBM -SPSS Statistics version 20 computer program. The student T-test and one way analysis of variance (ANOVA) was used to determine the prevalence of Escherichia coli and Salmonella species contaminations among the two different feeds (broiler starter feed and finisher feed) collected from four different farms. The significant difference were considered between and within variables.

ResUlts
Ta b l e 1 S h owe d t h e p rox i m ate composition of poultry feeds. The dry matter of broiler starter feed (A) was 95.02%, Dry matter of broiler finisher feed (B) was 94.86 %, Ash content of broiler starter recorded 7.89 %, the Ash content of broiler finisher feed was 7.85 %. The protein content of broiler starter feed was 22.54 % , 23.99 % was recorded for protein content of broiler finisher. Crude fat/Oil content indicated 5.99 % for broiler starter feed, 6.11 % was recorded for crude fat/oil content. The crude fiber content recorded for broiler starter was 4.92%, crude fiber content recorded for broiler finisher feed was 3.78 %. Nitrogen Free Extract recorded for broiler starter feed was 57.22 % and 55.24 % was recorded for finisher. Table 2 Showed the estimation of total viable bacteria count of chicken feeds. The mean microbial load of broiler starter from AS 1 and AS 2 ranged between 3.0x10 5 to 5.0x10 4 CFU/25g . While the mean microbial load for broiler finisher feed from AF 1 and AF 2 ranged between 5.0x10 5 to 8.0 x10 4 CFU/25g. BS 1 and BS 2 mean microbial load for broiler starter feed ranged between 3.0 x10 5 to 4.0x10 4 CFU/25g. While BF 1 and BF 2 broiler finisher feed was recorded as 4.0 x10 5 and to 6.0 x10 4 CFU/25g. CS 1 and CS 2 broiler starter feed indicated the present of microbial load between 6.0x10 5 to 10.0 x10 5 CFU/25g. While CF 1 and CF 2 of the broiler finisher feed was recorded as 6.0x10 5 to 12.0 x10 4 CFU/g. DS 1 and DS 2 broiler starter feed ranges from 3.0 x10 4 , to 8.0 x10 5 CFU/25g and the DF 1 and DF 2 ranges from 3.0 x10 4 to 9.0 x10 5 CFU/25g for each of the two dilutions ( 10 3 , 10 4 ) respectively. Table 3 showed characterization and identification of bacteria isolates. Isolate from AS 1 feed was Gram negative rod shape, methyl red positive ,catalase positive, citrate negative, indole positive, Voges-Proskauer negative and TSI (Acid production, no gas produced and buttom     4 positive occurrence with 25% in the two feeds analyzed from the four farms as the less predominant pathogen. Table 5 shows the result of antibacterial susceptibility test of Salmonella species and Escherichia coli to the antibiotic disc which indicated that Salmonella sp. and E.coli were either sensitive (R) , moderately sensitive (MS) and resistant (R) at different concentrations (ranging from 10 to 30 µg) respectively.

DisCUssiON
The ingredient percentage composition of broiler starter feed was higher than that of broiler finisher feed. This could be attributed to the chicks needs for higher amount of nutrient to enable them grow. The broiler finisher feed has less percentage composition of the feed ingredients as compared to broiler starter feed. This might be as a result of the chicken having the ability to have acquired the essential nutrients for their growth and so do not need more nutrients for growth but need just enough nutrient to keep them healthy. Dry matter has the highest ingredient percentage composition of the feed of 95.02 % for broiler starter feed and 94.86 % for broiler finisher feed making. This is to enable the chicken pick the feed easily. Nitrogen Free Extract (NFE) has the second high value of percentage composition of the feed ingredient. This shows the sugar and starch content of the feed which are essential for the meat content of the chicken. Ash content recorded the lowest % composition of the feed ingredient. This is because it is trace minerals which are needed only in a small quantity 54 . Crude fat, protein content and crude fiber were present in small quantities. Crude fat provide essential fatty acids and energy, protein content that maintains the growth in the chicken while fiber is associated with the reduced energy availability. This is similar to the research conducted in Zaria Kaduna State by Bukar et al 9 who reported proximate composition of chicken feed for crude fiber content with the lowest composition of 1.70 while dry matter recorded was with the highest percentage composition of 90.02. The statistical analysis of the ingredient for broiler starter and finisher feeds, indicates that there were no significant difference (p>0.05) between broiler starter and finisher feeds in all the proximate percentage compositions. Total viable count of the isolates showed the 10 3 dilution samples with higher colony forming unit and 10 4 dilution having less colony forming unit. This could be because colonies of microorganism varies with the dilution factor as reported by Pattabhiramaiah and Mallikarjunaiah 42 . The CF 1 feed recorded the highest number of colonies of 12 CFU/g while CS 1 feed recorded the second highest colony of 10 CFU/g The possible reason why CF 1 feed indicated the highest number of colonies could be due to the fact that it contains higher nutrient composition as compared to other feeds. BS 1 feed recorded the least number of colonies of 3CFU/g which could also be as a of result of low nutritional composition of the feed. The statistical analysis indicated Heeraman 24 reported that cereal ingredients for animal feeds were contaminated with Salmonella and Escherichia coli at the farm level, whereas 92% of the meat and bone meal samples tested in the US were contaminated with Escherichia coli and Salmonella sp., and 31% of Salmonella contamination was recorded in fishmeal samples in the Netherlands 50 . The poultry feeds from the four (4) farms showed higher contamination with Salmonella species than with E. coli even though there was no significant different (p>0.05) between them. A possible explanation for this may be due to the increased use of antibiotics in the feed for treatment which made poultry feed a major reservoir of antimicrobial resistant Salmonella and E. coli. This agrees with the work of Atere et al 6 and Brown et al 8 who reported the increased use of antibiotics in feed result in the contamination of feed by Salmonella and Eschericia. coli that develop antibiotic resistance due to transposons. The sensitivity, moderate sensitivity and resistant of Salmonella sp. and E.coli at different concentrations of the antibiotics ( ranging from 10 to 30 µg) respectively which could be attributed to the efficacy of the antibiotics on bacterial and/or resistant genes that are possessed by the bacterial genome as the case may be towards some of the antibiotics. due to the indiscriminate use of antibiotics by farmers This antibacterial susceptibility pattern is similar to reports of Alabi et al 2 who conducted a research in Abeokuta, Nigeria and reported a higher prevalence and antibacterial susceptibility pattern of salmonella isolates from chicken carcasses while Atere et al 6 and Silva et al 48 both also reported isolation and antibiotic susceptibility pattern of Salmonella and E. coli from livestock feeds.

CONClUsiON
The result of proximate analysis indicated dry matter having the highest percentage and crude fiber having the least percentage. The isolation and identification of the collected chicken feeds indicated Salmonella species having high number (6 positive samples) of contaminations in broiler starter and broiler finisher feeds than E. coli having lesser number (4 positive samples)