Probiotics based on Enterococcus Faecium NCIMB 10415 for the control of Salmonella Minnesota in broiler chickens

Currently, Brazil occupies a prominent place in the production of broiler chicken and is the third largest producer and the largest exporter of chicken in the world. To achieve this position, it took great efforts to increase productivity and biosecurity on farms, with the aim of satisfying the quality required by both the internal and external markets. Thus, the control of microbiological contamination of chicken meat with microorganisms that cause food-borne infections and food poisoning in human beings has become essential, as in the case of those infections produced by Salmonella. Salmonellosis poses a serious public health problem in both the developing and developed countries (Cardoso and Carvalho, 2006). The increase in biosecurity on farms involved intensification of analysis of flocks and processing plants, in search of contamination. In recent years, this has led to an increase in the number of serological varieties isolated (serovars). According to Shinohara et al. (2008) the presence of several serotypes of Salmonella has been reported on pig farms, where years ago had not been described. This phenomenon was also noted by Back (personal communication), who described an increase in the number of serovars detected in poultry samples, including Salmonella Minnesota (SM), which occupies an important place. In addition to biosecurity, the use of food additives for the control of Salmonella has increased; and this is the case of probiotic products, which have been defined as cultures of micro-organisms living either alone or in the form of mixtures , when applied to animals or humans, a beneficial effect on the host, in improving the properties of the endogenous microbiota (Havenaar et al., 1992). Enterococcus faecium (EF) is a bacterium producing lactic acid, which has inhibitory effects against Escherichia coli and Salmonella spp. (Lewenstein et al., 1979). Recently, it was reported that EF was able to improve the performance and feed conversion of the flocks (Mallo et al., 2010). Thus, the objective of this study was to assess the efficiency of a product based on EF in the control of SM.

60 broiler chickens were lodged from day 1 to day 35 of age, divided into three groups, as shown in Table 1. The animals were kept in negative pressure rooms, air-conditioned to ideal comfort temperature, according to the age of the birds. A bed of wood ss, sterilized in autoclave was used, and water and food were provided ad libitum. Before the start of the experiment, the bed and rations were examined for the presence of Salmonella spp. The diet was formulated using equal or higher levels than those recommended by the National Council of Research (NRC) (1994) of the United States. All the food was pelletized. Upon arrival of the animals, five of them were slaughtered and autopsied to collect the liver and caeca, which were subjected to analysis for Salmonella. All the other animals were weighed individually and distributed in a homogeneous way, according to their weight, among the different treatments. Birds of the T2 and T3 groups were inoculated after 15 days with an SM solution at a concentration of 10⁸ colony forming colonies (CFU)/ml of SM, orally. Cloacal swab samples were taken 48 hours after inoculation, at a rate of five samples per treatment (set or "pool" of 3 animals), to perform the Salmonella count. At 35 days of age, 10 animals per treatment were slaughtered, and autopsied to harvest the crop and the caeca, aseptically, and these organs were examined in search of Salmonella. On days 21 and 35 of age five aliquots of 10 g of bed from the rooms where the birds were housed were taken (5 samples per treatment) for analysis and count of Salmonella.

Table 1. Description of the Treatments

To perform the Salmonella counting procedure, the cloacal swabs, the crops, the caeca and the bed samples were diluted in peptone water at 2% and rediluted in vials with peptone water at 0.1%, to achieve the concentration of 10^-3. Later, 100 μl of each dilution were planted on plates, in duplicate, in Xylose lysine dexosycholate agar medium (XLD). These plates were incubated on a stove regulated at 35° C for 24 hours and, subsequently, they underwent the typical colonies count (adapted procedure of Desmidt et al., 1997). The initial peptone water solution at 2% was maintained at 35° C for 24 hours and, if no growth of typical colonies of Salmonella was detected on the XLD plates, they were placed in 100 μl of initial peptone water solution at 2%, in a vial containing 10 ml of Rappaport-Vassiliadis broth, incubated on a stove regulated at 42° C for 24 hours, for confirmation. If a sample was negative to the direct count in XLD gelose, but positive after the process using enrichment and selective media, the sample was considered positive for statistical analysis. The results of colony counting were expressed according to the Colony Counting Procedure (Procedures Contagem Colônia), Standard No. 6, published on August 26, 2003 by the Ministry of Agriculture, Livestock and Food (MAPA, 2003). The counting of colonies of Salmonella was transformed to Log 10 for statistical analysis using the ANOVA system, and the Fischer test to 5% probability. To compare the reduction in the SM count with regard to the control group, the Chi square test (P<0.05) was used.

All the bed and food samples collected prior to the arrival of the birds, the liver- and caeca samples collected on the first day and the samples collected from the negative control group in the different periods tested, tested negative to the analysis of Salmonella, showing the control under the experimental conditions.

Based on the analysis of the swabs collected 48 hours after inoculation, there was a reduction of 61.8% in the Salmonella counts, with regard to the positive control. In the bed and caeca count at 35 days there was a reduction compared to the positive control. This reduction was 47.2% and 73.4%, respectively. However, the use of the probiotic had no effect on the count of SM in bed at 21 days or in the crop at 35 days (Table 2). According to Lund et al. (2002), EF is capable of surviving the intestinal transit, being isolated in the feces of persons who consumed it in the diet. In addition to this, one of the characteristics of Enterococus is its ability to survive in the environment and on dry surfaces (Wendt et al., 1998; Neely and Maley, 2000). These two characteristics, associated with the production capacity of lactic acid by this bacterium, can explain the reduction in isolation of SM in the caeca and bed of poultry at 35 days of life.

Table 2. Colony counts of Salmonella in cloacal swabs 48 hours after inoculation, bed samples counts on days 21 and 35 and crop and caeca counts on day 35, in different groups of animals, 35 days of age (results expressed in Log10 CFU/g)

The use of a probiotic based on Enterococcus faecium was able to reduce the count of Salmonella Minnesota in the caeca and in the bed of poultry that consumed the product for 35 days, Thus proving it to be an important tool for the control of SM, associated to biosafety practices.

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