Association of organic acids, bacillus subtilis and a mannan oligosaccharide in the control of salmonella minnesota in broiler chickens

Introduction

Salmonellosis poses a serious public health problem in both the developing and developed countries (Cardoso and Carvalho, 2006). Furthermore, some serotypes of Salmonella which have been under diagnosed in the past have increased in prevalence and, currently, represent a serious public health problem (Shinohara et al., 2008). This phenomenon was also noted by Back (personal communication), who showed an increase in the number of serovars detected in bird samples. These new types include Salmonella Minnesota. With the intention of reducing the damage caused by this bacterium, research has been conducted to find new products that are capable of controlling the enterobacteria and improving the development of birds. The use of organic acids provided better performance in birds, better feed conversion (Maiorka et al., 2004; Viola et al., 2008) better average weight and better eggs production in layer hens (Gama et al., 2000). One more function attributed to organic acids is the reduction in the count of Salmonella Enteritidis (Bassan et al., 2008). According to these authors, the combination of organic acids and mannan oligosaccharides (mannanoligosacharides, MOS) manages to reduce in a similar way, and in some cases, even more, the Salmonella Enteritidis counts. Another alternative for the control of salmonella is the use of probiotics such as Bacillus subtilis, along with mannan oligosaccharides. This combination made it possible to achieve villi of greater height and deeper crypts in 21-day old birds (Pelican et al., 2005), possibly improving the birds' performance. Therefore, the objective of this study was to assess the efficiency of the association of organic acids, a mannan oligosaccharide as a prebiotic, and a probiotic based on Bacillus subtilis in the control of Salmonella Minnesota.

Materials & Methods

60 broiler chickens were lodged from day 1 to day 35 1 of age, divided into three groups, as shown in Table 1. The animals were kept in negative pressure rooms, which were disinfected and heated to ideal confort 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 euthanasia was practiced and the autopsy of five of them to collect the liver and the caeca, which were subjected to analysis for the presence or absence of Salmonella. All the other animals were weighed individually for homogenous distribution, by weight, among the different treatments (T); at 15 days of age the birds in groups T2 and T3 were inoculated a SM solution with a concentration of 10⁸ colony forming units (CFU)/ml orally. Cloacal swabs 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 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

Treatment 1 (T1)	Basal food not inoculated with Salmonella Minnesota
Treatment 2 (T2)	Basal food inoculated with Salmonella Minnesota
Treatment 3 (T3)	Basal food with a mixture of organic acids (Neoacid^®), a probiotic based on Bacillus subtilis and mannan oligosaccharides, + inoculation with Salmonella Minnesota

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 plate, 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, 100 μl of initial peptone water solution at 2% were placed 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.

Results and Discussion

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, all of them, negative to the analysis of Salmonella, showing the control under the experimental condition.

There was a reduction of 29.83% in the SM count, which represents a CFU number which is statistically lower than the inoculated control group (T2) (Table 2). The bed and caeca count on day 35 showed reductions of 40.00% 1 11.62%, respectively, in the number of SM CFUs, compared to the non inoculated control group (T2), which represents statistically different counts in this group. In the bed samples count on day 21 and in the crop samples count on day 35, the reduction in the counts was 22.10% and 42.52%, respectively, with no statistically significant difference between the inoculated control group and the inoculated and treated control group. According to Byrd et al. (2001), the incorporation of lactic acid in drinking water is able to reduce the contamination with Salmonella Enteritidis in the crop of animals before slaughter. The administration of organic acids in the ration of birds has proven to be especially effective in reducing the count of Salmonella spp.; therefore, this bacterium is intolerant to acids (Dibner and Buttin, 2002). According to Vilà et al. (2009) Bacillus cereus var Toyoi was able to reduce the number of Salmonella Enteritidis isolates in broiler chickens and improve their zootechnical performance.

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)

	*LOG10, CFU of Salmonella* according to the sample and the age of birds**
Treatment	Swabs 48h PI	Bed 21 days	Bed 35 days	Crop 35 days	Caeca 35 days
Treatment 1 (T1)	0.00±0.00^a	0.00±0.00^a	0.00±0.00^a	0.00±0.00^a	0.00±0.00^a
*Salmonella* (T2)	3.95±2.24^c	4.30±0.07^b	3.60±0.22^c	0.87±0.50^b	4.30±4.28^c
*Salmonella* + Probiotic + organic acid + Prebiotic (T3).	2.07±1.47^b	3.35±1.32^b	2.16±1.00^b	0.50±0.74^b	3.80±1.61^b

^{Mean ± standard deviation. a, b, c Different letters in the same column are significantly different (P≤0. 05).}

Conclusions

The use of organic acids combined with probiotics and a mannan oligosaccharide was able to reduce significantly, in comparison to the inoculated control group (T2), the SM count in cloacal swabs 48 hours after infection, and in bed- and caeca samples, on day 35 of life. Based on the results achieved, it is concluded that the association of a probiotic based on B. cereus and a prebiotic based on MOS, in combination with organic acids, is effective in reducing the SM count in broiler chickens.

Bibliography

Bassan JDL, Flôres ML, Antoniazzi T, Bianchi E, Kuttel J, Trindade,MM. 2008. Controle da infecção por Salmonella Enteritidis em frangos de corte com ácidos orgânicos e mananoligossacarídeo. Ciênc. Rural 38(7):1961-1965.

Byrd JA, Hargis BM, Caldwell DJ, Bailey RH, Herron KL, McReynolds JL, Brewer RL, Anderson RC, Bischoff KM, Callaway TR, Kubena LF. 2001. Effect of lactic acid administration in the drinking water during preslaughter feed withdrawal on Salmonella and Campylobacter contamination of broilers. Poult. Sci. 80:278-283.

Cardoso TG & Carvalho VM. 2006. Toxinfecção por Salmonella spp. Rev. Inst. Ciênc. Saude 24(2):95-101.

Desmidt M, Ducatelle R, Haesebrouck F. 1997. Pathogenesis of Salmonella enteritidis phage type four after experimental infection of young chickens. Vet. Microbiol. 56(1-2):99-109.

Dibner JJ & Buttin P. 2002. Use of organic acids as a model to study the impact of gut microflora on nutrition and metabolism. J. Appl. Poult. Res. 11:453-463.

Gama NMSQ, de Oliveira MBC, Santin E, Berchieri Junior A. 2000. Ácidos orgânicos em rações de poedeiras comerciais. Ciênc. Rural 30(3):499-502.

Maiorka A, Santin AME, Borges SA, Opalinski M, Silva AVF. 2004. Emprego de uma mistura de ácidos fumárico, lático, cítrico e ascorbico em dietas iniciais de frangos de corte. Arc. Vet. Sci. 9(1):31-37.

MAPA (Ministério da Agricultura, Pecuária e Abastecimento). 2003. Métodos analíticos oficiais para análises microbiológicas para controle de produtos de origem animal e água. Publicado no Diário Oficial da União de 18/09/2003. Seção 1, p.14. Brasil.

NRC (National Research Council). 1994. Nutrient requirements of poultry. National Academy Press, Washington, USA.

Pelicano ERL, Souza PA, Souza HBA, Figueiredo DF, Boiago MM, Carvalho SR, Bordon VF. 2005. Intestinal mucosa development in broiler chickens fed natural growth promoters. Braz J Poult. Sci. 7(4):221-229.

Shinohara NKS, de Barros VB, Jimenes SMC, Machado ECL, Dutra RAF, Filho JLL. 2008. Salmonella spp., importante agente patogênico veiculado em alimentos. Ciênc. Saúde Coletiva 13(5):1675-1681.

Vilà B, Fontgibell A, Badiola I, Esteve-garcia E, Jimenez G, Castillo M, Brufau J. 2009. Reduction of Salmonella enterica var. Enteritidis colonization and invasion by Bacillus cereus var. toyoi inclusion in poultry feeds. Poult. Sci. 88:975-979

Viola ES, Vieira SL, Torres CA, de Freitas DM, Berres J. 2008. Desempenho de frangos de corte sob suplementação com ácidos lático, fórmico, acético e fosfórico no alimento ou na água. R. Brás. Zootec. 37(2):296-302.