Use of a probiotic product based on lactobacillus added to water for control of Salmonella Minnesota in broilers

Introduction

Poultry production has increased considerably in the last decade. This increase has also raised concerns about food safety and, consequently, there have been increasingly rigid laws on the production of these foods from the farm to the industry, in order to control pathogens with zoonotic features such as Salmonella. This bacterium is responsible for food-borne infections in humans associated with consumption of chicken meat and contaminated eggs (Rossi Júnior et al., 2009). In particular, the medical community generally shows greater concern for the serological varieties (serovars) Enteritidis (SE) and Typhimurium (ST). However, at present, according to data presented by Back (personal communication), serotyping of isolates from commercial poultry between 2007 and 2010 shows a reduction in these serovars and an increase in others, as is the case of serovar Minnesota (SM). This has created an urgent need for studies to control other serovars in addition to SE and ST.

Besides, there are concerns in society about the development of bacterial strains resistant to antibiotics used in animal production. Some countries like Sweden (1986) and the rest of the European Union (1997) began the process of eliminating antibiotics in animal production on the basis of the "Precautionary Principle". Therefore, there is increased interest in alternatives to antibiotics to control pathogens in various areas of livestock production (Connoly, 2001).

Among the alternatives currently proven are probiotics, according to Fuller (1989), dietary supplements are composed of living organisms that affect the host animal, optimizing its intestinal microbial balance. Puupponen-Pimiä et al. (2002) suggest that the use of probiotics stimulates the growth of beneficial bacteria, causing a reduction in the proliferation of potentially harmful bacteria, and strengthens the natural defense mechanisms. Lactobacillus are the main microorganisms used as probiotics, probably because they are part of the normal intestinal microbiota of birds. Gabriel et al. (2006) found that Lactobacillus, as well as Streptococcus and some coliforms may be found in the small intestine (duodenum, jejunum and ileum), where the pH tends to be more neutral. The cecae holds a large number of Gram positive and Gram negative bacteria, such as Bifidobacterium, Bacteroides, Streptococcus and Clostridium.

The aim of this study was to evaluate the use of Lactobacillus-based probiotic, administered in water, for control of Salmonella enterica enterica serovar Minnesota.

Material and Methods

54 broilers from 1 to 28 days old were used, divided into three groups of 18 animals each. The experimental design was completely randomized and each animal was a repetition, as described in Table 1.

Table 1. Experimental samples with their respective treatments

Group	Description
Inoculation	Salmonella Minnesota (SM) 10⁸UFC/ml/bird
T1	Control without inoculation and without the addition of products
T2	Inoculated control and without the addition of products
T3	Commercial probiotic via drinking water on days 1, 19, 20 and 21 of life of the bird with inoculation

The day of arrival six animals were sacrificed and underwent necropsy to aseptically collect samples of cecum, liver and heart, to investigate Salmonella spp. All birds were negative. The animals were kept at the ideal comfort temperature for birds, providing food and water ad libitum. A balanced food with levels equal to or higher than those recommended by the National Research Council (NRC, 1994) of the United States were used. At 20 days of age, all animals (except group 1) were orally inoculated with 1 mL of the Salmonella Minnesota (SM) solution at a concentration of 10⁸ colony-forming units (CFU)/mL.

The commercial probiotic product used was FloraMax B11^® (Vetanco) consisting of Lactobacillus bulgaricus, L. casei, L. cellobiosus, L. fermentum and L. helveticus, offered in the drinking water on days 1, 19, 20 and 21 of life of the birds at a rate of 30 g of product diluted in 5 liters (L) of distilled water.

Swab samples were taken from the cloaca of all birds at 48 hours post-inoculation (PI) separating them in groups ("pools") of 4 birds, then the SM were counted.

At 28 days, 7 days after inoculation with SM, 18 birds per treatment were sacrificed and underwent necropsy to aseptically collect samples from crop and cecae, and perfom the count of Salmonella (individual for each animal) totaling 18 analysis of ceca and 18 analysis of crop, per group.

For counting the Salmonella, the samples of cloacal,ceca and crop swabs were diluted in 2% peptone water in a 1:9 ratio. 1 mL of each 2% peptone water solution, pouring it into another tube containing 9 mL of 0.1% peptone water, and so on, until obtaining the dilutions 10^-2 and 10^-3. Subsequently, 100 mL of each dilution were take to plant in triplicate agar xylose lysine deoxycholate (XLD) plates using a Drigalsky loop to expand the liquid on the plate. The plates were incubated in an oven at 35° C for 24 hours (h) and then subjected to counting typical colonies (adapted from Desmidt et al., 1998).

The initial solution of 2% peptone water was incubated at 35° C for 24 h. In case of failure to obtain growth of typical colonies of Salmonella after 24 h of incubation in any sample, 100 μl were removed from the initial 2% peptone water solution and added to a tube containing 10 mL of Rappaport-Vassiliadis broth. This was incubated in an oven at 42° C for 24 h, for confirmation. When a sample was negative to direct counts on XLD medium, but was positive after the process using the selective medium, the sample was considered positive in the order of log 10 = 1 for statistical analysis. The results of the colony counts were expressed according to the colony count procedures in accordance with Regulation No. 6 issued on August 26, 2003 (MAPA, BRAZIL).

The colony counts of Salmonella were transformed to Log10 for statistical analysis and were subjected to T test (P≤0.05).

Results and Discussion

It is important to underscore that no birds had clinical signs related to experimental infection with SM. The results of this study are shown in Table 2. The cloacal swabs sampled at 48 h PI showed no significant differences with respect to the removal of SM, but in the microbiological tests performed at 7 days PI a significant reduction in the presence of SM in the crop and the cecum of birds receiving treatment with Lactobacillus-based probiotic can be seen, compared with positive control group, being this reduction of 56 and 51.1%, respectively.

Table 2. Counting of Salmonella colonies in the different groups (mean±standard deviation)

Treatments	*Salmonella* count in each sample (log CFU)
Cloacal swabs 48 hours	Crop 7 days	Cecum 7 days
Negative Control	0.00±0.00^B	0.00±0.00^C	0.00±0.00^C
Positive Control	5.05±0.37^A	3.85±1.44^A	4.73±1.06^A
FloraMax B11^® in water + SM	5.26±0.54^A	1.69±1.19^B	2.31±1.72^B

^{A, B, C: different letters in the same column indicate significant differences (P<0.05) under T-test}

These results indicate that the probiotic was able to reduce the excretion of SM in the crop and the cecum of the animals. Patterson and Burkholder (2003) cite that microorganisms such as Lactobacillus can inhibit pathogens by mechanisms such as competition for binding sites, competition for nutrients, production of toxic compounds and stimulation of immunocompetent apparatus (immune system). These mechanisms can be called bacterial antagonism, bacterial interference, barrier effects, resistance to colonization or competitive exclusion. Another factor to take into account in the action of these bacteria to reduce intestinal pathogens,is the immunoregulatory effect of probiotics on the immune system of birds, which can improve the resistance against intestinal pathogens such as Eimeria acervulina (EA). Dalloul et al. (2003) found an increase in intestinal intraepithelial lymphocytes in birds supplemented with probiotics made with Lactobacillus, compared with a control group without the addition of the product. These cells play an important role in protecting the intestinal mucosa during infection and, in this case, resulted in a reduction in the excretion of the number of oocysts of EA.

Since in the present study we did not evaluate the immune response, we cannot conclude with certainty whether the reduction in the excretion of SM was due to the direct effect of competitive exclusion or if probiotic can be attributed to an effect associated with immunomodulation induced by Lactobacillus in birds. Further studies are needed to better determine the mechanism of action.

Conclusions

The use of these Lactobacillus-based probiotics can reduce the excretion of Salmonella Minnesota and may be helpful in controlling this infection.

Bibliography

Brasil, Instrução Normativa nº 6 de 26 de Agosto de 2003. 2003. Ministério da Agricultura, Pecuária e Abastecimento. Diário Oficial da União.

Connoly A. 2001. Reagindo ao desafio da retirada dos antibióticos promotores de crescimento das rações e a forma como os oligossacarídeos específicos assumiram a dianteira.Feed compounder junho/julho:20-25.

Dalloul RA, Lillehoj HS, Shellem TA, Doerr JA. 2003. Enhanced mucosal immunity against Eimeria acervulina in broilers fed a Lactobacillus-Based probiotic. Poult Sci 82:62-66.

Desmidt M, Ducatelle R, Haesebrouck F. 1998. Serological and Bacteriological observations on experimental infection with Salmonella Hadar in chickens. Vet Microbiol 60:259-269.

Fuller R,1989. Probiotics in man and animals. J. Appl. Bacteriol. 66:365-378.

Gabriel I, Lessire M, Mallet S, Guillot JF. 2006. Microflora of the digestive tract: critical factors and consequences for poultry. World's Poult Sci J 62:499-511.

National Research Council. 1994. Nutrient requirements of poultry. 9th rev.ed. National Academy Press: Washington, D.C.

Patterson JA & Burkholder KM. 2003. Application of Prebiotics and Probiotics in Poultry Production. Poult Sci 82:627-631.

Puupponen-Pimiä R, Aura AM, Oksmancaldentey KM, Myllärinen P, Saarela M, Mattila-Sanholm T, Poutanen K. 2002. Development of functional ingredients for gut health.Trends Food Sci Technol 13:3-11.

Rossi Junior OD, Berchieri Júnior A, Felipe LM. 2009. Agentes de interesse em saúde pública associados a produtos de origem avícola. pp.1039-1053. In: Doenças das Aves. Berchieri Júnior A, Silva EN, Di Fábio J, Sesti L, Zuanaze MAF (eds.). Fundação APINCO de Ciência e Tecnologia Avícolas. Campinas, SP, Brasil.