Performance and intestinal health of broilers inoculated with nalidixic acid-resistant Salmonella Typhimurium and treated with organic acids

An experiment with 630 one-day-old chicks experimentally inoculated with Salmonella Typhimurium was performed to evaluate the effects of an organic acid blend (benzoic, fumaric and 2-hydroxi-4-methylltio-butanoic HMTBa) on intestinal health and performance. The experimental challenge with Salmonella Typhimurium occurred in two different routes of administration: via crop on the first day after hatching, and via feed, offered from seven to 14 days of age. These groups were treated with organic acids in a 3 × 2 (agent versus acid) factorial arrangement. Chicks treated with organic acids had higher weight gain, mean live weight and better feed conversion at 14 days of age, and higher weight gain at 28 days of age. Chicks treated with organic acids inoculated via crop had higher duodenum villi height than animals of the control group. Jejunum villi were higher in chicks of the negative control group compared with birds supplemented with organic acids. Small intestine pH in chicks of the inoculated group was lower compared with control group throughout the trial period. HMTBa acid in the dosage of 0.4% improves intestinal health and performance when nalidixic acid resistant Salmonella Typhimurium is experimentally inoculated.


Introduction
For the last years, paratyphic infections have been classified as of high risk for both Brazilian poultry industry and public health and Salmonella enterica subsp.enterica sorovar Typhimurium has been considered one of the most important of these diseases.Then, Salmonella Typhimurium control in poultry facilities is essential to avoid trading restrictions and to minimize the financial hazards of the Brazilian Industry, which developed some criteria to control these pathogens until the period before slaughtering and processing.
The presence of a contaminated stock, environment and feeds is the major ways to introduce Salmonella into the production system are (Tessari et al., 2003).By this means, contamination of diet, silos or feeders enables the transmission of Salmonella to the bird stock (Berends et al., 1996).Dibner & Buttin (2002) stated that organic acids can reduce Salmonella contamination and, consequently, minimize the subclinical infections in birds, and contribute to improve nutrient absorption, increasing the expression of the digestive and immune system potentialities.
It is hard to compare results among experiments that tested organic acids in blends without considering their particular way of action.Benefits depend on the organic acid which is presented in blends and their doses, so, the correct use of these compounds requires more information about their way of action on the different gastrointestinal pathogens.
Because of this necessity to control the pathogens, this experiment was proposed and conducted to observe the activity of a blend of organic acids (benzoic, fumaric and 2-hidroxi-4-metiltiobutanoic) on the performance and intestinal health of broilers inoculated with Salmonella enterica subsp.enterica sorovar Typhimurium.

Material and Methods
Chicks were allotted in the Avian Diseases Experimental Section with proper isolation facilities for the study of these pathogens.These facilities were built to assure that birds would not be contaminated, blocking insects, rodents, wild birds and people.The contact established between the chicks and people was restricted to individuals particularly dressed and with specific biosecurity care established for this experiment.The necropsy of the chicks considered negative control confirmed the absence of Salmonella.A total of 630 day-old chicks was distributed into six treatments and seven replicates of 15 birds each were used for a period of 28 days.A completely randomized design was used in a 3 × 2 factorial arrangement, with three inoculated agents (0.85% saline solution as a placebo, and Salmonella enterica subsp.enterica sorovar Typhimurium inoculated in the crop and via feed) and the use of the organic acids proposed (0 or 4 kg/T of the organic acid blend) (Table 1).
The inoculum was prepared as described by Fernández et al. (2001) and nalidixic acid resistant Salmonella Typhimurium was obtained from the broiler samples used by Rezende et al. (2008) in her experiment.Feed was formulated based on corn-soybean meal according to recommendations by Rostagno et al. (2005) and were offered ad libitum.The organic acid blend was composed of benzoic acid (22.44%), fumaric acid (41.34%) and 2-hidroxi 4-methyltiobutanoic acid (28.40%), offered to the birds through the ration during all the experimental period.The contaminated feed was made at the dose of 0.4% of total ration.In the group challenged via ration, Salmonella inoculum (0.5 mL of the inoculum/500 g feed) was prepared by means of manual homogenization in the plastic bags.
Chicks and feed were weighed on the 1 st , 7 th , 14 th and 21st days of age mean body weight, weight gain, feed intake and feed conversion were calculated.On the 7 th , 14 th and 21 st and 28 th days of age, chicks fasted for four to six hours before the necropsy for elimination of the chime.One bird per experimental unit was weighed and necropsied.The small intestine relative weight and height of each bird was established.
Samples of duodenum and jejunum were collected for histomorphometrical evaluation and processed, following the standard methodology by Luna (1968), at 7, 14 and 28 days of age.At the same ages, the small intestine and jejunum content was collected from one bird per replicate and the pH values of the samples were determined according to the methodology used by Silva et al. (2000).
Results were submitted to ANOVA and means compared by Tukey test at 5% probability using SAS (2004).

Results and Discussion
Organic acid supplementation resulted in better results of feed conversion (P<0.05)compared with control treatments (Table 2).These results are in accordance with Partanen & Mroz (1999), who claimed that organic acids, associated with nutritional assessments, handling and biosecurity, can maintain intestinal health, which can affect feed conversion.Birds inoculated with Salmonella Typhimurium had worst feed conversion compared with the control group (Table 2), for both ways of inoculation tested.No challenge Yes, into the crop on the 1 st day of age No 1 0 5 5.0 × 102 CFU Yes, into the crop on the 1 st day of age Yes 1 0 5 5.0 × 10 2 CFU Yes, through feed from 7 th to 14 th days of age No 1 0 5 5.0 × 10 2 CFU/kg Yes, through feed from 7 th to 14 th days of age Yes 1 0 5 5.0 × 10 2 CFU/kg  The evaluation of the interaction between inoculated agents and organic acids (Table 3) showed the best mean weight and weight gain from 1 to 14 days of age for chicks inoculated with the bacteria via crop and treated with the organic acid blend.Le Ny (2005) reported that organic acids can, primarily, inhibit the pathogen establishment because of the intracellular pH reduction, which affects DNA replication and protein synthesis.
The group inoculated via crop with Salmonella Typhimurium (P<0.05)showed lower values for both mean weight at 14 days of age and weight gain from 1 to 14 days of age compared with the chicks inoculated via ration (Table 3).This result can be explained by the chick age at inoculation, because the birds were challenged via crop on the first day of age, and via ration at seven days of age.The age is an important factor for the microbiota composition, and in younger animals the bacteria can easily colonize the intestines.The microbiota is formed by the contact of the chick with the microorganisms presented in the hatching chamber, in the transportation boxes or in the facilities and equipments.Desmidt et al. (1997) reported that, by aging, the specific and non-specific immunological defense mechanisms are capable of impairing Salmonella infection, which can be attributed to the development of the cellular and humoral immune system on the first days of life.Beal et al. (2004) orally challenged chicks at one, three and six weeks of age with 10 8 CFU of Salmonella Typhimurium and verified that older birds, with a welldeveloped immune system, showed a faster response to eliminate the inoculated agents.Barrow (2000) confirmed that the invasion mechanism and capacity of Salmonella depends on the age of bird and the way the agent contaminates the organs.
By evaluating chick performance from 1 to 28 days of age (Table 4), it was verified that chicks that received Salmonella, via both inoculation ways, presented best feed conversion (P<0.05)and, on the other hand, worse feed conversion (P<0.05) when compared with the control birds.Similar results were observed by Xie et al. (2000), who verified significant performance reduction when birds were inoculated with a Salmonella Typhimurium lipopolysaccharide (LPS).
Birds treated with the organic acid blend showed increased efficiency in weight gain from 1 to 28 days of age compared with the control treatment (Table 4).Organic acids can reduce colonization by Salmonella (Van Immerseel et al., 2004) and increase performance (Snow et al., 2004;Rafacz-Livingston et al., 2005 a,b).In the evaluation of the activity of organic acids on broiler performance, Skinner et al. (1991) found a positive effect of the fumaric acid (0.125%) until 49 days of age with linear reduction of mortality until 0.5% of the diet.Viola & Vieira (2007) tested different mixtures of organic acids in diets and verified that the body weight gain was not affected by treatments.
In this study, chicks inoculated via crop at 7, 14 and 28 days of age which did not receive organic acids showed lower villus height in the duodenum compared with the group that received organic acids (Table 5).At 28 days of age, jejunum villi presented lower heights (P<0.05)compared with treatments which did not receive organic acids.These results suggest that the lower the contamination by non-desirable microorganisms in the intestine, the higher the villus height.Intestinal integrity and health can be related to the results obtained, because broilers which received organic acids had significant results for feed conversion at 14 days of age and weight gain at 28 days of age when compared with the group that did not receive the organic acid blend.
Table 3 -Development of interaction between inoculated agents and organic acids used for mean weight at 14 days of age and weight gain from 1 to 14 days of age for broilers inoculated with Salmonella Typhimurium via crop and via ration, treated with an organic acid blend due to the reduced microbiological challenges of the mucosa, increasing the capacity of feed use by the animals and reducing the maintenance wastes of intestinal tissues.According to Salazar et al. (2008), the way of action of the organic acids in the intestinal epithelium can reduce the pathogenic bacteria population because of their capacity to decrease intestinal medium pH, which hampers the bacteria adhesion to the mucosa, reducing epithelium damage.In the same way, Maiorka et al. (2004) observed that the presence of trophic agents, such as nutrients in the intestinal lumen stimulate the mucosa development, originating a mitotic process, in addition to increasing the number of cells and the villus height.At 21 days of age, the small intestine weight was lower (P<0.05)with the organic acid addition (Table 6).
Higher weight (P<0.05) was observed at 7, 14 and 28 days of age, for treatments inoculated with Salmonella, in all the inoculation ways compared with the placebo group.
The higher small intestine weight in the challenged groups does not seem to be related to a better intestinal health state or nutrient absorption capacity.This fact was confirmed by the data presented in Table 5 because at 7, 14, and 28 days of age, chicks inoculated via crop showed mean values (P<0.05) for duodenum villus height compared with the other analyzed groups.Another explanation is that the finding might be due to the Salmonella activity in the intestine, because microbiological challenges can result in intestinal inflammation and, sometimes, necrosis of the tissues can occur (Apajalahti, 2005).The group inoculated with placebo had higher small intestine length (P<0.05)compared with the groups inoculated with the pathogen via both tested ways at 7 and 14 days of age (Table 6).This fact can be due to Salmonella infection, which caused tissue and cell disorders which reduced the absorption area and worsened feed conversion at 7 and 14 days of age.
Through the analysis of the interaction between inoculated agents and organic acids of the small intestine weight at 14 days of age (Table 7), it was possible to verify that the small intestine weight of chicks that were inoculated via ration and received the organic acid blend was lower (P<0.05)compared with the birds that did not receive the organic acids in the diet.This is a possible result of the organic acids activity against the pathogenic agent.
The inclusion of the organic acid blend did not affect small intestine pH or ceca content (P<0.05) during the studied periods (Table 8).It was observed that the small intestine pH was statistically higher in the placebo group when compared with the inoculated chicks.The ceca pH was statistically different at 7 (Table 8) and 21 days of age (Table 9) and the placebo group showed the highest values.It is likely that S. Typhimurium determined higher intestinal fermentation and, as a consequence, reduced the pH.Lower pH indicates fermentation and production of volatile fatty acids (Huang et al., 2006).In a similar way, Chambers et al. (1997) verified that the samples with the highest pH values, in CFU of Salmonella were also the ones with lowest cecal pH.

Conclusions
Organic acids blend (benzoic, fumaric and 2-hidrox-4-methyltiobutanoic), offered at 0.4% in ration increases performance and intestinal health for broilers challenged with Salmonella Typhimurium between 1 and 28 days of age.

Table 1 -
Experimental design of the treatment

Table 2 -
Mean weight, weight gain, feed intake and feed conversion for broilers from 1 to 14 days of age inoculated with Salmonella Typhimurium, and treated with an organic acid blend Distinct letters on the column indicate difference (Tukey, 5%); 2 ST-crop: Salmonella Typhimurium inoculated via crop; 3 ST-ration: Salmonella Typhimurium inoculated via ration.CV = coefficient of variation.

Table 4 -
Mean weight, weight gain, feed intake and feed conversion of broilers from 1 to 28 days of age inoculated with Salmonella Typhimurium, and treated with an organic acid blend Negative control of the acid; c Inoculated via crop; d Inoculated via crop and treated with acid.CV = coefficient of variation.
Distinct letters on the same column and age differ by Tukey test (5%).a Negative control; b

Table 5 -
Villus height and crypt depth of duodenum and jejunum of chicks inoculated with Salmonella Typhimurium via crop Distinct letters on the same column indicate differences by Tukey test (5%).CV = Coefficient of variation.% WG = bird weight gain.

Table 6 -
Small intestine weight and height of broilers at 14, 21 and 28 days of age inoculated with Salmonella Typhimurium via crop and via ration and treated with an organic acid blend Distinct letters in the column and row indicate statistical differences by Tukey test (5%).

Table 8 -
Values of small intestine and ceca content pH of broilers at 7, 21 and 28 days of age inoculated with Salmonella Typhimurium via crop and via ration and treated with a blend of organic acids

Table 7 -
Development of statistical interaction for small intestine weight at 14 days of age for broilers inoculated with Salmonella Typhimurium via crop and via ration and treated with organic acids blend

Table 9 -
Development of statistical interaction between inoculated pathogenic agent and the use of organic acid blend in ration for ceca content pH at 21 days of age in chicks inoculated with Salmonella Typhimurium