Population of Escherichia coli in the ceca of broilers after water treatment with dichloroisocyanuric acid salts during pre-slaughter feed withdrawal

Pre-slaughter food withdrawal (fasting) is considered an important stage in broiler production, due to the fact that it has an influence in the quality and yield of meat. Such a procedure is performed in order to diminish contamination, because of residue, of the gastrointestinal tract at the slaughter house, avoiding the waste of carcasses, thus, improving production (Duke et al., 1997; Northcutt et al., 1997). Prolonged periods of fasting should be avoided, due to the fact that during such periods, birds have a further possibility of eating all available materials in the surroundings, such as excreta and litter debris, which increase the potential of contamination of the carcasses once in the slaughter house (Rasmussen & Mast, 1989; Lyon et al., 1991). At the beginning of industrial poultry, many studies were carried out in order to define a time period, in which these objectives could be reached, but without affecting the weight of the birds and the yield of the carcass. At that moment in time, several researchers defined such period as of 8 to 12 hours without food, as the optimal time to reduce the contamination incidence, without affecting the yield of the carcass (Smidt et al., 1964; Wabeck, 1972; Veerkamp, 1986; Lyon et al., 1991). Contamination occurs when the viscera of the digestive tract ruptures, or when the feces adhered to the feathers, skin, and legs, during transportation get in touch with the carcasses during processing. Transportation is the main stress cause before slaughter. In a contaminated batch, stress determines the increase in the elimination of fecal matter (Delazari, 2001). Delazari (2001) proved that after transportation, the level of Escherichia coli in the surface of chicken breasts, increases ten times more, during a period of only five hours. We can state that when we speak of contamination, at the slaughter house level, we are referring ourselves to the presence of intestinal content, both inside as well as outside an eviscerated carcass. Hence, we should try to diminish contamination of the gastrointestinal tract of birds, with the purpose of diminishing the contamination of the carcass, causing less risk to the consumer. The objective of this experiment was to test the efficiency of chlorine in drinking water of broilers, since the beginning of the feed withdrawal until the end of the fasting period, and the beginning of the collection for transportation, in order to reduce the quantity of Escherichia coli in the broiler ceca, diminishing the possibility of contamination of the carcass, thus, presenting a minor risk for public health.

Forty Cobb broilers were used, fed with commercial diets of Purina de Brasil^®. An initial diet was provided (Inicina^®), up to 21 days old and a growing diet (Nutriengorda^®), until slaughter, according to the recommendations of the manufacturer. Both water, as well as feed, were given ad libitum. Twelve hours before slaughter, which took place at 42 days of age, samples of the crop content were collected for a microbiologic analysis after slaughter of 10 birds. This same analysis was repeated at the end of the fasting period and water diet, 10 birds of each treatment were killed for sample collection. The design was a totally random study, four treatments (without fasting at the beginning of the pre-slaughter period, 12 hours fasting without the addition of chlorine in water, 12 hours fasting with the addition of chlorine in water and without fasting during the pre-slaughter period) with 10 repetitions per treatment group. Statistic Analysis methods were: Analysis of Variance by the F Test and the mean comparison by Tukey's Test at 1% and at 5% of probability. For the statistical analysis, the Agroestat computing program was used. Samples of the cecal content were collected, directly at the ceca of the slaughtered birds, in an aseptic manner using sterile materials. Samples of 25 g of cecal content were placed in flasks with 225 mL of buffered detonated water at 1% obtaining a 10^-1 dilution. From this dilution, consecutive decimal dilutions were done, using the same ratio. The presumptive test was performed as of the dilutions of 10^-1 and 10^-5 of the samples of the cecal content and three tubes were, respectively, inoculated with 1 mL of a lauric sulfate tryptose broth with a reverse Durham tube. After inoculation, these tubes were incubated at 35^oC during 24 to 48 hours, and those which presented bacteria growth characterized by turbidity of the means and gas production were considered as positive. From each tube with laurel tryptose sulfate broth, with a positive result in the presumptive test, corresponding pipes with  Fluorocult^® broth were inoculated by means of a loop and a reverse Durham tube. Incubation was done at 35ºC during 24 hours and the pipes presenting bacteria growth, the presence of gas and fluorescence, after the induction of ultraviolet light (366nm) were considered as positive. Results were obtained by comparison of the numbers of the positive pipes with the data of the table of Hoskins, always considering three consecutive dilutions from the main dilutions with three positive pipes. According to the number of positive pipes and with the use of the table of Hoskins, the MPN of  E. coli  per gram of cecal content (APHA, 2001) was determined. Salts of dichloroisocyanuric acid of Hidroall de Brasil Ltda. (Aviclor choque^â) in the drinking water of the treatment group of the birds on water diet with chlorine and fasting. In order to determine the concentration of free residual chlorine in the samples of the water measured at the sampling moment, reagent NN Diethyl paraphenylenediamine (DPD) and the electronic colorimeter (¹HI93710C-Hanna Instruments), starting in zeros, with 10 mL of the water simple without the DPD reagent were used, and the reading took place after the addition of the reagent to the bucket with 10 mL of the sample, homogenizing the sample, proceeding to the reading given in mg.L^-1.

Table 1. Most probable number (MPN) of Escherichia coli [y=log (x+5)] in the crop (MPN . mL^-1 of conservative solution of the swab) of broilers 12 hours before slaughter and subjected to fasting, fasting and chlorine addition in drinking water and without fasting during the pre-slaughter period (12 hours)

Means with different letters differ significantly at 5%. The amount of free residual chlorine of the container, fasting and chlorine immediately after the addition of 52 ppm. Free residual chlorine after 12 hours was of 43 ppm.

 

The transit velocity of food through the intestine is influenced by fasting, room temperature, level of activity of the birds and consumption standards before food withdrawal. The transit of food in chicken and turkeys is relatively fast (Hillerman et al., 1953); and according to Sibbald (1979), liquids have a faster transit in the intestines that solids, carrying microorganisms present in the cecal content. Nonetheless, the MPN of Escherichia coli did not present a significant difference when comparing broilers subjected only to fasting, with those 12 hours before slaughter (without fasting), probably, because of the virulence factors which provide E. coli with a further capacity of remaining in the intestinal environment, such as andesine, pili or fimbria (Sussman, 1997). No significant difference was found when comparing the cecal content of birds subject, just and only, to fasting (without the addition of chlorine in water) with those of birds subject to fasting with the addition of chlorine to water; probably, and for the same above mentioned reason, related to the virulence mechanisms of E. coli (Sussman, 1997). As expected, there was no significant difference when comparing birds 12 hours before slaughter (without fasting) with birds not subjected to fasting, nor to the chlorine treatment of drinking water during 12 hours pre-slaughter. In spite of these already expected results, data was collected due to the fact that the transit velocity of food, and the consequent profile of microorganisms are influenced by several factors, such as room temperature, diminution of the bird's activity due to the diminution of the amount of light, amongst others (May et al., 1988).

The addition of chlorine to the drinking water of broilers during the pre-slaughter fasting period, did not alter the amount of Escherichia coli in the ceca of broilers, probably due to the virulence mechanisms pathogenic E. Coli have in order to remain in the intestinal tract and because chlorine may not have reached enough activity so as to keep its disinfection capacity until that segment of the gastrointestinal tract.

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To the Amparo Foundation for research in the State of São Paulo (FAPESP) for granting the Masters scholarship and for the support to this research.