Bacteriological Investigation of Microorganisms ( Salmonella sp . and Other Enterobacteriaceae ) in Common Quails ( Coturnix coturnix ) Submitted to Different Forced-Molting Procedures

The aim of this study was to investigate the presence of Salmonella in common quails submitted to forced molting. A total of 240 quails were divided at 40 weeks of age into four groups: CG (control, quails not submitted to molting); FM (fasting method); WM (fed wheat midds ad libitum); and ZM (zinc oxide method). From each group, 10 cloacal swabs, 10 fecal samples, and 20 egg samples were collected before molting (two weeks) and after molting (two weeks). The microbiological procedures for Salmonella spp. identification were performed in four steps. The agglutination test, using somatic and flagellar antigens, was used to confirm Salmonella-suspected colonies. According to the methodology applied, none of the samples was positive for Salmonella spp. The results showed that 20.0% of the egg samples from birds submitted to forced molting were contaminated with enterobacteria. It was concluded that, under the conditions of the present experiment, the stress caused by forced molting did not induce infection by Salmonella spp. or increased Enterobacteriaceae contamination levels in the eggs.


IntRoduCtIon
Low egg quality is a common problem in the poultry industry and cause extensive losses.The main causes are related to environmental, health, and management issues (Cardoso et al., 2001).Among health factors, egg contamination by Salmonella spp. is extremely important in the poultry production chain (Hafez, 2005), because this genus is one of the main causes of food poisoning in humans (Chernaki-Lefferet al., 2002;Tirolli & Costa, 2006).
Microorganisms may contaminate internal egg contamination by several pathways: through the ovary, when the agent remains inside the egg during its formation; through the uterus, when the agent is present in the oviduct epithelium or in the serosa of the air sacs; through the cloaca at the time of lay, when the egg may get in contact with contaminated feces; and through the external environment after lay, which is the most frequent form of microorganism invasion (Soncini & Bittencourt, 2003).
Despite the significant role played by Salmonella spp. in food poisoning around the world and the economic losses caused in the poultry industry, other enterobacteria that may potentially cause foodborne diseases in humans should be considered, as several studies report their occurrence in eggs used for human consumption.The most frequent enterobacteria are Enterobacter sp., Klebsilella sp., Citrobacter sp., Serratia sp.Proteus sp., Pseudomonas sp., Hafnia and Providencia sp.(Adesiyunet al., 2006;Salles, 2007;Musgrove et al., 2008;Siqueira et al., 2008).

Bacteriological Investigation of Microorganisms (Salmonella sp. and Other Enterobacteriaceae) in Common Quails (Coturnix coturnix) Submitted to Different Forced-Molting Procedures
The main source of contamination of poultry products by food pathogens are animal feces (Rasmussen et al., 2004), particularly in the case of eggs.One of the most frequent forms of egg contact with feces is when birds present watery feces due to pathological (e.g., enteritis) or physiological conditions (e.g., changes in feed formulation), leading to egg contamination at the time of lay (Soncini & Bittencourt, 2003).
In order to prevent bacterial contamination problems, good management practices need to be applied in egg production, providing hens proper health conditions that prevent the spread and replication of microorganisms.A common management practices applied in the egg industry is forced molting by fasting, aiming at obtaining a second laying cycle.However, this frequently affects hens' health.The stress caused by feed fasting results in an increase in blood corticosterone levels (Webster, 2003).It is known that stress hormones, such as corticosterone, have anti-inflammatory properties, thereby impairing the immune function (Golden et al., 2008).Therefore, molting induced by fasting increases hen susceptibility to several pathogenic microorganisms, including Salmonella (Holt, 2003).Holt (2003) asserted that chickens submitted to forced molting are 100 to 1000 times more susceptible to Salmonella Enteritidis (SE) infection and may easily spread the bacterium to the non-infected hens in neighboring cages.This demonstrated that fasting to induce forced molting in commercial layers may eventually cause severe consumer health problems.This hypothesis was confirmed by Golden et al. (2008), who demonstrated that layers submitted to forced molting by feed fasting produced SE-contaminated eggs more frequently.
Despite these health issues involving the fasting method, forced molting has still been successfully used in commercial layer production.Alternative forced molting methods have been researched and used in poultry industry during the last decades to provide animal welfare and to prevent health problems.Among the employed methods, the supply of high dietary zinc levels is the most frequently studied and applied in the field, particularly in the United States (Mesquita Filho, 2008).Forced-molting methods using wheat-based feeds have also shown good results (Biggs et al., 2004;Dalanezi, 2007).
Forced molting is still not commonly applied in quail egg production (Garcia et al., 2001), although a few published studies have reported its effects on production and physiological aspects (Zamprônio et al., 1996;Garcia et al., 2002.;Teixeira et al., 2007;Mesquita Filho, 2008;Faitarone et al., 2008).Scientific data on the effects of forced molting on quail health are lacking.Therefore, the objective of the present study was to evaluate the effect of different forced molting methods on the presence of Salmonella spp. in the eggs, feces, and cloacal swabs of common quails (Coturnix coturnix).

Molting methods
In this experiment, 240 common quails at the end of their production cycle (40 weeks old) were used.Three forced molting methods were used: wheat method (WM), zinc oxide method (ZM), and fasting method (FM).The control group (CG) was composed of quails that were not induced to molting and were offered a commercial laying feed (Table 01) and water ad libitum during the entire experimental period.In Molting was induced in WM and ZM quails by feeding wheat midds and a laying feed containing 25000 ppm zinc oxide, respectively.FM birds were submitted to complete feed fasting until the end of the treatment.Independently of treatment group, all birds were offered water ad libitum.Birds were submitted to the treatments until the body weight loss (BWL) specific for each method was achieved, as follows: WM (25% BWL), and ZM and FM (30% BWL).

Microbiological evaluation and sample collection
The microbiological evaluation consisted in the investigation of the presence of Salmonella spp. in cloacal swabs, fresh fecal samples, and eggs two weeks before and two weeks after quails were submitted to forced molting.The same procedure was applied to investigate the presence of other Enterobacteriaceae in egg samples.Ten fecal samples and ten cloacal swabs (pool of three birds) were collected from each experimental group.Twenty egg samples, consisting of two eggs each, were evaluated.

Microbiological procedures
Cloacal swabs and fresh fecal samples (1g) were collected, transported to the Ornithological Studies Lab and immediately processed.Eggshells were disinfected by immersion in ethanol at 70%, and then broken.Egg content was homogenized by agitation in glass flasks, and 1-mL aliquots were used as sample unit.Enterobacteria isolation and identification were performed according to the following steps: Pre-enrichment, selective enrichment, plating and presumptive identification, biochemical tests, serology, and typification.During the pre-enrichment phase, 10 mL buffered peptone water at 1% were added to the collected clocal swab, fecal, and egg samples.In selective enrichment, 0.1 mL and 1 mL aliquots of the pre-enriched culture were transferred to tubes containing 10 mL Rappaport-Vassiliadis broth and 10 mL selenite cystine broth, respectively.Samples were then transferred to selective liquid media, using a platinum loop, to plates containing the selectiveindicator media brilliant green agar and MacConkey agar to perform the presumptive identification.Colonies with morphological characteristics suggesting Salmonella spp.were inoculated in tubes with TSI (triple sugar iron) agar slant, LIA (lysine iron agar) slant, and SIM (sulfide-indole-motility) agar.Colonies were biochemically characterized by glucose and sucrose fermentation properties, gas production, H 2 S production, indole production, and motility.All tests were carried out at 37 ºC incubation temperature for 24 h.Colonies biochemically identified as Salmonella spp.were submitted to serology, using rapid plate seroagglutination tests with the use of flagellar (H) and somatic (O) antisera.
The absolute frequency of Enterobacteriaceae was determined using the plating results of isolates suspected of Salmonella.The isolation of bacteria not belonging to the family Enterobacteriaceae were not considered in the study.The Chi-square test at 5% significance level was used to compare the numbers of egg samples contaminated with Enterobacteriaceae among treatments before and after forced molting was implemented.

ReSultS And dISCuSSIon
The microbiological procedures did not detect the presence of Salmonella spp. in none of the egg, fecal, or cloacal swab samples collected from all evaluated experimental groups.Despite the absence of Salmonella determined in the present study, several other authors demonstrated that laying hens submitted to forced molting by the method of feed fasting are more susceptible to this microorganism (Holt & Porter, 1992;Macriet al., 1997;Berchieri Jr., 2000;Holt, 2003;Berry, 2003).This was clearly shown in the study of Holt et al. (1995).The authors induced molting in commercial layers using fasting and orally infected these birds after four days with SE (5-10 x 10 6 ).They observed 24 and 96 hours after the experimental infection significantly higher SE counts in the colon, ceca, and feces in the molted birds compared with the hens not submitted to forced molting.In the ileum, SE counts were higher only 48h after the experimental infection.Nakamura et al. (2004) observed that layers submitted to forced molting by fasting shed more Salmonella in the feces until day 14 post-infection than the control birds.As to Salmonella shedding in the eggs, Golden et al. (2008) observed that layers submitted to fasting may produce SE-contaminated eggs more frequently than fed layers; however, shedding declines with time.

Bacteriological Investigation of Microorganisms (Salmonella sp. and Other Enterobacteriaceae) in Common Quails (Coturnix coturnix) Submitted to Different Forced-Molting Procedures
Salmonella isolation has also been reported in layers submitted to molting using alternative methods.McReynolds et al. (2006) found that alternative molting methods usually result in low Salmonella incidence and significantly reduce total bacterial counts in the ceca and other internal organs in layers submitted to forced molting and challenged with Salmonella Enteritidis.Therefore, the authors stress that alternative forced molting methods do not completely eliminate the possible risk of Salmonella infection.
Most studies involving Salmonella isolation in laying chickens submitted to forced molting used an inoculation procedure to evaluate bird sensitivity to that microorganism.In the present study, quails were not inoculated with Salmonella, which may explain why its presence was not detected.However, some studies in literature show the isolation of Salmonella spp in layers submitted to forced molting using epidemiological investigation.Garber et al. (2003) surveyed farms in 15 US states using fecal matter swabs collected in layers houses with different environments.The researchers observed that layers submitted to forced molting had more probability of being positive for Salmonella enterica serotype Enteritidis than contemporary layers not submitted to molting.Souza et al. (2002) performed an epidemiological survey on the incidence of Salmonella in layers submitted to forced molting and isolated Salmonella Give from cloacal and fecal samples before and 30 days after fasting.The authors detected the presence of that microorganism in samples of meat meal and feed supplied to the layers, and argued that the finding of the same serotypes in the birds indicated the intake of contaminated feed.Therefore, the presence or absence of Salmonella spp. in the environment where molting is induced may determine the spread of these bacteria.According to Hinton et al. (2000), fasting causes physical, chemical and microbiological changes in the bird's crop, and these changes may impair the natural capacity to inhibit the local colonization by Enterobacteriacea.
Table 2 shows the presumptive results relative to Enterobacteriacea isolated in quails submitted to forced molting by different methods.
There was no significant difference (p> 0.05) among treatments as to absolute frequencies of samples positive for Enterobacteriaceae before and after forced molting.The highest absolute frequency in contaminated egg samples was observed in the WM group post-molting (25%), whereas the lowest frequency was 10% (n=2) and it was observed in the CG and WM group before molting.The presumptive results found in the present study relative to absolute numbers and percentages of eggs contaminated with enterobacteria are similar or much lower than those reported in literature.This is demonstrated in the study of Stepien-Pyśniak (2010).The author qualitatively analyzed Gramnegative bacteria, particularly those belonging to Enterobacteriaceae family in the eggshell, yolk, and albumen of fresh eggs produced in large and small farms, and found that Escherichia coli, Enterobacter, Citrobacter, and Klebsiella were isolated more often in the eggshell, whereas in the albumen and in the yolk, Escherichia coli was more common.The least frequently isolated bacteria were Proteus and Serratia.Considering the total percentage of enterobacteria, Adesiyun et al. (2006) evaluated fresh eggs from Trinidad farms and found that 28.3% of the eggshells and 15.2% of the egg content were contaminated.Therefore, total egg contamination was higher than those found in quail eggs before or after forced molting.There are few studies on Enterobacteriaceae in quail eggs.However, in the metropolitan area of Fortaleza, CE, Brazil, Siqueira et al. (2008) carried out a study on the contamination of quail eggs by Enterobacteriaceae.The researchers found a significant contamination rate (45.59%) in quail eggs collected in supermarkets -Enterobacter and Shigella were the most frequent agents -and attributed these results to possible hygiene issues along the quail egg production chain.Therefore, the lowest contamination rate found in quail eggs before or after forced molting may be explained by the fact that the eggs were evaluated immediately after lay, and not after processing and preservation steps in a supermarket shelf.

ConClusions
Under the conditions of the present experiment, no Salmonella spp. was detected in cloacal swab, fecal or egg samples neither before nor after molting induced by different methods (feed fasting, feed based on wheat midds, or feed with high zinc oxide level).Therefore, it was not possible to determine if forced molting had any influence on the presence of that pathogen.Further studies are required involving previously infected quails to understand this relationship better.
The results demonstrated that the amount of quail eggs contaminated by bacteria belonging to the family Enterobacteriaceae was low before and after molting, and that this was not influenced by the evaluated forced molting methods.

Table 1 -
Ingredient composition and nutritional levels of the feed supplied to the experimental quails.

Cardoso WM2, Lopes ES, Rocha-e-Silva RC, Albuquerque AH, Horn RV, Salles RPR Bacteriological Investigation of Microorganisms (Salmonella sp. and Other Enterobacteriaceae) in Common Quails (Coturnix coturnix) Submitted to Different Forced-Molting Procedures total
, 60 birds were designated to each experimental group (WM, ZM, FM, CG).Birds were weighed and housed in conventional pyramid-type cages (64 birds/ m 2 ) belonging to the experimental houses of the School of Veterinary Medicine of the State University of Ceará, Brazil.

Table 2 -
Frequency of Enterobacteriaceae isolated from the eggs of common quails submitted to forced molting by different methods