Experimental Infection of Salmonella Enteritidis in Quails Submitted to Forced Molting by Feed Fasting

I Undergraduate of the Veterinary Medicine School, State University of Ceará (UECE), Brazil II Post-doctoral Student /CNPq/UECE III Doctoral Student of the Postgraduate Program in Veterinary Science, UECE / Fortaleza. IV BIOLAB Laboratory S/A Ltd V Master’s degree student of the Postgraduate Program in Veterinary Science, UECE / Fortaleza. VI Professor of the Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid. VII Professor of the Postgraduate Program in Veterinary Science, UECE / Fortaleza.


INTRODUCTION
The practice of forced molting to extend the longevity of laying hens has been widely studied and several methods have showed effective results in term of egg quality and quantity (Berry, 2003;Molino et al., 2009;Aygun, 2013).However, there a few studies with quails.Some recent papers have been published evaluated the effect of forced molting on production, reproduction, and health parameters (Garcia et al., 2001, Teixeira et al., 2007, Faitarone et al., 2008, Arora & Vatsalyia, 2011;Aljumaily et al., 2012, Teixeira et al., 2013).Interestingly, despite the close relation between molting and salmonellosis, this issue has not been explored yet.
The traditional forced-molting method involves submitting laying hens to several days of feed fasting, which debilitates birds and makes them susceptible to the infection by pathogens.The stress caused by fasting increases corticosterone plasma levels (Webster, 2003), and stress hormones are known to have anti-inflammatory properties, which reduce the effectiveness of the immune system in fighting diseases (Golden et al., 2008).Thus, birds with compromised immune system become susceptible to microorganisms, such as Salmonella Enteritidis (Holt, 2003), and may produce contaminated eggs (Gama et al., 2003), compromising public health.
Public health problems are still a concern in many countries that apply feed fasting as a forced-molting method.Brazil is a major egg producer, and there are few studies on the microbiological status of eggs produced by hens submitted to forced molting (Teixeira & Cardoso, 2011), particularly of quail eggs.Therefore, this study aimed at evaluating bacterial counts in cloacal swabs, feces, and eggs of quails submitted to forced molting by fasting and experimentally infected with Salmonella Enteritidis strains.

Birds
In total, 82 quails at the end of egg-production cycle (40 weeks of age) were used in this experiment.Quails were individually weighed, identified, and housed in battery cages (25 x 25 x 20 cm; three birds per cage) located at the experimental facilities of the Laboratory of Ornithological Studies, State University of Ceará, Fortaleza, Brazil.

Bacteriological monitoring before inoculation
Three days before the trial started, all birds were examined to ensure they were free of Salmonella spp.Birds were analyzed according to methodology of Zancan et al. (2000) with modifications.Fresh feces and cloacal swabs were collected per cage (pool of three birds) using sterile material.Samples were placed in tubes containing 2 mL selenite-cystine broth with novobiocin addition (40μg/mL) and incubated at 37° C in a bacteriological incubator for 24h.Samples were then plated on brilliant green agar and incubated under the conditions described above, after which the growth of colonies with morphological characteristics of Salmonella was observed in the plates.

Experimental groups
Each experimental unit consisted of a single cage, housing three quails.Quails were distributed into four treatments with seven replicates each, as follows: FI (group of quails induced to molt by fasting and inoculated with Salmonella Enteritidis -SE); CI (control group: quails fed layer feed and inoculated with SE); FNI (group of quails induced to molt by fasting and not inoculated with SE); CNI (quails fed layer feed and not inoculated with SE).

Induced molting method
Molting was induced by total deprivation of feed.Water was supplied ad libitum.The first day of feed deprivation was considered day zero (D0).Each experimental unit was daily weighed and when mean body weight loss 30% was obtained, birds were fed a layer commercial feed ad libitum.

Inoculum preparation
A strain of Salmonella Enteritidis (SE) resistant to nalidixic acid (SENal r ) isolated from chickens (Gallus gallus) was used in this study.The inoculum was prepared according to Berchieri et al. (2001) with modifications as follows: the bacterial culture was prepared in 5 mL buffered peptone water at 0.1% and incubated in a bacteriological incubator at 37 o C for 24 h.After incubation, serial dilutions were performed to determine the number of colony forming units (CFU) (Miles et al., 1938).The inoculum contained 10⁷ CFU of Salmonella Enteritidis Nal r /mL.

Inoculation
One day after the beginning of forced molting, FI and CI quails received 0.1 mL of the inoculum directly in the crop by gavage, using a probe coupled to a 1cc syringe.

Cloacal swab samples
On 1, 4, 7, and 14 days post-inoculation (dpi), cloacal swabs were collected (pool of three birds) per experimental unit, comprising 28 samples.Processing was carried out according to methodology of Zancan et al. (2000) with modifications, as follows: the cloacal material was collected from all birds with the aid of sterile swabs moistened in selenite-cystine broth with novobiocin addition (40μg/mL).Immediately after collection, samples were plated on brilliant green agar containing nalidixic acid (100μg/mL).Then, both broth and plates were incubated at 37°C in a bacteriological incubator for 24h, after which the growth of colonies with morphological characteristics of Salmonella was observed.An aliquot from each selenite-cystine tube from samples which plates were negative was seeded on brilliant green agar plates containing nalidixic acid (100μg/mL) and placed in a bacteriological incubator for 24h at 37°C.Samples were considered negative for Salmonella spp only after the second plating.

Feces samples
Aluminum trays were placed under all cages to collect fresh feces on 1, 4, 7, and 14 dpi.Fecal samples (0.5 g) were placed in tubes containing 5 mL peptone water, and serially diluted for subsequent CFU counting on plates containing brilliant green agar with nalidixic acid addition (100μg/mL) (Miles et al., 1938).Plates and fecal samples cultured in peptone water were incubated in a bacteriological incubator at 37°C for 24h.Negative samples after incubation were transferred to tubes containing 2 mL of selenite-cystine broth with novobiocin (40 uL) and, after 24 h at 37 o C , were plated on brilliant green agar containing nalidixic acid (100μg/mL).

Egg samples
Eggs from birds submitted to forced molting (FI and FNI) were collected for microbiological analysis during the first six days of the week and of the week they returned to lay (two weeks post-molting).The eggs produced by quails that were not submitted to molting were also analyzed according to the same selection procedure and sampling period.
Eggs were first immersed in a 70% alcohol solution for shell disinfection and processed according to Jahantigh & Nili (2010), with modifications, as follows: eggs were macerated and homogenized using spatula and sterile beaker.Samples were subsequently incubated in a bacteriological incubator at 37 o C for 24 h, after which they were transferred using sterile swabs to tubes containing 2 mL selenite-cystine broth supplemented with novobiocin (40μg/mL) and incubated at 37 o C for 24 h.Samples were then plated on brilliant green agar containing nalidixic acid (100μg/mL) and placed in a bacteriological incubator at the same aforementioned temperature and for the same time, and then observed for the presence of Salmonella Enteritidis.
Fecal microorganism count data were tested for normality by Shapiro-Wilk and Kolmogorov-Smirnov tests.Treatment means were compared by the nonparametric Mann-Whitney test (5%).Statistical analyzes were performed using the program ASSISTAT, beta version 7.6 (Silva et al., 2010).

RESULTS
All cloacal swab samples were negative for SE.However, 57.10% of the fecal samples of the quails submitted to forced molting were positive on 1 dpi.The CI group showed a higher number of positive birds (37.5%) on day 4 of observation (Figure 01).On day 7, none of the CI group fecal samples was tested positive, while a sample of the FI group (14.3%) still shed SE in the feces.After 14 days, SE shedding was not observed in none of the groups.None of fecal samples of the quails of the noninoculated groups (FNI and CNI) were positive for SE and therefore, no statistical comparison was performed.On the other hand, FI birds presented significantly higher (p<0.05)number of bacterial cells (SE) in the feces than CI group birds on 1 dpi.On 4 dpi, no significant difference was observed (p<0.05) in bacterial shedding between the FI and the CI groups.On 7 dpi, the FI group was the only one that shed the pathogen in the feces, and on 14 dpi, no group shed Salmonella (Figure 02).Quails submitted to forced molting completely ceased egg laying, which resumed only two weeks after feed supply.Microbiological analysis of all collected egg samples were negative for SE.

DISCUSSION
The negative SE results obtained in the microbiological examination of the cloacal swabs of infected quails are different from the findings of Barrow & Lovell (1991) obtained with laying hens orally infected with

Experimental Infection of Salmonella Enteritidis in
Quails Submitted to Forced Molting by Feed Fasting a higher concentration of SE (3x10 8 CFU / mL).Those authors observed that 100% of the cloacal swabs were positive in the first week post-inoculation, and that 12.5% of the evaluated birds remained positive in the fifth week.However, García et al. (2011) suggested that cloacal swabs are less efficient to detect SE infection in laying hens compared with fecal samples because in that study, while fecal samples were 92% positive for SE, only 4% of cloacal swabs were positive.
The high number of SE-negative cloacal swab samples may be associated with the sensitivity of this procedure for the identification of Salmonella sp.Some researchers have questioned the effectiveness of swabs for the detection of Salmonella in animals (Aho, 1992;Weiss et al., 2002).The intermittent shedding of Salmonella, associated with the fact that it can be shed in small amounts in the feces may impair detection, and therefore, cloacal swabbing presents limitations as a method to monitor Salmonella shedding (Andrade et al., 2007).Pereira et al. (2007) studied the presence of Salmonella in Greater Rhea (Rhea americana) and concluded that the use of cloacal swabs for the detection of birds infected with Salmonella has low sensitivity and therefore, the risk of indicating false negative results is high.This may explain why there were no positive cloacal swab samples obtained from inoculated quails in the present study, differently from the fecal samples.Higgins (1982), working with broiler chickens, asserted that the sensitivity of Salmonella detection in fresh feces is greater when compared with cloacal swabs.
Concerning the microbiological analyses of the feces, detected the presence of the inoculated pathogen was detected.A significantly higher (p<0.05)shedding of SE in the FI group on 1 and 7 dpi was recorded.However, on 4 dpi, no significant difference (p>0.05) in bacterial shedding was observed between the inoculated groups considering the period in which bacterial shedding in feces was observed.This shows that fasting affects bacterial shedding in birds submitted to induced molting.The results of the present study are consistent with the findings of Nakamura et al. (2004), who worked with laying chickens submitted to induce molting by fasting (Body weight loss of 30% body weight loss) and orally inoculated with Salmonella Enteritidis (2.4 x 10 9 of the SE).These authors verified that the pathogen was eliminated in the feces during nine observation moments post-inoculation until 42 dpi, after which bacterial shedding was no longer observed.In four of these moments, the group submitted to fasting presented significantly higher bacterial shedding when compared to the group of hens not inoculated.In the other five moments, no significant difference was observed between the groups.The longer period of SE shedding in hens in that study when compared towith the shedding period observed in the quails in this study may be explained by the higher bacterial concentration used to in the inoculate.
Molting induced by fasting may compromise the immune system of laying birds, making them more vulnerable to infections by various microorganisms (Holt, 2003).Onbaşılar & Erol (2007) asserted that fasting, because it causes severe physiological stress, is the least suitable method of molting in terms of bird welfare.Those authors compared fasting with other molting methods, such as the supply of zinc oxide or of barley grains in the feed, and found that the hens submitted to fasting presented higher corticosterone levels as well as impaired humoral immunity compared with other methods.The stress caused by fasting rises plasma corticosterone levels (Webster, 2003), which results in a decrease of circulating lymphocytes (Maerz & Davis, 2010), as well as of other leukocytes, and suppresses humoral immunity, weakening the resistance of birds to diseases (Mench et al., 1986).
As expected, non-inoculated quails submitted to molting by fasting were negative for Salmonella Enteritidis.This result shows that, although several studies in the scientific literature demonstrate that molting induced by fasting is closely associated with SE, the presence of this microorganism in the environment during the period of stress is essential for pathogen dissemination (Teixeira et al., 2013).
The finding of negative SE shedding in the eggs in the present study is different from the results of Golden et al. (2008), who reported that laying chickens submitted to the induced molting by fasting and orally inoculated with SE laid contaminated eggs during the first weeks post-molting.However, other studies show that hens orally inoculated with SE strains may present a low or absent prevalence of this bacterium in the eggs.Yang (1992) orally inoculated Japanese quails with SE (10 10 CFU) and found that out of 164 analyzed eggs, SE was detected in the albumen of seven eggs, in the yolk of 15 eggs, in the eggshell of 13 eggs, and in the shell membranes of 15 eggs, representing bacterial contamination of 10.9% of the eggs.In this case, the inoculum concentration was higher than that used in the quails of the present study, which may explain the absence of contaminated eggs.These results are Santos ICL, Teixeira RSC, Lopes ES, Albuquerque AH, Rocha e Silva RC, Salles RPR, Machado DN, Horn RV, Bezerra WGA, Buxade CC, Maciel WC Experimental Infection of Salmonella Enteritidis in Quails Submitted to Forced Molting by Feed Fasting

Figure 1 -
Figure 1 -Percentage of fecal samples positive for Salmonella Enteritidis obtained from quails induced to molting and experimentally infected.

Figure 2 -
Figure 2 -Fecal shedding of Salmonella Enteritidis in quails induced to molt and experimentally infected.*Significant differences among means on different days post-inoculation (p<0.05)