EVALUATION OF Salmonella pullorum SHEDDING IN JAPANESE QUAILS ( Coturnix coturnix )

Salmonella Pullorum is a pathogen that infects several species of birds, mainly offspring, and among them, quails. Therefore, this study was performed in order to evaluate the S. Pullorum shedding in experimentally infected Japanese quails (Coturnix coturnix) and egg contamination ability. S. Pullorum ATCC lyophilized was inoculated in all birds (60 quails at 160 days). Before the inoculation, rapid slide agglutination test was performed in all birds and individual cloacal swabs were collected randomly. Birds from group A and group B received 0.5mL of inoculum containing 1.5x102 CFU and 7.5 x 108 CFU of S. Pullorum, respectively, directly in the crop. The cloacal swab samples were collected on 3, 6, 9, 16 and 23 days post-inoculation (dpi) and all eggs were collected. Positive cloacal swab samples were initiated at the 3rd dpi. A total of 30 pool of eggs was analyzed, of which 73% positive for S. Pullorum until the 16th dpi. In conclusion, adult Japanese quails are possibly more resistant to the infection by S. Pullorum, but they can shed this pathogen in the environment as well.


Introduction
Salmonella causes diseases that affect the poultry industry.Among them, Pullorum disease, caused by Salmonella enterica subsp.enterica serovar Pullorum; fowl typhoid, caused by S. Gallinarum; and fowl paratyphoid, caused by any remaining serotype of Salmonella (1) .
Salmonella Pullorum causes a disseminated systemic infection in birds (2) , mainly young birds (3) .Pullorum disease stands out for its high economic cost to poultry production and, despite the low risk of infection, the surveillance program for S. Pullorum should be reliable (4) .
S. Pullorum can be vertically transmitted to chicks due to the location in the reproductive tract.The bacteria may also persist in the reproductive system and spleen of chickens for more than 40 weeks post-infection and can further expand horizontally in hatchery facilities (3) .
According to Shivaprasad (5) , several bird species are susceptible to pullorum disease, such as quails, pheasants, ducks, among others.Only a few differences in the susceptibility between them have been reported (6) .Mortality rates are very similar to that of chickens and turkeys (6) .
As the quail production ascends in the poultry market, both egg and meat businesses, special care to the specific health conditions concerning the National Program of Poultry Health (PNSA) becomes essential.Thus, this study was performed to evaluate the S. Pullorum shedding in Japanese quails (Coturnix coturnix) experimentally infected, and to assess the egg contamination.

Material and Methods
We used a strain of Salmonella enterica subsp.enterica serovar Pullorum ATCC lyophilized and stored at -20 °C, acquired from the Oswaldo Cruz Institute/RJ.The S. Pullorum was prepared in saline solution at 0.85%, then plated on nutrient agar and incubated at 37ºC/24h for preparation of the inoculum.
A single colony was removed from the nutrient agar and transferred to a test tube containing 10 mL of tryptic soy broth (Acumedia Manufacture, Inc. Lansing, Michigan, USA), which was incubated at 37 °C for 24 hours.After this period, 0.1 mL of this suspension was subjected to eight serial dilutions (10 -1 to 10 -8 ) containing 10 mL of tryptic soy broth.Of each tube, 0.1mL was plated on brilliant green agar (BGA) (Oxoid Ltd., Basingstoke, Hampshire, England) containing 20 μg/mL of novobiocin (NOV) (Sigma Chemical Co., St. Louis, MO, USA) and spread with Drigalski handle.These plates were incubated in a bacteriological incubator at 37 °C/24h and, after this period, the Colony Forming Units (CFU) were quantified and the concentration of each tube determined.Given the CFU's, two inocula were prepared with different concentrations (1.5 x10 2 UFC and 7.5 x10 8 UFC) of Salmonella Pullorum.
A total of 60 quails (Coturnix coturnix) at 160 days were used.The birds were housed in the experimental facilities of the Laboratory of Ornithological Studies (LABEO), State University of Ceará (UECE).Birds were divided into two groups (A and B) in cages measuring 20 x 15 x 15 cm, with four quails per cage.Birds received feed and natural mineral water ad libitum throughout the experiment.The local ethics committee approved this study under the registry number: 08627875-4.
Rapid Slide Agglutination Test (RSAT) was performed in all birds and 21 individual cloacal swabs were collected from randomly selected birds for bacteriological examination three weeks prior to ROCHA-E-SILVA, R.C. et al.

inoculation.
Birds in group A and group B were inoculated with 0.5 mL of inoculum containing 1.5 x 10 2 CFU and 7.5 x 10 8 CFU, respectively, orally through a 500 μL micropipette attached to an urethral tube.
On the 3 rd , 6 th , 9 th , 16 th , and 23 rd post-inoculation day, individual cloacal swabs were performed on all birds, totaling 300 samples, and eggs were collected aseptically with procedure gloves.Each pool of ten eggs corresponded to a sample, totaling 30 samples throughout the experiment.
The bacteriological procedure was performed according to the methodology adopted by Pinheiro et al. (7) , as follows: swab samples were transferred to tubes containing 9 mL of selenite-cystine broth (EM Science, Merck, Darmstadt, Germany) and incubated at 37 ºC/24h.After this period, they were plated on brilliant green agar with Novobiocin (20 µg / mL) (AVB-Nov) and again returned to the bacteriological incubator for 24 hours at 37 °C.Triple sugar iron agar slants (EM Science, Merck, Darmstadt, Germany), lysine iron agar slants (Oxoid Ltd., Basingstoke, Hampshire, Engalnd), and semi-solid SIM medium (sulfide, indole, and motility) (Oxoid Ltd., Basingstoke, Hampshire, England) were used for biochemical identification.Subsequently, the samples were confirmed by serological tests with antiserum somatic "O" and flagellar "H" (Difco Laboratories, Becton Dickinson Microbiology Systems, Sparks, Maryland, USA).
From each egg sample, composed by a pool of 10 eggs (eggshell and egg internal content) crushed in a sterile beaker, an aliquot of 1 mL was transferred to selenite-cystine broth (Oxoid Ltd., Basingstoke, Hampshire, England) and microbiological procedures were performed as described previously.
The frequency data for Salmonella isolation on swabs were compared by chi-square, and egg samples by Fisher's exact test.All data were subjected to a significance level of 5%.

Results
The rapid slide agglutination test was negative in all birds.There was no isolation of Salmonella spp.from cloacal swabs of any birds sampled before the start and no mortality throughout the experiment.
Group A showed significant difference in isolation on the 6 th , 9 th , and 16 th dpi with the highest number of positive samples compared to 3 rd and 23 rd dpi.However, group B showed no significant difference between days post-inoculation.
The frequency of positive samples isolated from cloacal swabs was high on the 3 rd dpi, and the isolation rate was statistically higher in group B when compared to group A (Table 1).On the 6 th , 9 th , 16 th , and 23 rd dpi there was no significant difference between the groups.
Thirty egg samples were analyzed.Of these, 22 were positive for S. Pullorum until the 16 th dpi.There was no significant difference between the groups because all the samples were positive on the 3 rd and 6 th dpi.A 50% decrease of positive egg samples on the 9 th dpi was observed, while on the 23 rd dpi all the samples were negative (Figure 1).

Discussion
No inoculated birds showed clinical symptoms, unlike the results obtained by Buchholz & Fairbrother (6) , in which the bobwhite quails (Colinus virginianus) presented difficulty breathing, head drawn down, eyes closed, drooping wings, anorexia, weight loss and profuse white diarrhea after four days of infection.Pinheiro et al. (7) observed brown chickens inoculated with S. Pullorum showed clinical signs characterized by drowsiness, weakness, loss of appetite, and drooping wings after four weeks, but there was no mortality and all birds recovered completely, while white chickens showed no symptoms.This demonstrates some birds have greater resistance to infection by Salmonella Pullorum and asymptomatic carriers can spread to other birds.
According to Calenge et al. (8) , commercial varieties of chicken are very homogeneous concerning production, but they are not homogeneous for disease resistance; therefore, resistance to salmonellosis and carrier state varies with strain and age.The resistance or susceptibility of carrier status in both age groups (young and adult) and in all organs varies according to the genotypic characteristics of the bird (9) .
No mortality was observed in the groups inoculated with Salmonella Pullorum throughout this experiment, in agreement with the study by Berchieri Jr. et al. (10) , who used the White Leghorn chicken strain, and observed no mortality on birds inoculated with 10 9 CFU's of S. Pullorum during the experiment.Buchholz & Fairbrother (6) reported high mortality on 10-day-old quails infected by S. Pullorum with the concentration of 10 3 and 10 10 , reaching at the end of the experiment (14 days) 75% mortality.Pourciau & Springer (11) also reported 50% mortality on bob white quails at three weeks of age inoculated with paratyphoid salmonellae (1x10 7 and 5x10 6 ).The birds used in this experiment were not young, which may have contributed for a reduction in mortality, since the disease affects more severely young birds.
In this experiment, higher shedding of S. Pullorum was observed in cloacal swabs from the 3 rd to the 9 th dpi in both groups.Buchholz & Fairbrother (6) also isolated it from cloacal swabs on the first few days post-infection (6 th dpi) from bobwhite quails inoculated with S. Pullorum.Pourciau & Springer (11) found positive quails inoculated with S. Braenderup at 53 days post-inoculation.Wigley et al. (3) observed that chickens inoculated with S. Pullorum had negative cloacal swabs.Berchieri Jr. et al. (10) showed isolation of S. Pullorum in laying hens from four days post-inoculation until the end of the experiment (21 weeks of age).The presence of Salmonella in fecal samples of challenged chickens may be dose dependent.According to Ishola (12) , birds that received the largest inoculum of S. Enteritidis (1.3x10 8 CFU's/mL) were able to excrete the bacteria for longer when compared to birds that received lower inoculum (1.3x10 4 CFU's/mL), demonstrating that the oral dose has significant effects on important parameters in Salmonella infection (13) .Contaminated eggs were found until the 9 th dpi, which differs from the results found by Berchieri Jr. et al. (14) , where 4-days-old birds inoculated with S. Pullorum persisted shedding in eggs until 21 weeks of age.According to Jones et al. (15) , the bird age may affect the integrity of the eggshell, reducing the quality and contributing to the microorganism penetration into the egg.
The infection affected the production of eggs.At the beginning of this experiment, the production was approximately 80%, and suffered a reduction of 40%.Barrow & Lovell (16) observed that hens at 40 weeks of age inoculated intraperitoneally with S. Enteritidis, also lowered the production by 10.5% within two weeks after infection, whereas oral infection in chickens at 24 weeks of age had no effect on egg production.
In a study performed by Wigley et al. (3) with a total of 1,243 eggs of hens experimentally infected with S. Pullorum resulted in 81 positive eggs (6.5%).In addition, the pathogen was present in follicles, developing eggs and ovarian tissue, which indicates that vertical transmission occurred through the infection of eggs in development.Salmonella can penetrate through the eggshell and associated membranes leading to contamination of chicks in the hatchery (17) , being the contamination of eggs of unvaccinated birds quite high during the three weeks after oral inoculation of S. Pullorum (18) .Pinheiro et al. (7) reported a reduced number of infected eggs (1%) from chickens inoculated with 1.2x10 8 CFU's / mL of S. Pullorum for nine months.Berchieri Jr. et al. (14) noted that for six weeks following the experimental infection with 10 6 CFU's of S. Enteritidis to commercial strain chickens, 360 eggs were tested and none was positive, with low isolation from the liver, spleen, caeca, and ovarian interstitial tissue.After the invasion and colonization of the ovary and oviduct tissues, it is possible that species of the genus Salmonella are incorporated to the internal components of the egg during the formation, causing the production of contaminated eggs (19) .

Conclusion
Given the results of this study, we suggest that adult Japanese quails are more resistant to infection by S. Pullorum than commercial hens.In addition, they may function as asymptomatic carriers, eliminating the bacteria in the environment through the feces, infecting other birds, as well as their own progeny, through eggs.Therefore, establishing strict programs of surveillance, control and eradication of S. Pullorum in quails is of great importance to prevent the spread and contamination of products.