Recovery of Salmonella Gallinarum in the Organs of Experimentally-Inoculated Japanese Quails (Coturnix coturnix)

ABSTRACT

Salmonellosis is an infection caused by specific or non specific serotypes of theSalmonella genus, responsible for losses in the poultry industry. Fowl typhoid, caused by S. Gallinarum (SG) is important because it causes elevated mortality in adult birds, leading to economic losses in the poultry industry. This study aimed at quantifying the number of viable SG cells in the liver, spleen, lung, cecum, and reproductive tract (ovary and testicles) of experimentally inoculated Japanese quails (Coturnix coturnix), as well as SG shedding in their feces. One hundred and two Japanese quails, with four months of age at the beginning of the experiment, were used. The birds were inoculated with three bacterial cultures containing different concentrations (6x10⁴ CFU/0.1mL, 2x10⁵ CFU/0.4mL, or 5x10⁶ CFU/0.2mL) of SG resistant to nalidixic acid. On days 1, 4, 7, and 14 after the inoculation (dpi) individual cloacal swabs were collected from six birds per group, which were subsequently sacrificed for organ sampling. The swab samples were streaked directly on plates containing brilliant green agar and nalidixic acid (VBNal). Samples that were negative after 24h, were streaked again. The collected organs were individually macerated and transferred to buffered peptone water at 0.1%. The solutions were immediately diluted serially for CFU counting in VBNal. SG was successfully recovered from one quail, which was inoculated with 2x10⁵ CFU/0.4mL, and from five quails of the group inoculated with 5x10⁶ CFU/0.2mL inoculum. All of the analyzed cloacal swab samples were negative. Therefore, this study demonstrated it was difficult to isolate SG from the analyzed organs and that it was not possible to recover thepathogen in the cloacal swabs collected from inoculated quails. These results may be explained by the absence of flagella in SG, inducing weak intestinal immune response in the beginning of the infection and preventing its isolation in cloacal swab samples. The low positivity rate of the analyzed organs may be due to the immune status of the euthanized birds, since the SG dissemination in the animal organism occurs mostly close to death, which was observed in the birds found dead during the experiment.

Keywords:
Cloacal swabs; gallinaceous; organs; reisolation

INTRODUCTION

Salmonella Gallinarum is a pathogenic serotype ofSalmonella specific of birds (Setta et al. 2012),and it is characterized by its capacity to grow in systemic locations (Chadfield et al., 2003). This serotype has been isolated from several poultry species, such as chickens, turkeys, pheasants, ducks, guinea fowl, and quails (Shivaprasad, 2000).

Quails have been commercially reared both for egg and meat production (Schmid & Wechsler, 1997), and its production is currently expanding in Brazil. Some researchers have reported both the presence of Salmonella spp. (Bacci et al., 2012) and increasing severity of its infection in quails (Rocha e Silva et al., 2013).

Most avian salmonellosis infections are transmitted through the fecal-oral route. The small intestine is the main site of invasion, which occurs through the gut-associated lymphoid tissues (GALT) and Peyer's patches. The ceca are also colonized, although Salmonella Gallinarum and S. Pullorum are auxotrophic (Chappell et al., 2009).

The adhesion and/or colonization of the avian intestinal tract is initially associated with LPS (lipopolysaccharides), flagella, fimbriae, and some external membrane proteins present in Salmonella spp. (Berndt et al. , 2007). Several studies suggest that the presence of flagella is involved in the inflammatory response at the adhesion site and the absence of these structures may avoid the recognition of the pathogenic agent by the immune system. This contributes for the development of systemic infections (Chappell et al., 2009) due to the difficulty in mounting an adequate immune response (Lahiri et al., 2010), culminating in the dissemination of the agent in the environment or in high bird mortality.

The disease caused by SG (fowl typhoid) causes significant economic losses in poultry production (Proux et al., 2002). Due to the expansion of quail rearing in the last few years and the lack of studies on this species, the knowledge of the behavior of the infection of these birds by SG is essential. This study aimed at quantifying the number of viable SG cells in the liver, spleen, lung, cecum, and reproductive tract (ovary or testicle) of experimentally infected Japanese quails (Coturnix coturnix), as well as to evaluate its shedding in the feces.

MATERIAL AND METHODS

Birds

In total, 102 (51 female and 51 male) Japanese quails (Coturnix coturnix) were used. Birds were 16 weeks old at the beginning of the experiment, and were randomly assigned to three groups (G1, G2 and G3) with 32 birds each. Six birds were kept as controls. Two quails (one male and one female) were housed per cage (22x21x16cm) in batteries placed in a pyramid at the inoculation facilities of the Sector of Ornithological Studies of the State University of Ceará. Water and feed were provided ad libitum. The feed did not contain any antibiotics, and birds were not submitted to vaccination or internal parasite control. House temperature (25.5ºC) and photoperiod (16h light/day) were controlled. This project was approved by the local Ethics Committee for the Use of Animals under protocol number 10244779-9/26.

Bacteriological pre-inoculation monitoring

Before the beginning of the experiment, birds were tested for the presence ofSalmonella spp. in order to ensure that they were free from this pathogen. The procedure was performed according to Zancan et al. (2000), with modifications, as follows: individual cloacal swabs impregnated with selenite-cystine and novobiocin (40µg/mL) (SCNov) were directly streaked onto brilliant green agar plates containing nalidixic acid (100µg/mL) (VBNal) and incubated at37ºC for 24h. Negative samples for Salmonellaspp. were again plated in VBNal and incubated at 37ºC for 24h to confirm the absence of the pathogen in the birds.

The control quails were euthanized and their organs (liver, spleen, lung, cecum, and ovary or testicle) were collected for microbiological examination. The fragments of these organs were macerated, inoculated in 0.1% buffered peptone water, and incubated at 37ºC for 24h. A loopful of each material was then transferred to tubes containing SCNov and incubated at 37ºC for 24h. Subsequently, samples were streaked onto plates containing VBNal and incubated at 37ºC for 24h.

Inoculum preparation

Salmonella Gallinarum strain resistant to nalidixic acid (SGNal^r) isolated from chickens (Gallus gallus) was used to prepare the inoculum. This strain was donated by the Department of Veterinary Pathology of UNESP, Jaboticabal, Brazil. The inoculum was prepared according to Rocha e Silva et al. (2013) and three distinct concentrations of the inoculum (6x10⁴CFU/0.1mL, 2x10⁵CFU/0.4mL e 5x10⁶ CFU/0.2mL ofSalmonella Gallinarum Nal^r/mL) were obtained.

Salmonella Gallinarum inoculation

All birds received 0.1mL of the inoculum via gavage, directly in the crop, with the aid of a cannula linked to a 1mL syringe. Group 1 birds (G1) received 6x10⁴CFU/0.1mL, group 2 (G2) received 2x10⁵CFU/0.4mL, and group 3 (G3) received 5x10⁶ CFU/0.2mL.

Post-inoculation monitoring

During the experimental period, mortality and clinical signs were observed daily. Dead birds were submitted to necropsy for gross evaluation and their organs (liver, spleen, lung, cecum, and reproductive tract) were collected for microbiological analyses according to Rocha e Silva et al.(2013).

Fecal material was collected with the aid of sterile cloacal swabs impregnated in SCNov from euthanized birds on 1, 4, 7, and 14 days post-inoculation (dpi) and directly streaked onto VBNal. Both the broth and the plates were incubated in a bacteriological incubator at 37ºC for 24h, after which samples negative forSalmonella spp. were again plated onto VBNal.

On 1, 4, 7, and 14 dpi, six birds per group were randomly selected and euthanized by neck dislocation. At necropsy, the spleen, liver, lung, ceca, and reproductive tract (ovary ortesticle) were submitted to gross examination and any changes were duly recorded. Samples of the examined organs were aseptically collected for CFU counting. The microbiological procedure was performed according to Rocha e Silva et al. (2013).

Results

The microbiological analyses showed that the cloacal swabs from all quails, as well as organ samples (spleen, liver, lung, ceca and reproductive tract) of the birds euthanized before the beginning of the experiment were negative forSalmonella spp.

It was not possible to countCFU/mL in the organ samples because no colony growth was observed. However, SG was recovered from 4.16% (1/24)of G1 birds and 20.83% (5/24) of G2 and G3 birds on 4 dpi and 7 dpi (G3), respectively, after selective enrichment inselenite-cystine broth (Table 1). The only positive G2 quail on 4 dpi presented SG in liver and lung samples. In theG3 group, on 4 dpi, SG was successfully recovered from the liver, spleen, and lung of two quails, and from testicle of one bird. On 7 dpi, SG was isolated from the ovaries of three quails, and from the liver, spleen, and lung samples of one quail.

Table 1
Isolation of Salmonella Gallinarum fromthe organs of experimentally infected and euthanized Japanese quails(Coturnix coturnix)

• L: Liver; S: Spleen; C: Ceca; Lg: Lung; RT: Reproductive Tract

The pathogen was also isolated from several organs of birds that died during the experiment (Table 2). In G1, one bird presented SG in the liver, spleen, ceca, lung, and testicle, while the other positive bird presented SG in liver and ovary. In G2 birds found dead, all organs were positive (liver, spleen, ceca, lung, and reproductive tract), with the exception of a single bird that presented negative ceca and liver. All organs of the eight G3 birds that died during the experiment were positive for SG, except for four birds, which presented negative cecum samples.

Table 2
Isolation of Salmonella Gallinarum from the organs of experimentally infected Japanese quails (Coturnix coturnix) that were found dead during the 14 days of the experiment

• L: Liver; S: Spleen; C: ceca; Lg: Lung; RT: Reproductive Tract

Birds showed typical fowl typhoid clinical signs, including ruffled feathers, closed eyelids, diarrhea, apathy, typically remained quiet in a cage corner, and were frequently found dead after such signs were observed.

Bird mortality began on 5 dpi and was observed until 10 dpi. The main macroscopic changes observed in the birds found dead were hepatomegaly and splenomegaly, dilated gallbladder, and hemorrhages in the liver, intestines, and deformed ovarian follicles. On the other hand, the main findings in euthanized birds were hemorrhagic or icteric liver with or without petechiae, dilated gallbladder, splenomegaly, hemorrhagic or icteric spleen with or without petechiae, and hemorrhagic ovarian follicles (Table 3).

Birds that remained alive at the end of the experiment (24 dpi) were euthanized and cloacal swab and organ samples were collected. All samples were negative for SG.

DISCUSSION

The percentage of SG isolated from the organs of the euthanized quails was much lower when compared with the quails that naturally died during the experiment. This may be explained by the fact that the infectivity of that microorganism, which is enhanced by its high concentration in the tissues when the bird is near death (Buxton & Davies, 1963).

Salmonella spp. stimulates receptors in the intestinal cells known as toll-like receptor 5 (TLR5). Once activated, these receptors initiate the production of pro-inflammatory interleukins (IL-1, IL-6 and IL-8), which together with macrophages, heterophils, and natural killer cells (NK), are part of the innate immune response, an initial line of defense against the invasion ofSalmonella in the host's body. Innate immunity helps preventing the systemic infection and triggers the humoral and the cell-mediated acquired immune responses (Berchieri Júnior & Freitas Neto, 2009). However, the ineffective immune response caused by SG due to the low or absent IL-6 production, a pro-inflammatory cytokine, may have favored the penetration of the pathogen without any tissue damage and, in the absence of adequate immune response, the systemic infection developed without alerting the host (Kaiser et al., 2000).

Table 3
Macroscopic changes of the organs of quails experimentally inoculated with Salmonella Gallinarum

Although almost all organs of the quails that were found dead were positive for SG, the ceca was less frequently affected. Evaluating different Salmonella serotypes,Setta et al. (2012) found that S. Enteritidis and S. Infantis colonize the ceca more efficiently than SG and S. Pullorum, and Barrow et al. (1999) mentioned that it is difficult to isolate SG from the cecum because of its poor intestinal colonization, which explains the high mortality caused by this serotype in this study. SystemicSalmonella infections induce both cellular and humoral responses and the persistence of the bacteria in the gastrointestinal tract maintains a more prolonged immune response (Wigley et al., 2005). In chickens, the activity of macrophages may play an important role in systemic resistance to Salmonella (Wigleyet al. , 2002).

All cloacal swab samples analyzed were negative for SG, which was expected, because it is very difficult to isolate SG from cloacal swabs. According to Proux et al. (2002), this serotype does not seem to be shed in large numbers in the feces, thereby limiting environmental contamination and consequently reducing the risk of horizontal infection. Nevertheless, Berchieri et al. (2000) asserted that the transmission of infection among susceptible birds is essentially horizontal by the ingestion of feces and mucus containing SG or yet by cannibalism.

In the present study, SG shedding did not present a dose-response behavior relative to the three concentrations of the inoculum applied, as occurs with otherSalmonella trains. When challenged with S. Enteritidis, laying hens shed this pathogen for longer periods when high concentrations of inoculum were used, since oral exposure significantly affects important parameters of exposure and infection by Salmonella, such as the number of bacteria in internal tissues (Gast et al., 2011).

Several studies with chickens have reported some of the clinical signs that were observed in the present study, such as closed eyes (Freitas Neto et al., 2007), ruffled feathers and diarrhea (Alvarez et al., 2003), and apathy (Garcia et al., 2010). Also, some of the gross changes observed during necropsy were consistent with those found in chickens, including hepatomegaly and hemorrhagic ovarian follicles (Berchieri et al., 2000), as well as deformed and congested liver (Hossain & Islam, 2004).

CONCLUSION

Salmonella Gallinarum was not recovered from any of the evaluated organs of the experimentally infected quails. However, it was successfully quantified in birds that died of the disease during the experimental period. Therefore, we suggest that the immune system of the birds that survived may have cleared the infection, hindering the recovery of the pathogen in birds that did not die of the infection. It is difficult to recover SG in fecal samples of experimentally inoculated quails, which suggests that infected quails that do not present clinical signs do not contribute for the horizontal transmission of the pathogen via feces.

ACKNOWLEDGEMENTS

The authors are grateful to CAPES for the financial support and to the Laboratory of Ornithological Studies (LABEO/FAVET/UECE) for their support to this study

REFERENCES

• Alvarez MT, Ledesma N, Téllez G, Molinari JL, Tato P. Comparison of the immune responses against Salmonella enterica serovar Gallinarum infection between naked neck chickens and a commercial chicken line. Avian Pathology 2003;32(2):193-203.
• Bacci C, Boni E, Alpigiani I, Lanzoni E, Bonardi S, Brindani F. Phenotypic and genotypic features of antibiotic resistance in Salmonella enterica isolated from chicken meat and chicken and quail carcasses. International Journal of Food Microbiology 2012;160(1):16-23.
• Barrow PA, Lovell MA, Murphy CK, Page K. Salmonellainfection in a commercial line of ducks; Experimental studies on virulence, intestinal colonization and immune protection. Epidemiology and Infection 1999;123(1):121±132.
• Berchieri Jr A, Freitas Neto OC. Salmoneloses. In: Berchieri JR A, Silva EN, Di Fábio J, Sesti L, Zuanaze MAF, editors. Doença das aves. São Paulo: FACTA; 2009. p.435-454.
• Berchieri JrA, Oliveira GH, Pinheiro LAS, Barrow PA. ExperimentalSalmonella Gallinarum infection in light laying hen lines. Brazilian Journal of Microbiology 2000;31(1):50-52.
• Berchieri Jr A, Murphy A, Marston K, Barrow PA. Observations on the persistence and vertical transmission of Salmonella entericaserovars Gallinarum and Pullorum in chickens: effect of bacterial and host genetic background. Avian Pathology2001;30(3):221-231.
• Berndt A, Wilhelm A, Jugert C, Pieper J, Sachse K, Methner U. Chicken cecum immune response to Salmonella enterica serovars of different levels of invasiveness. Infection and Immunity 2007;75(12):5993-6007.
• Buxton A, Davies JM. Studies on immunity and pathogenesis of salmonellosis. II. Antibody production and accumulation of bacterial polysaccharide in the tissues of chickens infected withSalmonella Gallinarum. Immunology 1963;6(6):530-538.
• Chadfield MS, Brown DJ, Aabo S. Comparison of intestinal invasion and macrophage response of Salmonella Gallinarum and other host-adapted Salmonella enterica serovars in the avian host. Veterinary Microbiology 2003;92(1-2):49-64.
• Chappell L, Kaiser P, Barrow P, Jones MA, Johnston C, Wigley P. The immunobiology of avian systemic salmonellosis. Veterinary Immunology and Immunopathology 2009;128(1-3):53-59.
• Freitas Neto OC, Arroyave W, Alessi AC, Fagliari JJ, Berchieri Jr A. Infection of Commercial Laying Hens with Salmonella Gallinarum: Clinical, Anatomopathological and Haematological Studies. Brazilian Journal of Poultry Science 2007;9(2):133-141.
• Garcia KO, Santana AM, Freitas Neto OC, Simplício KMMG, Alessi AC, Berchieri Jr A, Fagliari JJ. Experimental infection of commercial layers using aSalmonella enterica sorovar Gallinarum strain: blood serum components and histopathological changes. Brazilian Journal of Veterinary Pathology 2010;3(2):111-117.
• Gast RK, Guraya R, Guard J, Holt PS. Frequency and magnitude of internal organ colonization following exposure of laying hens to different oral doses of Salmonella Enteritidis. International Journal of Poultry Science 2011;10(4):325-331.
• Hossain MA, Islam MA. Seroprevalence e mortalityin chicken causad by pullorum disease and fowl typhoidin certain government poultry farm in Bangladesh. Bangladesh Journal of Veterinary Medicine 2004;2(2):103-106.
• Kaiser P, Rothwell L, Galyov EE, Barrow PA, Burnside J, Wigley P. Differential cytokine expression in avian cells in response to invasion bySalmonella Typhimurium, SalmonellaEnteritidis and Salmonella Gallinarum. Microbiology 2000;146(12):3217-3226.
• Lahiri A, Lahiri A, Iyer N, Das P, Chakravortty D. Visiting the cell biology of Salmonella infection. Microbes and Infection 2010;12(11):809-818.
• Proux K, Humbert F, Jouy E, Houdayer C, Lalande F, Oger A, Salvat G. Improvements required for the detection of Salmonella Pullorum and Gallinarum. The Canadian Journal of Veterinary Research 2002;66(3):151-157.
• Rocha e Silva RC, Cardoso WM, Teixeira RSC, Albuquerque ÁH, Vasconcelos RH, Cavalcanti CM, Lopes ES, Gomes Filho VJR.Salmonella Gallinarum virulence in experimentally-infected Japanese quails (Coturnix japonica). Brazilian Journal of Poultry Science2013;15(1):233-238.
• Setta AM, Barrow PA, Kaiser P, Jones MA. Early immune dynamics following infection with Salmonella enterica serovars Enteritidis, Infantis, Pullorum and Gallinarum: cytokine and chemokine gene expression profile and cellular changes of chicken cecal tonsils. Comparative, Immunology Microbiology and Infectious Diseases 2012;35(5):397-410.
• Shivaprasad HI. Fowl typhoid and pullorum disease. Office International des Epizooties, Scientific and Technical Review 2000;19(2):405-424
• Schmid I, Wechsler B. Behaviour of Japanese quail (Coturnixjaponica) kept in semi-natural aviaries. Applied Animal Behaviour Science 1997;55(1-2):103-112.
• Wigley P, Hulme SD, Bumstead N, Barrow PA. In vivo and in vitro studies of genetic resistance to systemic salmonellosis in the chicken encoded by the SAL1 locus. Microbes Infection 2002;4(11):1111-1120.
• Wigley P, Hulme S, Powers C, Beal R, Smith A, Barrow P. Oral infection with the Salmonella enterica serovar Gallinarum 9R attenuated live vaccine as a model to characterise immunity to fowl typhoid in the chicken. BMC Veterinary Research 2005;1(2):1-6.
• Zancan FT, Berchieri J A, Fernandes AS, Gama NMSQ.Salmonella investigation in transport boxes of day-old birds. Brazilian Journal of Microbiology2000;31(3):230-232.

Publication Dates

• Publication in this collection
  Sept 2015

History

• Received
  Oct 2014
• Accepted
  Mar 2015