Salmonella Enteritidis Infection Research Articles

Temporal changes in the expression of avian β-defensins in the chicken vagina during sexual maturation and Salmonella infection

Avian β-defensins (AvβDs) constitute a family of antimicrobial peptides that are critical to innate immunity in chickens, providing protection against microbial pathogens including Salmonella Enteritidis (SE). As apart from the digestive tract another main route of SE colonization in birds is via infection of the oviduct and specifically of the vagina, the aim of this study was to investigate the expression of the complete family of AvβDs, in the chicken vagina in vivo, to determine whether sexual maturation affects their mRNA abundance and to investigate whether SE infection alters the vaginal AvβDs expression. Expression analysis revealed that 11 members of the AvβD family were expressed in the chicken vagina. Quantitative real-time PCR analysis revealed that the mRNA abundance of five AvβDs was up regulated and of one AvβD was down regulated with respect to sexual maturation. In addition SE infection resulted in a significant induction of AvβD5, 7, 10, 11, 12 and 14 in the vagina of sexually mature birds, and in a significant induction of AvβD5 and 11 in the vagina of aged birds. These findings provide strong evidence to suggest that an AvβD-mediated immune response mechanism exists in the chicken vagina providing protection against bacterial pathogens including Salmonella species.

Evaluating risk factors for endemic human Salmonella Enteritidis infections with different phage types in Ontario, Canada using multinomial logistic regression and a case-case study approach

BackgroundIdentifying risk factors for Salmonella Enteritidis (SE) infections in Ontario will assist public health authorities to design effective control and prevention programs to reduce the burden of SE infections. Our research objective was to identify risk factors for acquiring SE infections with various phage types (PT) in Ontario, Canada. We hypothesized that certain PTs (e.g., PT8 and PT13a) have specific risk factors for infection.MethodsOur study included endemic SE cases with various PTs whose isolates were submitted to the Public Health Laboratory-Toronto from January 20th to August 12th, 2011. Cases were interviewed using a standardized questionnaire that included questions pertaining to demographics, travel history, clinical symptoms, contact with animals, and food exposures. A multinomial logistic regression method using the Generalized Linear Latent and Mixed Model procedure and a case-case study design were used to identify risk factors for acquiring SE infections with various PTs in Ontario, Canada. In the multinomial logistic regression model, the outcome variable had three categories representing human infections caused by SE PT8, PT13a, and all other SE PTs (i.e., non-PT8/non-PT13a) as a referent category to which the other two categories were compared.ResultsIn the multivariable model, SE PT8 was positively associated with contact with dogs (OR=2.17, 95% CI 1.01-4.68) and negatively associated with pepper consumption (OR=0.35, 95% CI 0.13-0.94), after adjusting for age categories and gender, and using exposure periods and health regions as random effects to account for clustering.ConclusionsOur study findings offer interesting hypotheses about the role of phage type-specific risk factors. Multinomial logistic regression analysis and the case-case study approach are novel methodologies to evaluate associations among SE infections with different PTs and various risk factors.

Use of bacteriophage for biological control of Salmonella Enteritidis infection in chicken

Bacteriophage ΦCJ07 with broad host ranges for Salmonella strains isolated from sewage effluent were used to reduce Salmonella Enteritidis (SE) infection in chickens. One-day-old chicks challenged with 5×107 colony-forming units/bird of SE were cohabitated with contact chicks and treated with three concentrations (105, 107 and 109 plaque forming units (PFU)/g) of bacteriophage prepared as a feed additive for 21days after challenge. Salmonella in the intestine was quantified and environmental contamination level was examined at 1, 2 and 3weeks after challenge. All treatments reduced intestinal SE colonization in challenged and contact chickens and reduced the environmental contamination level, but the reductions produced by 107 and 109PFU/g of bacteriophage were significant (P<0.05) as compared with untreated controls. In addition, seven out of 10 (70%) contact chickens treated with 109PFU/g of bacteriophage had no detectable intestinal Salmonella at 3weeks after treatment, suggesting that bacteriophage therapy significantly prevented the horizontal transmission of SE. These results provide important insights into preventive and control strategies against SE infection in poultry and indicate that the use of bacteriophage could reduce the incidence of Salmonella food poisoning.

Comportamento de células do sistema imune frente ao desafio com Salmonella Enteritidis em aves tratadas e não tratadas com ácidos orgânicos

A Salmonelose é uma importante zoonose, considerada a principal causa de infecções bacterianas, sendo associada ao consumo de produtos avícolas. Como alternativa de controle, ácidos orgânicos têm sido amplamente usados. No entanto, pouco se conhece sobre o estado imunológico de aves de produção, e uma avaliação deste status é necessária para proteger frente a enfermidades e para garantir à aplicação segura de agentes terapêuticos ou imunização profilática. Este trabalho teve como objetivo verificar o comportamento do sistema imunológico das aves previamente infectadas com Salmonella Enteritidis (SE) tratadas com um composto de ácidos orgânicos em diferentes concentrações administrado via água e ração comparando com as aves infectadas e não tratadas. Foram inoculados 120 frangos de corte com 1mL de SE, via oral, na concentração de 1,0 x 108 UFC/mL, no 1º e 2º dia de idade, divididos em seis tratamentos com duas repetições, utilizando 200, 400, 500 e 1000ppm do ácido orgânico. Aos 35 dias de vida das aves, foram coletados, de todos os grupos, alíquotas de sangue de 3mL em tubo contendo EDTA para a avaliação das células imunes através de citometria de fluxo. Foram analisadas as porcentagens circulantes de células CD4+, CD8β+, MHC I+, MHC II+, TCRVβ1+, TCRVβ2+ e CD28+. Para análise microbiológica foram coletadas tonsilas cecais destas aves. Observou-se com esse estudo que os ácidos orgânicos nas dosagens 1000ppm na água e 500ppm na ração durante, dois e sete dias respectivamente antes do abate, foram eficazes na redução da infecção por SE em frangos de corte, comprovadas pelo método microbiológico e demonstradas através do comportamento das células do sistema imune. No presente estudo as aves infectadas apresentaram uma proporção menor de células T auxiliares circulantes quando comparadas às aves infectadas, mas tratadas com o AO ou com o grupo não infectado. A mesma tendência pode ser observada para as células CD28+, TCRVβ1+ e MHC IIbright+, e, com menor resolução, para CD8β+.

Functional analysis and induction of four novel goose (Anser cygnoides) avian β-defensins in response to salmonella enteritidis infection

In the current study, four novel avian β-defensins (AvBDs) (AvBD2, 5, 9, and 10) were identified in tissues from the Chinese goose (Anser cygnoides). The antibacterial activity of the AvBDs showed that all of these AvBDs exhibited antibacterial activity against most of the bacteria investigated (P<0.01). In addition, antibacterial activity of all of the AvBDs against Staphylococcus aureus and Pasteurella multocida decreased significantly or was completely abolished at 150mM NaCl (P<0.01). None of the AvBDs showed hemolytic activity. AvBD2 and AvBD10 were expressed widely, whereas AvBD5 and AvBD9 mRNAs were expressed in a limited number of geese tissues. AvBD9 was significantly induced in some immune tissues from geese after Salmonella enteritidis infection. The others were significantly upregulated in small intestine and some immune tissues of the geese (P<0.01). The present results suggest that the AvBDs are part of the host defense mechanism of the goose.

Changes in the expression of Toll-like receptors in the chicken testis during sexual maturation and Salmonella infection

Rooster infertility is a major concern in the poultry industry and chicken male reproductive organs are the infectious tissues of various pathogenic microorganisms. Protection of the chicken male reproductive organs from pathogens is therefore an essential aspect of reproductive physiology. Recently Toll-like receptors (TLRs) have been identified as one of the key components of innate immunity in vertebrate species and have been reported to be expressed in the reproductive organs in various female species, including the chicken. However, mechanisms of antimicrobial protection of male reproductive organs mediated by TLRs are poorly understood. The objectives of this study were to determine the expression profile of the entire family of the ten chicken TLR genes in the chicken testis, to investigate whether sexual maturation affects their testicular mRNA abundance and to determine the changes in their expression levels in response to Salmonella enteritidis (SE) infection. RNA was extracted from the testis of healthy pre-pubertal, sexually mature and aged birds, and from sexually mature SE infected birds. RT-PCR analysis revealed that all TLRs, apart from TLR1-1 (TLR6), were expressed in the chicken testis. Quantitative real-time PCR analysis revealed that the testicular mRNA abundance of certain TLRs was developmentally regulated with respect to sexual maturation, while SE infection resulted in a significant induction of TLR2-1, 4, 5, 15 and 21 in the testis of sexually mature birds compared, to healthy birds of the same age. These findings provide strong evidence to suggest a key role of TLRs in the innate immune responses of chicken testis against Salmonella colonization.

Effects of sexual maturation and Salmonella infection on the expression of Toll-like receptors in the chicken vagina

Toll-like receptors (TLRs) are a critical component of the innate immune response in many vertebrates, including avian species. The recent findings of chicken TLRs (cTLRs) expression in ovarian follicles and in the chicken ovary in vivo, as well as the changes in their expression in response to lipopolysaccharide or Salmonella enteritidis (SE) infection, have broad implications for reproductive physiology and for the prevention of transmission of zoonotic diseases to humans through the consumption of contaminated poultry eggs. Because the main route of egg contamination is from infection of the oviduct and mainly from the vagina, the aim of this study was to investigate the expression of the ten cTLRs identified to date in the chicken oviduct in vivo, to determine whether sexual maturation affects their mRNA abundance and to investigate whether SE infection alters the expression of TLRs in the chicken vagina. RNA was extracted from the vagina of healthy prepubertal, sexually mature and aged birds, and from sexually mature and aged SE infected birds. RT-PCR analysis revealed that all types of cTLRs apart from TLR1-1 were expressed in the vagina of sexually mature birds. Quantitative real-time PCR analysis revealed that the mRNA abundance of TLR2-1, 2-2 and 4 differ with respect to sexual maturation in the chicken vagina. SE infection resulted in a significant induction of TLR5 and 15 in the vagina of sexually mature birds, and in a significant induction of TLR2-1, 4 and 15 in the vagina of aged birds, while a significant down-regulation was observed for TLR7 in the vagina of sexually mature birds. These findings suggest that a TLR mediated immune response mechanism exists in the chicken vagina, playing a crucial role in preventing microbial pathogens from being incorporated into newly forming eggs.

Transcriptional profiling of antimicrobial peptides avian β-defensins in the chicken ovary during sexual maturation and in response to Salmonella enteritidis infection

Avian β-defensins (AvβDs) are antimicrobial peptides that play significant roles in the innate immune system in chickens. The aim of this study was to identify the types of AvβDs expressed in the chicken ovary, to investigate the effects of sexual maturation in the ovarian mRNA abundance and to determine the changes in their expression levels as a result to Salmonella enteritidis (SE) infection. RNA was extracted from the ovary of healthy prepubertal, sexually mature and aged birds, as well as from sexually mature and aged SE infected birds. Real-time PCR analysis revealed that 11 AvβDs genes were expressed in the chicken ovary. A significant up regulation of AvβD1, 3, 4, 5, 7 and 11 was observed in the ovary of sexually mature and aged birds. Furthermore, a significant up-regulation of AvβD4, 5, 7, 11 and 12 was observed in the ovary of SE infected sexually mature birds. These results suggest that the mRNA expression of at least six AvβDs increase with age in the ovary of laying hens, and that at least five AvβDs show an induction in their expression in response to SE infection, indicating an AvβD-mediated immune response mechanism in the chicken ovary.

β 1-4 mannobiose enhances Salmonella-killing activity and activates innate immune responses in chicken macrophages

Salmonella spp. is one of the major causes of food-borne illness in humans, and Salmonella enteritidis (SE) infection in commercial poultry is a world-wide problem. Here we have investigated the in vitro immune-modulating effects of β 1-4 mannobiose (MNB), which was previously found to prevent SE infection in vivo in chickens, using chicken macrophage (MQ-MCSU) cells. Treatment of MQ-NCSU cells with MNB dose-dependently increased both phagocytic activity and Salmonella-killing activity of macrophages, with the highest reduction in SE viability observed at a concentration of 40 μg/ml at 48 h post-infection. Likewise, both hydrogen peroxide (H 2O 2) and nitric oxide (NO) production were increased in a dose-dependent manner by MNB. Gene expression analysis of MNB-treated macrophages revealed significant increases in the expression of iNOS, NOX-1, IFN-γ, NRAMP1, and LITAF, genes critical for host defense and antimicrobial activity, when compared to untreated cells. This data confirms that MNB possesses potent innate immune-modulating activities and can up-regulate antibacterial defenses in chicken macrophages.

Transcriptional profiling of Toll-like receptors in chicken embryos and in the ovary during sexual maturation and in response to Salmonella enteritidis infection

One of the key members of the innate immune system in many vertebrate species is the family of Toll-like receptors (TLRs). These molecules, which initiate the innate immune response and mount an anti-microbial response in both vertebrates and invertebrates, have recently been identified in the chicken genome. The recent findings of chicken TLRs (cTLRs) expression in ovarian follicles during follicular growth and in response to lipopolysaccharide (LPS) infection are very important for reproductive physiology due to the transovarian transmission of Salmonella enteritidis (SE) in laying hens. The aim of this study was to investigate the expression of the ten cTLRs identified to date, in the chicken ovary in vivo and embryos during early embryonic development, to investigate whether sexual maturation affects their ovarian mRNA abundance and to investigate the transcriptional changes of TLRs in the chicken ovary in response to SE infection. RNA was extracted from embryos from day 3 to day 10 of embryonic development as well as from the ovaries of healthy prepubertal, sexually mature and aged birds, and from sexually mature and aged SE infected birds. RT-PCR analysis revealed that all TLRs apart from TLRs 1–1 and 2–2 were expressed in the ovary of sexually mature chickens, while all TLRs apart from TLR1–1 were expressed in the chicken embryos during embryonic development. Quantitative real-time PCR analysis revealed that the ovarian mRNA abundance of TLRs differ with respect to sexual maturation. SE infection resulted in a significant induction of TLR4, and 15 in the ovary of sexual mature birds, and in a significant induction of TLR15 in the ovary of aged birds, while a significant down-regulation was observed for TLR3 in the ovary of aged birds. These findings suggest that a TLR-mediated immune response mechanism exists in the chicken ovary.

A time-course study of gene responses of chicken granulosa cells to Salmonella Enteritidis infection

Consumption of eggs contaminated with Salmonella Enteritidis (SE) has been recognized as one of the important causes of human foodborne salmonellosis. Chicken granulosa cells (cGCs) comprise the last tissue layer surrounding the yolk in preovulatory follicles and are a preferred site for SE invasion. To understand the cGC response to SE infection, we conducted an in vitro time-course study to identify cGC transcriptional changes using chicken whole genome microarrays. The expression of 135 (4 h postinfection) and 120 cGC genes (48 h postinfection) were altered ( P < .01) compared to uninfected cells. Many of the altered genes were related to immune response, physiological processes, signal transduction, and transcription. Furthermore, we also found that the Jak-STAT pathway, which is essential in the regulation of cellular cytokines and growth factors, was highly active in this study. Among the genes identified by microarray, the mRNA levels of TLR15, IL-6, CXCLi1, CXCLi2, and K203 were shown to be upregulated by real-time RT-PCR (qRT-PCR). In contrast, the mRNA levels of RASD1 and HB-EGF decreased according to both microarray and qRT-PCR analyses. These results suggest that during the SE infection, cGCs recruit cells of the innate immune responses; the infection may also induce suppression of cGC cell proliferation, which alters follicular development and ovulation.

EVALUATION OF PEFLOXACIN FOR TREATMENT PEKIN DUCKLINGLING SALMENLLOSIS AT SHARKIA GOVERNORATE WITH SPECIAL REFFERENCE TO ITS RESIDUCE IN TISSUES

A total of 120 apparently healthy, one day old Pekin duckling were used to investigate the effect of pefloxacin on Salmonella enteritidis infection. Effect of Salmonella enteritidis and pefloxacin on body weight gain and hemato-biochemical changeswas recorded as well as the residue of pefloxacin in muscles, skin and some organs were determined. Duckling were divided into four equal groups 30 for each). The 1st group apparently healthy duckling (control group), 2nd group was apparently healthy duckling and treated with pefloxacin (5mg / kgm b.wt.) in drinking water for 5days, while the duckling in 3rd & 4th groups were infected with Salmonella enteritidis at the 10th day of age. The 3rd group was kept as infected non treated duckling; Meanwhile the 4th group (infected duckling) was treated with pefloxacin (5mg kgm b.wt.) in drinking water for 5 day. Five duckling from each group were weighed individually for calculation weight gain for calculation of feed conversion rate at 1st and 21th day post treatment.At 7th and 14th day post treatment 5 duckling from each group were sacrificed for collection of 2 blood samples. The 1st sample was collected for estimation of erythrogram and the 2nd sample was collected to obtain clear serum for estimation some biochemical parameters. Another 5 duckling from each group were sacrificed samples from thigh muscle, liver, kidneys, gizzard and skin were collected at 1st,3th,7th and 10 thday post treatment for determination drug residuce Salmonella enteritidis induced 40% mortality in infected non treated pekin duckling. and 6.67% in infected group treated with pefloxacin.Healthy ducklings treated with pefloxacin revealed significant increase in the body gain, AST, ALT, alkaline phosphatase, uric acid, creatinine and significant decrease in feed conversion rate,erythrocytic count, hemoglobin content,packed cell volume, total protein and albumin. Infected ducklings with Salmonella enteritidis and non treated evoked a reduction in body weight gain, albumin and a significant increase in feed conversion rate, AST, ALT, alkaline phosphatase, uric acid, creatinin and globulin. These parameters were improved towards the normal levels when treated infected duckling with pefloxacin. Pefloxacin residue in the examined samples of liver, kidney, muscles, gizzard,skin ISSN 1110-7219 Mansoura, Vet. Med. J. (217 - 229) 218 Emam, E. E.; et al... Vol. XI, No. 2, 2009Mansoura, Vet. Med. J. INTRODUCTION Duck are a source of protein to human beings. Duck industry in Egypt has been well established; therefore any problems may be common, bacterial diseases are one of the most important problems facing duck industry (Moustafa, et al. 2008). Salmonellosis is one of the most important serious problems threatening poultry industry, where it causes serious economic losses due to high mortality (Seo, et al. 2000). Virulence factors of Salmonella include three general toxins which play role in their pathogenicity, endotoxins associated with cell wall lipopolysaccharides when liberated to circulation during bacte-rial deaths produce fever, liver and spleen lesions (Turnbull and Snoeyenbos, 1974). Antimicrobial is an important tool in reducing the losses in poultry industry. Among these antimicrobials are fluoroquinolones, which widely used in clinical practice because of their excellent antibacterial effect (Avril, et al. 1995), they act by inhibiting the activity of bacterial DNA-gyrase which responsible for supercoiling of bacterial DNA (Sun, et al. 2001). Pefloxacin is one of a 3rd generation of fluoroquinolones with broad spectrum activity against G+ve, G-ve bacteria and Mycoplasma (Garcia, et al. 1999). Pefloxacin active against all of the Salmonella strains (Wille et al. 1988). Antibiotic residues in meat derived from treated animals and chickens could pose health threats to consumers, while the constant exposure of some microorganisms to these drugs may manifest itself in development of antibiotic resistant bacteria, allergic reactions in sensitized persons and possible direct toxic effects of minute amounts over long periods of time (Corry et al., 1983). This work was designed to evaluate the activity of pefloxacin against salmonella enteritidis infection in pekin duckling. Furthermore, effects of pefloxacin and salmonella enteritidis on some hemato-biochemical,parameter as well as drug residuce determination MATERIAL AND METHODS Drug : Pefloxacin (Peflodad 10%)® solution was obtained from Dar Al Dawa Veterinary and Agricultural Industrial Co. Itd. Jordan. Each ml contains 100mg of pefloxacin base. Experimental duckling: A total of 120 apparently healthy one day old white pekin ducklings were obtained from and fat were very high at 1st day post treatment then become very low at 5th day,Moreover the pefloxacin completely disappeared from all the examined samples after 7 thdays post treatment days The highest levels of pefloxacin residues were recorded in the liver followed by skin and fat then gizzard and muscles but the lowest levels was observed in the kidneys samples. from this study we concluded that, pefloxacin has some reversible hepatotoxicity and nephrotoxicity in healthy duckling. Salmonella enteritidis in duckling resulted in adverse effect in both erythrogram and biochemical parameters

Inhibition of NF-kB 1 (NF-kBp50) by RNA interference in chicken macrophage HD11 cell line challenged with Salmonella enteritidis

The NF-kB pathway plays an important role in regulating the immunity response in animals. In this study, small interfering RNAs (siRNA) were used to specifically inhibit NF-kB 1 expression and to elucidate the role of NF-kB in the signal transduction pathway of the Salmonella challenge in the chicken HD11 cell line. The cells were transfected with either NF-kB 1 siRNA, glyceraldehyde 3-phosphate dehydrogenase siRNA (positive control) or the negative control siRNA for 24 h, followed by Salmonella enteritidis (SE) challenge or non-challenge for 1 h and 4 h. Eight candidate genes related to the signal pathway of SE challenge were selected to examine the effect of NF-kB 1 inhibition on their expressions by mRNA quantification. The results showed that, with a 36% inhibition of NF-kB 1 expression, gene expression of both Toll-like receptor (TLR) 4 and interleukin (IL)-6 was consistently and significantly increased at both 1 h and 4 h following SE challenge, whereas the gene expression of MyD88 and IL-1β was increased at 1 h and 4 h, respectively. These findings suggest a likely inhibitory regulation by NF-kB 1, and could lay the foundation for studying the gene network of the innate immune response of SE infection in chickens.

Passive Immunity of Progeny from Broiler Breeders Vaccinated with Oil-Emulsion Bacterin Against Salmonella Enteritidis

Young poultry are very susceptible to Salmonella Enteritidis (SE) infections because of the absence of complete intestinal flora colonization and an immature immune system. This study evaluated the role of passive immunity on the resistance of young birds against early infections caused by SE. The progeny of broiler breeders vaccinated with an oil-emulsion bacterin was compared to the progeny of unvaccinated birds. Efficacy was determined by challenging birds at 1 and 14 days of age with SE Nal Spc strain, phage type 4. After challenge at 1 day of age, the progeny of vaccinated birds presented a significantly lower number (log10) of SE Nal Spc reisolation (P < 0.05) in liver (2.21), spleen (2.31), and cecal contents (2.85) compared with control groups (2.76, 3.02, and 6.03, respectively). The examination of the internal organs, 3 days after infection, revealed that 28% of the birds (7/25) from vaccinated breeders were positive, whereas 100% (25/25) of the chicks derived from unvaccinated birds were positive. Birds challenged at 14 days of age presented a lower number of positive samples compared with those challenged at 1 day of age, and the progeny of vaccinated birds presented statistically lower numbers (log10) of colony-forming units/ml of SE Nal Spc only in the cecal contents compared with nonvaccinated breeder progeny (2.11 vs. 2.94). Age seems to influence the susceptibility of birds to SE infections: in control groups, the number of positive birds at 14 days of age (9/25) was lower when compared with the group infected at 1 day of age (25/25). The number of positive fecal samples of the progeny of vaccinated birds was significantly lower (36) than those of the control group (108) after challenge at 1 day of age. Unchallenged progeny of vaccinated birds presented passive antibodies detectable by enzyme-linked immunosorbent assay (ELISA) up to 21 days of age. On the other hand, antibodies of the control group were detected by ELISA 14 days after challenge. These results show a significant contribution of breeder vaccination by increasing the resistance of the progeny against early SE infections. However, the bacteria were not completely eliminated, suggesting that additional procedures are needed to effectively control SE infections.

Cellular Assessment of Crop Lymphoid Tissue from Specific-Pathogen-Free White Leghorn Chickens After Salmonella Enteritidis Challenge

The crop may be an important site along the upper alimentary tract in which a humoral immune response against Salmonella Enteritidis (SE) is elicited locally. The mucosal immune response within the crop (ingluvies) of specific-pathogen-free (SPF) white leghorn (WL) chickens against SE was investigated. Three trials were conducted using SPF WL pullets at age 5-6 wk. Trial 1 consisted of 77 birds evaluated for 10 wk post-SE infection (pi), trial 2 was composed of 72 birds monitored through 8 wk pi, and trial 3 was made up of 30 birds assessed for 5 wk pi. Birds were challenged per os with 10(8) colony-forming units/ml SE phage type 13. Crop lavage samples, crop tissues, ceca, and/or liver-spleen were collected preinfection and then at weekly intervals post-SE infection. Bacteriologic examination of cecal contents and/or liver-spleen occurred weekly to monitor progression of SE infection. Crop lavages were analyzed for SE-lipopolysaccharide (LPS)-specific immunoglobulin A (IgA) by enzyme-linked immunosorbent assay to assess humoral immune response. General histologic staining (hematoxylin and eosin [H&E] and methyl green-pyronin [MGP]) and immunohistochemical (IHC) staining (monoclonal antibodies CD45 and Bu-1) were applied to serial sections of crop to evaluate lymphoid tissue via light microscopy, to grade isolated lymphoid follicles (ILFs) by using score 0 (minimal, < 50 microm in diameter) to score 5 (sizable, > 200 microm in diameter) scale, and to characterize the cellular population of ILFs. Results revealed that cecum samples and liver-spleen samples were 100% SE culture positive at 1 wk pi, and then the percentage of SE positives progressively declined over time. Markedly increased crop SE-LPS-specific IgA antibodies were detected in crop samples by 2-3 wk pi, and the humoral response remained elevated above week 0 baseline for the duration of each trial. Crop ILFs of score 3 to 5 were observed in H&E-stained tissues, with an increased proportion of ILFs in post-SE-infected crops vs. uninfected. MGP staining showed plasma cells scattered within and at the periphery of ILFs. IHC staining revealed CD45 (pan-leukocyte) and Bu-1 (B-lymphocyte)-positive cells within crop ILFs. The chicken crop seems to be an organ in which lymphoid tissue may arise in response to enteric SE infection, and a site in which a humoral response may be generated against the SE pathogen.

A Study of the Distribution Patterns and Levels of Salmonella Enteritidis in the Immune Organs of Ducklings After Oral Challenge by Serovar-Specific Real-Time PCR

The objective of this study was to understand the distribution patterns and levels of Salmonella Enteritidis (SE) in the immune organs of ducklings after oral challenge. We conducted serovar-specific real-time polymerase chain reaction (PCR) for SE to detect the genomic DNA of SE in the blood and immune organs, including the bursa of Fabricius, thymus, spleen, and Harderian gland, from ducklings after oral challenge at different time points. The results showed that SE was consistently detected in all the samples. The Harderian gland and spleen tested positive at 8 hr postinoculation (PI). The organism was detected in the blood, bursa of Fabricius, and thymus at 10 hr PI. The copy number of SE DNA in each tissue reached a peak at 24-36 hr PI. The spleen, blood, and Harderian gland contained high concentrations of SE, whereas the thymus and bursa of Fabricius had low concentrations. SE populations began to decrease and were not detectable at 2 days PI, but they were still present up to 9 days PI in the spleen, without producing any apparent symptoms. To validate these results, the indirect immunofluorescent antibody (IFA) technique was used, and the IFA results were similar to those of the fluorescent quantitative-PCR. In conclusion, the results provided insight into the SE life cycle in the immune organs; furthermore, the Harderian gland and spleen were determined to be the primary sites of invasion among the immune organs of normal ducklings after oral SE challenge. This study will help in understanding the pathogenesis of SE infection in vivo and may help in the development of a live Salmonella vaccine in the future.

Salmonella Enteritidis–Induced Alteration of Inflammatory CXCL Chemokine Messenger-RNA Expression and Histologic Changes in the Ceca of Infected Chicks

To understand better the events in early avian host immune responses to Salmonella Enteritidis (SE), we examined messenger-RNA (mRNA) expression for eight genes: CXCLi1[K60], CXCLi2 [IL-8/CAF], interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6, IL-12alpha, IL-12beta, and gallinacin (Gal)-2 in the ceca of young chicks 1 wk postinoculation with SE. Cecum tissue sections were stained and evaluated for the presence of macrophages, lymphocytes, heterophils, and apoptotic cells following SE infection. With the use of quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), SE infection was associated with a significant (P < 0.01) upregulation of cecal CXCLi1 and CXCLi2 mRNA expression. Infection with SE was also associated (P < 0.05) with increased staining for macrophages and decreased apoptosis (single-stranded DNA [ssDNA]) in cecal tissue sections when these sections were compared with those of uninfected animals. Changes in chemokine expression and cell population dynamics are a direct result of SE infection, as uninfected animals do not show these alterations. Thus, these SE-induced changes reflect the host immune response to SE in young chickens.

Serovar-Specific Real-Time Quantitative Detection of Salmonella Enteritidis in the Gastrointestinal Tract of Ducks After Oral Challenge

The objective of this study was to identify and understand the regular distribution pattern and primary penetration site for Salmonella Enteritidis (SE) in the gastrointestinal tract of ducks. An assay based on the serovar-specific DNA sequence of SE from GenBank, a serovar-specific real-time, fluorescence-based quantitative polymerase chain reaction, was developed for the detection of SE. We used this assay to detect genomic DNA of SE in the blood and gastrointestinal tract, including duodenum, jejunum, ileum, cecum, rectum, esophagus, stomach muscularis, and stomach glandularis, from ducks after oral challenge at different time points. The results showed that SE was consistently detected in all segments of the gastrointestinal tract. The jejunum and ileum were positive 8 hr postinoculation (PI). The organism was detected in blood 12 hr PI, while the final organ to show a positive result was the stomach at 24 hr PI. The copy number of SE DNA in each tissue reached a peak at 24-36 hr PI, with the jejunum, ileum, and cecum containing high concentrations of SE, whereas the blood, duodenum, rectum, stomach, and esophagus had low concentrations. SE populations began to decrease and were not detectable at 2 days PI, but were still present up to 9 days PI in the jejunum, ileum, and cecum without causing apparent symptoms. By 3 days PI the cecum had significantly higher numbers of SE than any of the other areas (P < 0.01), and this appeared to reflect its function as a repository for SE. In conclusion, the results provided significant data for understanding the life cycle of SE in the gastrointestinal tract and showed that the jejunum, ileum, and cecum were the primary sites of invasion in normal ducks after oral challenge. This study will help to understand the mechanisms of action of SE infection in vivo.

Crop Immune Response Post–Salmonella Enteritidis Challenge in Eight Commercial Egg-Layer Strains and Specific-Pathogen-Free White Leghorn Chickens

The crop immune response against Salmonella Enteritidis (SE) challenge in eight commercial egg-layer strains (five white-egg layer and three brown-egg layer) and specific-pathogen-free (SPF) White Leghorn (WL) hens was investigated. Pre- and post-SE challenge mucosal immune responses within the crops were evaluated. Commercial layers and SPF WL hens were orally challenged with 10(8) CFU/ml SE PT13a and SE nalR PT13, respectively. Crop lavage samples were collected at weekly intervals from day 0 (pre-challenge) to day 25-27 postinfection (PI), and bacteriological examination was performed to monitor progression of SE infection. Crop lavage samples were analyzed for SE-lipopolysaccharide (LPS)-specific IgA using enzyme-linked immunosorbent assay (ELISA). H&E-stained slides of crop sections from day 34 PI and uninfected controls were assessed for lymphoid tissue via light microscopy. Lymphoid areas were graded based on morphology, size, and cellularity using a score 0 to 5 scale. The 0 to 5 (low to high) numerical values represented progressive increases in size and cellular density of lymphoid tissue. Bacterial culture results showed the highest percentage of SE-positive crop lavage samples from all hen groups at day 5-6 PI and day 11-12 PI. A progressive decline in percentage of SE-positive crop lavage samples did occur as time PI lengthened; however, at day 25-27 PI SE persisted in crop lavage samples from SPF WL hens and three commercial white-egg layer strains. A marked increase in SE-LPS-specific IgA was measured in crop lavage samples between day 0 and day 11-12 PI for all hen groups. Crop SE-LPS-specific IgA response remained elevated above day 0 baseline for the duration of the experiment. Well-defined score 3 to 5 lymphoid tissue aggregates were observed in crop tissue sections harvested at day 34 PI. Comparison of crop sections determined a 1.2-4.0 times increase in ratio of lymphoid tissue in day 34 PI SE-challenged hens vs. uninfected control hens.

Bacteriophage Treatment Reduces Salmonella Colonization of Infected Chickens

Three different lyric bacteriophages (BPs) were isolated from the sewage system of commercial chicken flocks and used to reduce Salmonella Enteritidis (SE) colonization from experimental chickens. Ten-day-old chickens were challenged with 9.6 x 10(5) colony-forming units (CFU)/ml of a SE strain and treated by coarse spray or drinking water with a cocktail of the three phages at a multiplicity of infection (MO1) of 10(3) plaque-forming units (PFU) 24 hr prior to SE challenge. Chickens were euthanatized at day 20 of age for individual SE detection, quantitative bacteriology, and phage isolation from the intestine and from a pool of organs. SE detection was performed by both bacteriologic culture and genome detection by polymerase chain reaction (PCR). Qualitative bacteriology showed that aerosol-spray delivery of BPs significantly reduced the incidence of SE infection in the chicken group (P = 0.0084) to 72.7% as compared with the control group (100%). In addition, SE counts showed that phage delivery both by coarse spray and drinking water reduced the intestinal SE colonization (P < 0.01; P < 0.05, respectively). BPs were isolated at 10 days postinfection from the intestine and from pools of organs from BP-treated chickens. We conclude that the phage treatment, either by aerosol spray or drinking water, may be a plausible alternative to antibiotics for the reduction of Salmonella infection in poultry.