RIPK3-Dependent Recruitment of Low-Inflammatory Myeloid Cells Does Not Protect from Systemic Salmonella Infection.

Regulated macrophage death has emerged as an important mechanism to defend against intracellular pathogens. However, the importance and consequences of macrophage death during bacterial infection are poorly resolved. This is especially true for the recently described RIPK3-dependent lytic cell death, termed necroptosis. Salmonella enterica serovar Typhimurium is an intracellular pathogen that precisely regulates virulence expression within macrophages to evade and manipulate immune responses, which is a key factor in its ability to cause severe systemic infections. We combined genetic and pharmacological approaches to examine the importance of RIPK3 for S. Typhimurium-induced macrophage death using conditions that recapitulate bacterial gene expression during systemic infection in vivo Our findings indicate that noninvasive S. Typhimurium does not naturally induce macrophage necroptosis but does so in the presence of pan-caspase inhibition. Moreover, our data suggest that RIPK3 induction (following caspase inhibition) does not impact host survival following S. Typhimurium infection, which differs from previous findings based on inert lipopolysaccharide (LPS) injections. Finally, although necroptosis is typically characterized as highly inflammatory, our data suggest that RIPK3 skews the peritoneal myeloid population away from an inflammatory profile to that of a classically noninflammatory profile. Collectively, these data improve our understanding of S. Typhimurium-macrophage interactions, highlight the possibility that purified bacterial components may not accurately recapitulate the complexity of host-pathogen interactions, and reveal a potential and unexpected role for RIPK3 in resolving inflammation.IMPORTANCE Macrophages employ multiple strategies to limit pathogen infection. For example, macrophages may undergo regulated cell death, including RIPK3-dependent necroptosis, as a means of combatting intracellular bacterial pathogens. However, bacteria have evolved mechanisms to evade or exploit immune responses. Salmonella is an intracellular pathogen that avoids and manipulates immune detection within macrophages. We examined the contribution of RIPK3 to Salmonella-induced macrophage death. Our findings indicate that noninvasive Salmonella does not naturally induce necroptosis, but it does so when caspases are inhibited. Moreover, RIPK3 induction (following caspase inhibition) does not impact host survival following Salmonella systemic infection. Finally, our data show that RIPK3 induction results in recruitment of low-inflammatory myeloid cells, which was unexpected, as necroptosis is typically described as highly inflammatory. Collectively, these data improve our understanding of pathogen-macrophage interactions, including outcomes of regulated cell death during infection in vivo, and reveal a potential new role for RIPK3 in resolving inflammation.

Combining Whole-Genome Sequencing and Multimodel Phenotyping To Identify Genetic Predictors of Salmonella Virulence.

Salmonella comprises more than 2,600 serovars. Very few environmental and uncommon serovars have been characterized for their potential role in virulence and human infections. A complementary in vitro and in vivo systematic high-throughput analysis of virulence was used to elucidate the association between genetic and phenotypic variations across Salmonella isolates. The goal was to develop a strategy for the classification of isolates as a benchmark and predict virulence levels of isolates. Thirty-five phylogenetically distant strains of unknown virulence were selected from the Salmonella Foodborne Syst-OMICS (SalFoS) collection, representing 34 different serovars isolated from various sources. Isolates were evaluated for virulence in 4 complementary models of infection to compare virulence traits with the genomics data, including interactions with human intestinal epithelial cells, human macrophages, and amoeba. In vivo testing was conducted using the mouse model of Salmonella systemic infection. Significant correlations were identified between the different models. We identified a collection of novel hypothetical and conserved proteins associated with isolates that generate a high burden. We also showed that blind prediction of virulence of 33 additional strains based on the pan-genome was high in the mouse model of systemic infection (82% agreement) and in the human epithelial cell model (74% agreement). These complementary approaches enabled us to define virulence potential in different isolates and present a novel strategy for risk assessment of specific strains and for better monitoring and source tracking during outbreaks.IMPORTANCESalmonella species are bacteria that are a major source of foodborne disease through contamination of a diversity of foods, including meat, eggs, fruits, nuts, and vegetables. More than 2,600 different Salmonella enterica serovars have been identified, and only a few of them are associated with illness in humans. Despite the fact that they are genetically closely related, there is enormous variation in the virulence of different isolates of Salmonella enterica Identification of foodborne pathogens is a lengthy process based on microbiological, biochemical, and immunological methods. Here, we worked toward new ways of integrating whole-genome sequencing (WGS) approaches into food safety practices. We used WGS to build associations between virulence and genetic diversity within 83 Salmonella isolates representing 77 different Salmonella serovars. Our work demonstrates the potential of combining a genomics approach and virulence tests to improve the diagnostics and assess risk of human illness associated with specific Salmonella isolates.

Efficacy of Bacillus methylotrophicus SY200 strain as feed additive against experimental Salmonella typhimurium infection in mice.

To investigate the effects of Bacillus methylotrophicus SY200 on Salmonella typhimurium (STM) infection in mice, a total of 36 three-week-old male mice were selected and randomly divided into 3 equal groups (N = 12). Group A and group B were fed with basal diet while group C was fed the basal diet supplemented with 0.1% (w/w) B. methylotrophicus SY200 during the 21 days experimental period. On the 14th day of the experiment, mice of group A were intragastrically administered with 0.5 ml of normal saline, group B and C were orally administered with 0.5 ml of STM suspension. On the first day and seventh day after STM challenge, the number of total white blood cells (WBCs) and neutrophils, relative weight of visceral organs, the number of Salmonella spp., Escherichia coli, Lactobacillus spp. and Bifidobacterium spp. in ileum and cecum, and diversity of cecal microflora were measured. The results showed that: on the first day and seventh day after STM challenge, the number of WBCs and neutrophils in the blood of the mice was the highest in group B, then followed by group C, and group A. On the first day after STM challenge, the relative weight of spleen in group C was significantly higher than that in group B (p < 0.05), moreover, compared with group B, B. methylotrophicus SY200 significantly reduced the number of Salmonella spp. and E. coli (p < 0.05), and increased the number of Lactobacillus spp. and Bifidobacterium spp. (p < 0.05) in the intestines of mice, and improved the Shannon-Wiener diversity (H), Simpson (E) and richness (S) indices of cecal flora of mice (p < 0.05). The results indicated that B. methylotrophicus SY200 could alleviate the inflammatory reaction after STM infection and resist the adverse effects of STM infection on mice intestinal flora.

Maintenance of Type IV Secretion Function During Helicobacter pylori Infection in Mice.

The Helicobacter pylori type IV secretion system (T4SS) encoded on the cag pathogenicity island (cagPAI) secretes the CagA oncoprotein and other effectors into the gastric epithelium. During murine infection, T4SS function is lost in an immune-dependent manner, typically as a result of in-frame recombination in the middle repeat region of cagY, though single nucleotide polymorphisms (SNPs) in cagY or in other essential genes may also occur. Loss of T4SS function also occurs in gerbils, nonhuman primates, and humans, suggesting that it is biologically relevant and not simply an artifact of the murine model. Here, we sought to identify physiologically relevant conditions under which T4SS function is maintained in the murine model. We found that loss of H. pylori T4SS function in mice was blunted by systemic Salmonella coinfection and completely eliminated by dietary iron restriction. Both have epidemiologic parallels in humans, since H. pylori strains from individuals in developing countries, where iron deficiency and systemic infections are common, are also more often cagPAI+ than strains from developed countries. These results have implications for our fundamental understanding of the cagPAI and also provide experimental tools that permit the study of T4SS function in the murine model.IMPORTANCE The type IV secretion system (T4SS) is the major Helicobacter pylori virulence factor, though its function is lost during murine infection. Loss of function also occurs in gerbils and in humans, suggesting that it is biologically relevant, but the conditions under which T4SS regulation occurs are unknown. Here, we found that systemic coinfection with Salmonella and iron deprivation each promote retention of T4SS function. These results improve our understanding of the cag pathogenicity island (cagPAI) and provide experimental tools that permit the study of T4SS function in the murine model.

Vaccination and Infection of Swine With Salmonella Typhimurium Induces a Systemic and Local Multifunctional CD4+ T-Cell Response.

The gram-negative facultative intracellular bacteria Salmonella Typhimurium (STM) often leads to subclinical infections in pigs, but can also cause severe enterocolitis in this species. Due to its high zoonotic potential, the pathogen is likewise dangerous for humans. Vaccination with a live attenuated STM strain (Salmoporc) is regarded as an effective method to control STM infections in affected pig herds. However, information on the cellular immune response of swine against STM is still scarce. In this study, we investigated the T-cell immune response in pigs that were vaccinated twice with Salmoporc followed by a challenge infection with a virulent STM strain. Blood- and organ-derived lymphocytes (spleen, tonsils, jejunal and ileocolic lymph nodes, jejunum, ileum) were stimulated in vitro with heat-inactivated STM. Subsequently, CD4+ T cells present in these cell preparations were analyzed for the production of IFN-γ, TNF-α, and IL-17A by flow cytometry and Boolean gating. Highest frequencies of STM-specific cytokine-producing CD4+ T cells were found in lamina propria lymphocytes of jejunum and ileum. Significant differences of the relative abundance of cytokine-producing phenotypes between control group and vaccinated + infected animals were detected in most organs, but dominated in gut and lymph node-residing CD4+ T cells. IL-17A producing CD4+ T cells dominated in gut and gut-draining lymph nodes, whereas IFN-γ/TNF-α co-producing CD4+ T cells were present in all locations. Additionally, the majority of cytokine-producing CD4+ T cells had a CD8α+CD27- phenotype, indicative of a late effector or effector memory stage of differentiation. In summary, we show that Salmonella-specific multifunctional CD4+ T cells exist in vaccinated and infected pigs, dominate in the gut and most likely contribute to protective immunity against STM in the pig.

Neuroimmunological Implications of Subclinical Lipopolysaccharide from Salmonella Enteritidis.

Mounting evidence has indicated that lipopolysaccharide (LPS) is implicated in neuroimmunological responses, but the body's response to subclinical doses of bacterial endotoxin remains poorly understood. The influence of a low single dose of LPS from Salmonella Enteritidis, which does not result in any clinical symptoms of intoxication (subclinical lipopolysaccharide), on selected cells and signal molecules of the neuroimmune system was tested. Five juvenile crossbred female pigs were intravenously injected with LPS from S. Enteritidis (5 µg/kg body weight (b.w.)), while five pigs from the control group received sodium chloride in the same way. Our data demonstrated that subclinical LPS from S. Enteritidis increased levels of dopamine in the brain and neuropeptides such as substance P (SP), galanin (GAL), neuropeptide Y (NPY), and active intestinal peptide (VIP) in the cervical lymph nodes with serum hyperhaptoglobinaemia and reduction of plasma CD4 and CD8 T-lymphocytes seven days after lipopolysaccharide administration. CD4 and CD8 T-lymphocytes from the cervical lymph node and serum interleukin-6 and tumour necrosis factor α showed no significant differences between the control and lipopolysaccharide groups. Subclinical lipopolysaccharide from S. Enteritidis can affect cells and signal molecules of the neuroimmune system. The presence of subclinical lipopolysaccharide from S. Enteritidis is associated with unknown prolonged consequences and may require eradication and a deeper search into the asymptomatic carrier state of Salmonella spp.

Blood serum acute phase proteins and iron dynamics during acute phase response of Salmonella enterica serotype Dublin experimentally infected buffalo calves.

The study aimed to evaluate clinical signs, blood serum acute phase proteins (APP) and iron dynamics during the acute phase response (APR) of Salmonella Dublin experimentally infected Murrah buffalo calves. Six buffalo calves constituted the control group (CNT) and six were orally inoculate with 108 CFU of S. Dublin (INF). Clinical evaluation was performed, rectal swabs to detect S. Dublin strains were collected and venous blood was sampled before and throughout seven days after inoculation. The APP fractions ß-haptoglobin, α-haptoglobin, ceruloplasmin and transferrin were analyzed by 1-D and 2-D electrophoresis. Proteins were identified using LC/ESI-MS/MS and NCBI database. Plasma fibrinogen, serum iron and serum haptoglobin concentrations were measured. The inoculation of 108 CFU of S. Dublin was effective in inducing clinical signs of Salmonellosis, such as hyperthermia and diarrhea. 1-DE showed that ß and α-haptoglobin increased 204% (p = 0.008) and 184% (p = 0.022) 48 h after inoculation (HAI), respectively, with highest concentrations 120 HAI (498% increased, p = 0.012; 431% increased, p = 0.011) and 168 HAI (492% increased, p = 0.019; 523% increased, p = 0.028). 2-DE showed that the expression of two spots, identified as ß-haptoglobin, were increased 693% (p = 0.0006) and 580% (p = 0.0003) 168 HAI, respectively, while one spot, identified as α-haptoglobin, increased 714% (p = 0.040). Haptoglobin concentrations increased 1339% (p < 0.0001) 168 HAI. 1-DE showed that ceruloplasmin increased 42% (p = 0.034) 48 HAI, with highest concentration 120 HAI (133% increased, p = 0.022). 2-DE showed that the expression of two spots, identified as ceruloplasmin, were increased 218% (p = 0.0153) and 85% (p = 0.0143) 168 HAI, respectively. Fibrinogen increased 78% (p = 0.012) 96 HAI, with highest concentration 120 HAI (increased 114%, p = 0.002). Iron decreased 33% 24 HAI (p = 0.015) and 37% 72 HAI (p = 0.029), and began to be restored 96 HAI. 1-DE showed that transferrin decreased 23% 120 HAI (p = 0.047), and that values were restored 168 HAI. 2-DE showed that expression patterns of transferrin comparing 0 h and 168 HAI were similar, evidencing that values were restored 168 HAI. In conclusion, the inoculation of 108 CFU was effective in inducing hyperthermia and diahrrea. ß and α-haptoglobin, ceruloplasmin and fibrinogen worked as positive APP during the APR to S. Dublin infection and are potential biomarker candidates. Concentrations of iron and transferrin decreased during the infection, highlighting the fact that mechanisms for restricting iron availability are part of the APR triggered against S. Dublin infection in buffalo calves.

High Dietary Iron Differentially Influences the Iron Distribution in the Livers and the Spleens of Laying Hens After Salmonella Typhimurium Infection.

Salmonella and the host battle for iron (Fe), due to its importance for fundamental cellular processes. To investigate Fe redistribution of Salmonella-infected hens and the effects of high dietary Fe on it, Salmonella-free hens were randomly assigned to 1 of 4 treatments in 2 (two dietary Fe level) × 2 (Salmonella-inoculation or -noninoculation) factorial assignment. After feeding a basal diet supplemented with 60 (adequate, control) or 300 mg Fe/kg (high-Fe) for 4 weeks, 59-week-old Salmonella-free hens were orally inoculated with 5 × 107 colony-forming units of Salmonella Typhimurium (infection) or PBS (vehicle). Blood, spleen, and liver samples (n = 8) were collected at 14 days post-inoculation to determine Fe concentration and Fe transporters expression. Salmonella infection decreased (P < 0.05) hematocrit, serum Fe concentration, and splenic Fe concentration regardless of high-Fe or control hens, whereas increased (P < 0.05) Fe centration in the livers of high-Fe-treated hens. High dietary Fe increased hematocrit and serum Fe concentration, but did not affect (P = 0.11) splenic Fe concentration in Salmonella-infected hens. Salmonella infection did not influence (P = 0.31) liver Fe centration in control hens, but increased (P = 0.04) it in high-Fe-treated hens. High dietary Fe decreased (P < 0.01) the mRNA abundance of divalent metal transporter 1 and transferrin receptor, but increased (P < 0.02) ferroportin-1 (FPN1) mRNA and protein in the spleens and the livers regardless of Salmonella-infected or vehicle hens. Salmonella infection increased (P < 0.02) FPN1 mRNA and protein expression in the spleens, but did not influence its expression in the livers. These results suggested Salmonella infection and high dietary Fe differently influence the Fe distribution in the spleen and the liver of Salmonella-infected hens.

The effect of heat stress on intestinal integrity and Salmonella invasion in broiler birds.

The intestinal mucosa works as a barrier to protect the internal environment of the animal from bacteria and bacterial toxins found in the gut lumen. Heat stress may harm this function. Therefore, we designed the current experiment to investigate the effect of heat stress on intestinal integrity, physiological and immunological responses and Salmonella invasion in broiler chickens. At 26 days of age, 72 birds were randomly distributed into 3 treatments, with 8 replicates per treatment and 3 birds per replicate. The three treatments were control treatment; kept at thermoneutral environmental conditions (20 ± 2°C), chronic heat stress treatment (exposed to 30 ± 2°C; 24h/day) and acute heat stress treatment (exposed to 35 ±2°C from 09:00 to 13:00 and kept at 20 ± 1°C from 13:00 to 09:00). The heat stress exposure was conducted for 10 successive days. Compared with the control treatment, birds subject to chronic and acute heat stress had reduced (P < 0.05) body weight and body gain and increased (P < 0.05) feed conversion ratio. However, feed intake and mortality rate were only increased (P < 0.05) in the acute heat stress treatment. Rectal temperature and Δ rectal temperature (°C/h) increased (P < 0.05) sharply during the first 2 days of exposure followed by gradual decreases until a plateau was achieved. Heat-stressed birds had increased (P < 0.05) serum concentrations of corticosterone, endotoxin lipopolysaccharide and the systemic inflammatory cytokine: TNF-α and IL-2, as well as a higher (P < 0.05) prevalence of Salmonella spp. in meat and livers, as compared with control treatment. It can be concluded that heat stress impaired intestinal integrity which resulted in increased intestinal permeability to endotoxin, translocation of intestinal pathogens (Salmonella spp.) and serum inflammatory cytokines. Therefore, avoiding thermal dysfunction of intestinal barrier is a significant factor in maintaining welfare, immune status and meat safety of broiler birds.

Characterization of adaptive immune responses induced by a new genetically inactivated Salmonella Enteritidis vaccine.

The superior conservation of antigenic determinants on the surface of genetically inactivated bacterial ghosts makes them attractive immunogenic inactivated vaccine candidates. The efficacy of Salmonella Enteritidis (SE) ghost vaccination was evaluated in chickens by characterizing the nature of the adaptive immune response. Chickens from the immunized group demonstrated significant increases in SE-specific plasma IgG, intestinal secretory IgA, and lymphocyte proliferative response. The populations of CD4, CD8, and TCR γδ T-cells in immunized chickens were significantly greater than in the controls. Increased levels of IFN-γ, IL-2, IL-6 and IL-10 were observed in peripheral blood mononuclear cells stimulated with SE specific antigen. After virulent SE challenge, the immune system of immunized chickens was rapidly stimulated, as indicated by significantly increased population of CD4 and CD8 T-cells. Furthermore, the immunized group exhibited decreased challenge strain recovery of the internal organs compared to the non-immunized group. Together, these data indicate that the immunization induced humoral and cell-mediated immunity might be responsible for significant reduction of the virulent challenge strain load in the internal organs of immunized chickens.

Phenotypic characterisation of the monocyte subpopulations in healthy adult pigs and Salmonella-infected piglets by seven-colour flow cytometry.

The present study describes the distinct bone marrow (BM) and peripheral blood (PB) monocyte subpopulations detected by seven-colour flow cytometry. Mononuclear phagocytes were identified as viable CD172a(+) SWC8(-) CD203a(-) mononuclear leukocytes. After that, monocyte subpopulations were differentiated by using CD14, CD163 and SLA-DR markers. Four distinct monocyte subpopulations were found in the BM and PB. Based on the discovered populations two possible maturation pathways have been proposed. The first pathway was characterised by release of CD14(hi) CD163(-) SLA-DR(-) BM monocytes into the PB where they matured into CD14(low) CD163(+) SLA-DR(+) monocytes. In the alternative pathway the monocytes finalised their phenotypical maturation in the BM and then they were released into the PB as CD14(low) CD163(+) SLA-DR(+) cells. In Salmonella-infected piglets, the population of CD14(low) CD163(+) SLA-DR(+) monocytes was elevated in the BM and mesenteric lymph nodes (MLN), suggesting the role of this population in pathogenesis of Salmonella infection in pigs.

[Comparing the results of the serological detection of Salmonella antibodies in blood serum and meat juice from different muscles from slaughter pigs]. / Untersuchungen zur Vergleichbarkeit der Ergebnisse des serologischen Nach- weises von Salmonella-Antikörpern bei Schlachtschweinen in Blutserum und Fleischsaft aus verschiedenen Muskelpartien.

The German Salmonella Monitoring Programme started by the QS-System in 2002 (Blaha, 2004) is mandatory due to the so-called "Salmonella Regulation for Pigs" since 2007 (Anonym, 2007). The Regulation does not clearly prescribe the specific muscle which is to be taken as source of the meat juice. Thus, at different slaughter plants meat samples are also taken from different muscles and several scientific papers describe various muscles as source of the meat juice, too. The objective of this study was to compare the serological results of meat juices from three different locations (diaphragm pillar, neck, belly muscle) to each other and to those of the blood serum from exactly the same animals. All samples were simultaneously tested for Salmonella antibodies by two serological tests (Salmo-type Pig Screen, LDL, Germany; HerdChek Swine Salmonella, IDEXX, Germany). Comparisons were carried out between the various sample kinds per animal and between the two test systems. The analysis of all results of the meat juices revealed in both test systems a clear decline of the OD% values from the diaphragm pillar to the neck to the belly muscle. The average OD% values of all samples were higher when measured by the HerdChek ELISA (IDEXX, Germany) than by the Salmotype ELISA (LDL, Germany), especially in blood serum. Since the results of the meat juice samples gained from the diaphragm pillar were in both test systems by far the closest to the results of the corresponding serum blood samples, it is recommended to amend the "Salmonella Regulation for Pigs" by prescribing meat from the diaphragm pillar as the only muscle for gaining meat juice.

Stronger in vitro phagocytosis by monocytes-macrophages is indicative of greater pathogen clearance and antibody levels in vivo.

Monocytes-macrophages are crucial players in specific and nonspecific immune responses to protect organisms from invasion of bacteria or viruses. In this study, monocytes in circulation from 2 lines of Silky and Starbro chickens with different disease resistance were separated and cultured in vitro. After identification with acridine orange (AO), Giemsa staining, and CD14 immunostaining, monocytes-macrophages were used for adherence and phagocytosis test. The overall percentages of adherence of Silky monocytes was 1.5 times greater than that of Starbro (P < 0.01), which were 26.85% +/- 8.24% and 18.34% +/- 8.15%, respectively (mean +/- SD). The monocytes-macrophages phagocytic index, phagocytic product, and percentage of phagocytosis in Silkies were greater than in Star-bros, respectively. The difference of phagocytic index was significant (P < 0.05), that is, 3.70 +/- 1.75 and 1.97 +/- 0.31, respectively (mean +/- SD). Then, 20 Silkies were divided into 2 groups according to phagocytic index: high phagocytic index (HPI) group and low phagocytic index (LPI) group, to study the relationship between phagocytic activity in vitro and pathogen clearance. After being challenged against Salmonella Pullorum C79-13, the Silky birds with HPI produced a 3-fold greater level of specific antibodies compared with those with LPI (P < 0.01), 50.21 +/- 6.67 and 16.85 +/- 4.52, respectively (mean +/- SD). In contrast to LPI birds, HPI birds shed less Salmonella Pullorum bacteria (P < 0.05), that is, 168.98 x 10(8) +/- 294.74 x 10(8) compared to 385.40 x 10(8) +/- 399.94 x 10(8) (mean +/- SD), and the shedding peak of Salmonella Pullorum in the test span appeared 4 d earlier. These results indicated that phagocytosis of monocytes-macrophages had strong effects on antibody titer and bacteria shedding postchallenge, which could be used to predict the disease resistance in animals.

Prevalencia serológica de Salmonellaenteritidis en la población canina del Municipio de Tunja, Colombia / The serological prevalence of Salmonella enteritidis amongst Tunja's canine population (a large Colombian city)

Objetivo Determinar la prevalencia serológica de Salmonella enteritidis en la población canina de Tunja (Boyacá), durante julio y octubre del 2006 y establecer la relación entre los factores predisponentes y la serorreactividad en la población canina. Materiales y Métodos Sobre la población canina estimada en 9 623 animales (censo de 2002), y asumiendo una prevalencia critica preestablecida de 3 por ciento y un nivel de confianza del 95 por ciento, se recolectaron 72 muestras sanguíneas caninas distribuidas en cinco zonas de la ciudad, se aplicó una encuesta para cada animal para determinar la presencia de factores predisponentes y un examen clínico completo. Con los sueros obtenidos, se tituló el nivel de anticuerpos mediante la prueba de microaglutinación en placa para Salmonella enteritidis (MAG). Resultados La prevalencia serológica a Salmonella enteritidis fue del 41,7 por ciento, las prevalencias zonales fueron: 10 por ciento Norte, 10 por ciento occidente, 16,7 por ciento Oriente, 36,7 por ciento sur y 26,6 por ciento centro. Se determinó una correlación directa con la presentación de problemas entéricos anteriores (p<0,05) (OR 3,5) y en menor grado con el acceso a desechos orgánicos (p<0,05) (OR 0,4); otros factores como la convivencia con otros animales y la salud mostraron niveles de riesgo positivo (OR 1,4 Y OR 2). Conclusiones Existe una alta prevalencia serológica a Salmonella enteritidis en la ciudad de Tunja, con mayor incidencia en la zona sur, siendo el principal factor predisponente la presentación de problemas entéricos anteriores, el cual es un factor de alto riesgo en la diseminación de esta entidad.

Objective Determining the serological prevalence of Salmonella enteritidis in the city of Tunja's canine population (in Boyacá) from July to October 2006 and establishing the relationship between predisposed factors and seroreactivity within this canine population. Materials and Methods 72 canine blood samples were gathered from all of the city's five areas from an estimated 9 623 canine population (2002 census), assuming a 3 percent pre-established critical prevalence and 95 percent confidence level. A survey was made of each animal to determine the presence of predisposed factors and they were all given a complete clinical examination. The sera so obtained were used for titering antibody levels by means of the microagglutination plate test for Salmonella enteritidis (MAG). Results Salmonella enteritidis serological prevalence was 41,7 percent. Area prevalence within the city was 10 percent for the north, 10 percent for the west, 16,7 percent for the east, 36,7 percent the south and 26,6 percent for the city-center. A direct correlation was established with the presentation of previous enteric problems (p<0.05) (OR 3.5) and, to a lesser extent, with access to organic waste (p<0.05) (OR 0.4). Other factors presented positive risk levels, such as coexistence with other animals and health (OR 1.4 and OR 2) Conclusions Salmonella enteritidis had a high serological prevalence in the city of Tunja, most incidents occurring in the southern area. The presentation of previous enteric problems was the main predisposed factor, this being a high risk factor in this entity's dissemination.

Chicken cecum immune response to Salmonella enterica serovars of different levels of invasiveness.

Day-old chicks are very susceptible to infections with Salmonella enterica subspecies. The gut mucosa is the initial site of host invasion and provides the first line of defense against the bacteria. To study the potential of different S. enterica serovars to invade the gut mucosa and trigger an immune response, day-old chicks were infected orally with Salmonella enterica serovar Enteritidis, S. enterica serovar Typhimurium, S. enterica serovar Hadar, or S. enterica serovar Infantis, respectively. The localization of Salmonella organisms in gut mucosa and the number of immune cells in cecum were determined by immunohistochemistry in the period between 4 h and 9 days after infection. Using quantitative real-time reverse transcription (RT)-PCR, mRNA expression of various cytokines, chemokines, and inducible nitric oxide synthase (iNOS) was examined in cecum. As a result, all S. enterica serovars were able to infect epithelial cells and the lamina propria. Notably, serovar Enteritidis showed the highest invasiveness of lamina propria tissue, whereas serovars Typhimurium and Hadar displayed moderate invasiveness and serovar Infantis hardly any invasion capabilities. Only a limited number of bacteria of all serovars were found within intestinal macrophages. Elevated numbers of granulocytes, CD8+ cells, and TCR1+ cells and mRNA expression rates for interleukin 12 (IL-12), IL-18, tumor necrosis factor alpha factor, and iNOS in cecum correlated well with the invasiveness of serovars in the lamina propria. In contrast, changes in numbers of TCR2+ and CD4+ cells and IL-2 mRNA expression seemed to be more dependent on infection of epithelial cells. The data indicate that the capability of Salmonella serovars to enter the cecal mucosa and invade lower regions affects both the level and character of the immune response in tissue.

Hematology, plasma biochemistry, and serosurvey for selected infectious agents in southern giant petrels from Patagonia, Argentina.

In conjunction with reproductive and feeding ecology studies on southern giant petrels (SGP, Macronectes giganteus) blood samples were collected for baseline health evaluations. Twenty-five adult SGP from a breeding colony in Chubut, Argentina, were sampled during two consecutive breeding seasons, 1999-2000 (n = 15) and 2000-01 (n = 10). Values for hematology, plasma biochemistry, and minerals are described for 20 birds in apparent good physical condition. A serologic survey of exposure to selected infectious agents was also conducted on all 25 birds sampled. Southern giant petrels were serologically negative for evidence of exposure to infectious laryngotracheitis virus, avian encephalomyelitis virus, avian influenza virus, avian reovirus, infectious bursal disease virus, infectious bronchitis virus, paramyxovirus 1, 2, and 3 virus, Chlamydophila, and Aspergillus. Antibodies to avian adenovirus were found in 14% of SGP during the first sampling season, and 60% in the second year. Additionally, all birds were negative for antibodies to Salmonella pullorum at the first sampling date, but 90% had low titers the following breeding season. This study contributes to understanding the health status of South Atlantic seabirds and to establishment of baseline information for SGP. Long-term monitoring of pelagic predator-scavenger seabirds such as SGP should be established for the surveillance of marine ecosystem health.

Comparison of the effects of infection with Salmonella enteritidis, in combination with an induced molt, on serum levels of the acute phase protein, alpha1 acid glycoprotein, in hens.

Periods of inflammation due to infection, injury, or malignancy are marked by increases in serum constituents known as acute phase proteins (APP), and these proteins have been used as markers for early stages of disease. Four experiments were performed to examine whether levels in chickens of one such APP, alpha1 acid glycoprotein (AGP), would be affected by an infection with Salmonella enteritidis (SE) and if the added stress of induced molting via 14-d feed withdrawal would increase these effects. In all experiments but Experiment 1, hens were divided into four equal groups: molted infected, nonmolted infected, molted noninfected, nonmolted non-infected (Experiment 1 lacked this last group). Blood and intestinal samples were collected at various times from the hens and assayed for AGP and SE levels, respectively. Infection with SE significantly elevated serum AGP levels above those found in the noninfected groups of hens in two of four experiments, whereas in molted infected hens, serum AGP levels were significantly higher than those found in the noninfected counterparts in all four experiments. Comparison of AGP titer between the infected groups of hens revealed that significantly higher SE levels generally did not guarantee significantly higher AGP levels, although when individual values were plotted, a trend was observed toward increasing serum levels concomitant with increasing SE counts. Serum AGP levels show promise as a method to detect infection problems in hens, especially when the severity of the infection is exacerbated by stress situations. However, more work is needed to determine what other factors may elevate serum AGP levels and potentially confound the picture.

Early cytokine response of gnotobiotic piglets to Salmonella enterica serotype typhimurium.

Cytokine response against Salmonella Typhimurium is traditionally studied in conventional animals. Germ-free animals, however, enable to study response against infection without background effect of other microorganisms. Plasma and ileal inflammatory cytokines in germ-free piglets orally infected with virulent LT2 strain or, with a non-virulent SF1591 rough mutant were quantified by ELISA. In plasma and ileal washes, IFN-gamma levels significantly increased in both infected groups. TNF-alpha and IL-18 were mostly missing in plasma 24 h after infection. In the ileum, IFN-gamma, TNF-alpha, and IL-1beta were induced mainly by the virulent strain, whereas IL-18 was induced in highest quantity by non-virulent Salmonella. These data confirmed an important role of IFN-gamma, as well as other inflammatory cytokines in early stage of salmonellosis.

Acute phase responses of pigs challenged orally with Salmonella typhimurium.

This study evaluated responses of the systemic endocrine stress (cortisol) and growth (IGF-I, GH) axes, as well as those of inflammatory mediators (prostaglandin E2 [PGE2] and tumor necrosis factor alpha [TNFalpha]), to active infection with Salmonella typhimurium. Eighteen crossbred barrows were penned individually with ad libitum access to feed and water. After an acclimation period, jugular catheters were placed in all animals. Control pigs received sterile broth orally (CON, n = 7), whereas the treated pigs (S.TYP, n = 11) received 3 x 10(9) cfu of S. typhimurium orally. Plasma was collected at 6-h intervals from -48 to 120 h. Body weights, feed intake, and rectal temperatures also were monitored. Rectal temperatures were elevated in S.TYP pigs (P < .01) relative to CON pigs by 12 h, peaked at 42 h (P < .001), and remained elevated throughout the remainder of the study. Feed intake was reduced maximally in S.TYP pigs at 48 h (P < .001) and remained reduced through 120 h after the challenge. Daily body weight gain also was reduced during the 2 wk following infection (P < .001). Plasma cortisol concentrations increased (P < .05) at 18 h after the challenge in S.TYP pigs and remained elevated generally until 60 h after infection. A marked suppression of plasma IGF-I occurred in S.TYP pigs beginning at 30 h after infection (P < .001), and it remained lower through 108 h. Plasma GH was not affected consistently by treatment, nor did infection alter plasma TNFalpha and PGE2. Taken together, the results reveal that infectious processes produce profound alterations in the endocrine stress and the somatotropic axis, and this may occur in the absence of significant changes in systemic proinflammatory mediators.

The early immune response in the liver of BALB/c mice infected with S. typhimurium.

Gram-negative bacteria acquired through gastrointestinal infection can be a serious cause for the development of septic shock especially in immunosuppressed patients. Thus, the aim of this study was to examine the early events of the immune reaction against S. typhimurium. Bacteria were injected into mice at different concentrations. Four animals from each group were killed at five different points of time. Liver cytokine mRNA expression was determined by semiquantitative rt-PCR and liver histology was examined. Serum cytokine levels of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-4 and IL-10 were determined. intravenous (i.v.) infection with 109 bacteria led to lethal septic shock within 24 h. A delayed production of IFN-gamma, TNF-alpha, IL-18 and IL-10 and milder histological alterations in the liver were observed in these animals. The highest expression of cytokines in the liver and the strongest histological alterations were seen after infection with 107 bacteria. Here, an increased mRNA expression of all proinflammatory cytokines began 1 h after infection. Animals infected with 1 x 102 bacteria had the highest detectable serum levels of IL-6 and IL-10. These data indicate that the immediate events in the immune reaction within the liver after infection with S. typhimurium are associated with the outcome of the subsequent sepsis.