Infection dynamics of Salmonella Infantis strains displaying different genetic backgrounds - with or without pESI-like plasmid - vary considerably.

Food-borne infections with Salmonella are among the most common causes of human diseases worldwide, and infections with the serovar Infantis are becoming increasingly important. So far, diverse phenotypes and genotypes of S. Infantis have been reported. Therefore, the present study aimed to investigate the infection dynamics of two different S. Infantis strains in broilers. For this purpose, 15 birds were infected on day 2 of life with 108 CFU/ml of a pESI+ or a pESI- S. Infantis strain, respectively. Ten uninfected birds served as in-contact birds to monitor transmission. In both groups, an increase of infection was observed from 7 days of age onwards, reaching its peak at 28 days. However, the pESI+ strain proved significantly more virulent being re-isolated from most cloacal swabs and organs by direct plating. In contrast, the pESI- strain could be re-isolated from cloacal swabs and caeca only when enrichment was applied. Although the excretion of this strain was limited, the transmission level to in-contact birds was similar to the pESI+ strain. Differences in infection dynamics were also reflected in the antibody response: whereas the pESI+ strain provoked a significant increase in antibodies, antibody levels following infection with the pESI- strain remained in the range of negative control birds. The actual findings provide for the first time evidence of S. Infantis strain-specific infectivity in broilers and confirm previous observations in the field regarding differences in persistence on farms and resistance against disinfectants.

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.

Effects of two probiotic spores of Bacillus species on hematological, biochemical, and inflammatory parameters in Salmonella Typhimurium infected rats.

Salmonella infections have become a major health concern in recent decades. This pathogen has evolved to become resistant to antibiotics, which has caused problems in its treatment. As such, finding a novel preventive method is important in the treatment and management of this infection. In recent years, uses of probiotics, especially spore-former genera such as Bacillus spp. has become increasingly popular. In this study spores of two probiotic bacteria, Bacillus subtilis and Bacillus coagulans were fed to rats for three weeks through their daily water intake after which Salmonella Typhimurium was gavaged to the rats. On days 1, 3, 5 and 7 after gavaging, the number of Salmonella was counted in liver, spleen, mesenteric lymph nodes, feces and content of ileum and cecum. Hematological and biochemical parameters, inflammatory mediators, total antioxidant capacity and malondialdehyde were also measured. The results showed that B. subtilis and B. coagulans caused delation in infiltration of Salmonella into the lymph nodes, spleen and liver, reduction of the inflammatory mediators, and decreases in oxidative stress, hematological and biochemical changes. The overall count of Salmonella in the above mentioned parameters has also decreased and a faster return to normal base were also witnessed. The results showed that the use of B. subtilis and B. coagulans can potentially help boost the body's immune system, to combat the effects of exposure to the Salmonella pathogen.

Analyses of genetics and pathogenesis of Salmonella enterica QH with narrow spectrum of antibiotic resistance isolated from yak.

Salmonella is an important pathogen for public health due to food poisoning and acute infectious intestinal disease by zoonotic trait. We isolated Salmonella enterica QH which represents the normal growth condition in Luria-Bertani culture and displays a wide range of susceptibility for multiple antibiotics. To further investigate genetic and pathogenic traits of S. enterica QH, the sequencing genome of S. enterica QH and oral Salmonella infection in mice were performed in this study. Compared with other Salmonella strains, several large sequences containing prophages and genomic islands were inserted into S. enterica QH genome. Furthermore, nucleotide and synonymous codon usage patterns display mutation pressure and natural selection serving as drivers for the evolutionary trend of S. enterica QH at gene level. The unique codon usage pattern of S. enterica QH probably contributes to adaptation to environmental/host niches and to pathogenicity. In an early oral S. enterica QH infection, the levels of CD4+ and CD8+ lymphocytes significantly reduce in peripheral blood of mice, but the increasing transcription levels of some cytokines (IFN-ß1, IFN-γ and CXCL10) might have pleiotypic immune effects against S. enterica QH infection. Of note, IL10 displays significant enhancement at levels of transcription and translation, suggesting that immunosuppressive effects mediated by IL10 may function as an early oral S. enterica QH infection. The systemic investigations, including genomic and genetic characterizations and biological traits of S. enterica QH in vivo and in vitro may reflect the basic lifestyle of S. enterica QH, requiring intestine colonization, undergoing environmental stresses and performing dissemination.

Outbreak investigation of septicemic salmonellosis in calves.

INTRODUCTION: An early and accurate diagnosis of septicemic salmonellosis is critical for implementing timely and proper treatment, prevention, and control measures. METHODOLOGY: Here, we report a study on three outbreaks of septicemic salmonellosis in calves from Midwestern Brazil. RESULTS: the morbidity, mortality and lethality rates were of 10.55%, 2.79%, and 26.4%, respectively. Higher susceptibility was detected in Bos taurus than in Bos indicus cattle. Clinical manifestations consisted of apathy, hyperthermia, difficulty breathing and panting, and pallor of the mucous membranes. Chronic cases had necrosis of the tail tip and ears. Gross findings included enlarged liver, non-collapsed edematous lungs and diphtheritic enteritis. Significant histopathological changes included paratyphoid nodules in the liver and acute interstitial pneumonia. Salmonella enterica subsp. enterica serotype Dublin was detected by culture and by PCR from the blood of live calves, and from the spleen, liver, bile, mesenteric lymph node and lung samples of necropsied calves. CONCLUSIONS: We suggest that in clinical cases of septicemic salmonellosis, blood samples are better than fecal samples for detection of the agent, being a sound test to identify animal carriers in the herd.

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.

Assessment of the feasibility of serological monitoring and on-farm information about health status for the future meat inspection of fattening pigs.

Current macroscopic meat inspection cannot detect the most common pork-borne pathogens (Salmonella spp., Yersinia enterocolitica and Toxoplasma gondii). Furthermore, food chain information (FCI) may not provide sufficient data for visual-only inspection, which is supposed to be the common way of inspection of pigs in the European Union. Our observational study aimed to evaluate the serological monitoring and the clinical evaluation of on-farm health status of pigs and assess the feasibility of these data as part of the FCI in meat inspection. We studied the serological status of Salmonella spp., Yersinia spp. and T. gondii in pigs during the fattening period. Additionally, we evaluated the association between on-farm health status and meat inspection findings. On 57 indoor fattening pig farms in Finland, we collected blood samples (mean of 20 pigs/farm) and assessed the on-farm health (coughing, tail biting, lameness) at the end of the fattening period. We visited 34 of these farms also at the beginning of the fattening for sampling and on-farm health evaluation of the same pigs. Meat inspection results were obtained after slaughter for all 57 farms. Salmonella seroprevalence was low at the end of the fattening period: it was 17.6%, 10.6% or 1.9%, with the cut-off values of OD15% (recommended by the test manufacturer), OD20% (used by Danish monitoring programme) and OD40% (used by German monitoring programme), respectively. The overall seroprevalence of Salmonella spp. and Yersinia spp. increased significantly (P < 0.001) during the fattening period (from 8.1% to 17.2% and from 30.3% to 72.3%, respectively), while the seroprevalence of T. gondii remained low (<1%). The within-farm seroprevalences of Salmonella spp. and Yersinia spp. differed significantly between the farms and this farm-level serological data could be used as FCI for risk-based decisions to improve food safety. Such potentially feasible decisions could include additional carcass testing, carcass decontamination, carcass processing, slaughtering arrangements and improved biosecurity measures at the farm. However, risk mitigation targets and procedures must be carefully adjusted for each pathogen regarding also economic aspects. Tail biting observed on farm was associated with partial carcass condemnations and arthritis at slaughter. This information could be included in the FCI and used when making decisions regarding meat inspection procedure: visual-only or additional inspections.

Antibody Responses to Salmonella in Pigs from Weaning Up to Marketing and Presence of Salmonella at Slaughter.

Salmonella is estimated to be one of the leading causes of enteric illness worldwide. Human salmonellosis is most frequently related to contaminated food products, particularly those of animal origin, such as pork. Pigs are often asymptomatic carriers of Salmonella, highlighting the importance of identifying high-prevalence farms and effective detection methods. The objectives of this study were to investigate Salmonella antibody responses and their association with on-farm shedding and Salmonella isolation at slaughter. Fourteen groups of pigs from eight farrowing sources were followed from birth to slaughter (totaling 796 pigs). Information about farm management was collected through a questionnaire. Blood and fecal samples were collected four times at different stages of production, and palatine tonsils/submandibular lymph nodes were obtained at slaughter. Sera were tested for Salmonella antibodies by enzyme-linked immunosorbent assay, and fecal/tissue samples were cultured for Salmonella. Data were analyzed using a mixed-effect multivariable modeling method with farm, litter, and pig as random effects. Salmonella seropositivity rates were 20.3%, 5.8%, 15.9%, and 37.3% at weaning, at the end of nursery, at end of grower, and at end of finisher, respectively. Salmonella seropositivity and shedding increased with age (p < 0.05), and pigs shedding Salmonella were more likely to test seropositive (p = 0.02). Antibody response and shedding on-farm had no significant association with isolation of Salmonella from tissues harvested at slaughter. The variation in Salmonella seropositivity due to farm was 28.9% of total variation. These findings indicate that on-farm intervention may be a more effective approach to control Salmonella and to reduce the presence of Salmonella at slaughter. Additionally, the observation that some pigs in this study were Salmonella-negative throughout production and at slaughter is promising with regard to food safety, and studies are needed to explore the genotypes of those pigs.

Protein expression in the liver and blood serum in chickens in response to Salmonella Enteritidis infection.

Events occurring in the chicken caecum following Salmonella Enteritidis infection are relatively well-described. However, mechanisms of the immune response and defence beyond the intestinal tract are less well-described. In this study, we therefore determined changes in protein abundance in the liver and blood serum in response to S. Enteritidis infection using the unbiased approach of shotgun proteomics. Complement and coagulation cascades, TNF signalling, antigen processing and presentation was activated in the liver following infection with S. Enteritidis. Chicken proteins that decreased in the liver were involved in glycolysis, the citrate cycle, oxidative phosphorylation and fatty acid metabolism. No functional category was significantly activated or suppressed in the serum. Concerning individual proteins, VNN1, SAA, AVD, SERPINA3, SERPINB10, AGT, MRP126 or CP increased in abundance both in the liver and serum. MT4, MT3, PTGDS, GLRX and TGM4, though highly inducible in the liver, did not increase in the serum. PIGR, SERPINF2 and IGJ increased in the serum but not in the liver. SERPINA4, apoAIV, CLEC3B, SERPINF1, HRG, AHSG and ALB decreased both in the liver and serum. Avidin-like LOC431660, THRSP, GATM, GGACT, ACOX1, ALDOB or FABP7 decreased in the liver but not in the serum. Finally, CKM, CKB, PLTP, COMP, IGFALS, AMY1A or SERPIND1 decreased in the serum after S. Enteritidis infection but not in the liver. Differently abundant proteins characterise the chicken's response to infection and can be also used as markers of chicken health status.

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.

A bead-based suspension array for the detection of Salmonella antibodies in pig sera.

BACKGROUND: Slaughter pigs are monitored for the presence of the zoonotic pathogen Salmonella, using both serology and bacteriology. ELISAs used to investigate pig herds are based on the detection of antibodies against components of the Salmonella cell envelope. Nearly all Salmonella isolates in food-producing animals are serovars of Salmonella enterica subspecies enterica, distributed over various serogroups as determined by the composition of their lipopolysaccharide (LPS). ELISAs for Salmonella serology are usually based on serogroup B and C1 LPS, often combined with serogroup D or E LPS. Although C2 LPS may improve serology, use of C2 LPS in a broad ELISA was never achieved. RESULTS: To enable detection of serum antibodies against Salmonella in pigs, a bead-based suspension array was developed with five LPS variants (B, 2× C1, C2, D1), each conjugated to a different bead set using triazine chemistry. Reactivity of the beads was confirmed with rabbit agglutination sera and with experimental pig sera. With a mixture of bead sets, 175 sera from slaughter pigs were investigated for the presence of antibodies against Salmonella. With a combination of ROC analysis (B and D LPS) and a prevalence estimation based on historic data (C LPS), individual cut-offs were defined for each LPS-conjugated bead set, and assay performance was evaluated. Results of the suspension array (BC1C1C2D) suggest that more pigs are seroconverted than indicated by a commercial BC1D1-ELISA, and that most of these extra seropositive samples give a signal on one of the beads with C LPS. These results show that expansion of a standard panel with more C LPS variants improves antibody detection. CONCLUSIONS: A suspension array for Salmonella serology in pigs was developed, that detects more seropositive sera than ELISA, which is achieved by expanding the panel of Salmonella LPS variants, including C2 LPS. The results demonstrate that bead-based suspension arrays allow for testing of pig sera, with the advantage of being able to set cut-offs per antigen. Ultimately, this type of assay can be applied in routine veterinary serology to test for antibodies against multiple Salmonella serovars (or other pathogens) in one single serum sample, using up-to-date antigen panels.

Time course of Salmonella shedding and antibody response in naturally infected pigs during grower-finisher stage.

A longitudinal trial was conducted to determine the course of Salmonella shedding and antibody response in naturally infected grower-finisher pigs. Ten-week-old pigs (n = 45) were transferred from a farm with history of salmonellosis and housed at a research facility. Weekly fecal samples (weeks 1 to 11) as well as tissue samples at slaughter were cultured for Salmonella. Serum samples were tested for presence of Salmonella antibody by enzyme-linked immunosorbent assay (ELISA). Data were analyzed using a multilevel mixed-effects logistic regression model. Over 10 wk, 91% and 9% of pigs shed Salmonella ≤ 4 and > 5 times, respectively. The estimated median of Salmonella shedding duration was 3 to 4 wk but some pigs shed Salmonella for up to 8 wk. Salmonella shedding increased 1 wk post-arrival but followed a decreasing pattern afterwards up to week 11 (P < 0.05). Salmonella isolates (n = 29), which were recovered from 18 pigs at different occasions, were S. Typhimurium (28%), S. Livingstone (21%), S. Infantis (14%), S. Montevideo (7%), S. Benfica (3%), S. Amsterdam (3%), S. Senftenberg (17%), and S. I:Rough-O (7%). Of 11 pigs from which the first and last isolates were serotyped, 10 pigs were reinfected with a different serotype. At slaughter, Salmonella was isolated from 7 pigs, of which 5 (71%) had not tested positive for at least 7 wk prior to slaughter. Antibody response peaked 4 wk after the peak of Salmonella infection; Salmonella shedding reduced as antibody response elevated (P < 0.05). These findings indicate that pigs may shed Salmonella into the mid-point of the grower-finisher stage and may be reinfected with different serotypes.

Un essai longitudinal a été réalisé afin de déterminer la progression de l'excrétion de Salmonella et la réponse en anticorps chez des porcs en période de croissance-finition naturellement infectés. Des porcs âgés de 10 semaines (n = 45) ont été transférés d'une ferme avec une histoire de salmonellose et hébergés dans une installation de recherche. Des échantillons de fèces ont été prélevés à chaque semaine (semaines 1 à 11) de même que des échantillons de tissus lors de l'abattage et ont été cultivés pour Salmonella. Des échantillons de sérum ont été testés pour la présence d'anticorps contre Salmonella par une épreuve immunoenzymatique (ELISA). Les résultats ont été analysés par un modèle de régression logistique multiniveaux à effets mixtes. Au-delà de 10 semaines, 91 % et 9 % des porcs excrétaient Salmonella ≤ 4 et > 5 fois, respectivement. La médiane estimée de la durée d'excrétion de Salmonella était de 3 à 4 sem mais quelques porcs ont excrété Salmonella jusqu'à 8 sem. L'excrétion de Salmonella augmenta 1 sem après l'arrivée mais fut suivie par la suite d'un patron de diminution jusqu'à la semaine 11 (P < 0,05). Les isolats de Salmonella (n = 29) qui ont été obtenus de 18 porcs à différentes occasions, étaient S. Typhimurium (28 %), S. Livingstone (21 %), S. Infantis (14 %), S. Montevideo (7 %), S. Benfica (3 %), S. Amsterdam (3 %), S. Senftenberg (17 %) and S. I :Rough-O (7 %). De 11 porcs pour lesquels les premiers et les derniers isolats furent sérotypés, 10 porcs étaient réinfectés avec un sérotype différent. Lors de l'abattage, Salmonella a été isolé de sept porcs, parmi lesquels cinq (71 %) n'avaient pas eu de culture positive pour au moins 7 sem avant l'abattage. La réponse en anticorps a présenté un pic 4 sem après le pic d'infection par Salmonella; l'excrétion de Salmonella a diminué alors que la réponse en anticorps augmentait (P < 0,05). Ces résultats indiquent que les porcs peuvent excréter Salmonella jusqu'au milieu de leur période de croissance-finition et peuvent être réinfectés avec un sérotype différent.(Traduit par Docteur Serge Messier).

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.

Looking for new approaches for the use of serology in the context of control programmes against pig salmonellosis.

Most swine Salmonella national control programmes in Europe have been based on the categorization of herds according to risk levels based on serological results. However, none of the non-Scandinavian countries have reported of any significant success on Salmonella infection reduction in fattening pigs or the number of human cases attributable to pigs or pork. The limited accuracy of the tests used, the small number of animals sampled and the likely lack of herd representativeness of the samples used could be major factors affecting the suitability of these programmes. Focusing on minimizing Salmonella shedding at slaughter appears more important to prevent human infections than focusing on detection of seropositive pigs/herds at this stage. This study assessed whether performing on-farm serology may help to predict shedding at slaughter. Between 2010 and 2016, pigs from six cohorts from a Salmonella-positive herd were bled at 30, 60 and 90 days on fattening and before slaughter, and faecal samples collected at slaughter. Serology on days 60, 90 and before slaughter predicted somewhat shedding at slaughter with no significant differences among them. Pigs with higher OD% values at these point times would have higher risk of shedding when arriving to slaughter. The probability of shedding for a pig sampled on day 90 and showing an OD% value of 10 was 43%, and the risk increased up to 65% if the OD% was 40. Concluding, on-farm serology may help to determine to some extent the risk of Salmonella shedding at slaughter from seropositive fattening units, which would allow for prompt on-farm and slaughter interventions to reduce the likelihood of slaughter contamination with Salmonella.

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.

CASE SERIES: CLINICAL SALMONELLOSIS IN FOUR BLACK RHINOCEROS (DICEROS BICORNIS) CALVES.

Although Salmonella spp. infection has been identified in captive and free-ranging rhinoceros, clinical cases in black rhinoceros ( Diceros bicornis ) calves have not been described. This case series describes clinical salmonellosis in four black rhinoceros calves. Two calves developed self-limiting diarrhea, recovering after treatment. The other two cases were fatal. One of the fatal cases had a short clinical course, whereas the other case was protracted, with signs reflecting multiple organ system involvement. In all cases, diagnosis was by fecal culture and/or quantitative polymerase chain reaction. A variable clinical presentation, which is typical for salmonellosis in domestic hoofstock, was a feature of these rhinoceros cases. Similarly, postmortem pathology in black rhinoceros calves was consistent with domestic neonatal ungulates with salmonellosis. Potential predisposing factors for infection were considered to be primiparity of the dam and failure of passive transfer in the calf. The case investigation included attempts to identify the source of infection, which was aided by organism serotyping. In one case, the patient's dam and another conspecific in the facility were shown to be asymptomatic shedders of the organism strain responsible for disease in the calf. Further surveillance of captive rhinoceros Salmonella spp. carrier status is needed to inform screening recommendations for this taxa.