Early epithelial invasion by Salmonella enterica serovar Typhimurium in the swine ileum

The purpose of the experiment was to define the early cellular invasion of Salmonella enterica serovar Typhimurium in a swine gut-loop model. Ileal gut-loops were surgically prepared in 4-5 week old pigs and inoculated with sterile media or Salmonella serovar Typhimurium for 0 to 60 minutes. Loops were harvested and prepared for both scanning and transmission electron microscopy. Preferential bacterial adherence to M cells was observed by 5 minutes postinoculation and apical invasion was seen in M cells, goblet cells and enterocytes by 10 minutes postinoculation. This nonspecific invasion was observed throughout the course of infection. The results suggest early invasion by Salmonella serovar Typhimurium is not cell-specific and calls into question the concept of cell-specific invasion sites. In addition, scanning electron micrographs showed a specific affinity by Salmonella serovar Typhimurium to sites of cell extrusion. Transmission electron micrographs depicted bacteria around and in the crevices formed by extruded cells and the adjacent cells, and in the cytoplasm immediately beneath extruded cells. The combination of scanning and transmission electron micrographs suggests Salmonella serovar Typhimurium may utilize sites of cell extrusion for early invasion in swine

Early Epithelial Invasion by Salmonella enterica Serovar Typhimurium DT104 in the Swine Ileum

Salmonella enterica serovar Typhimurium is an important intestinal pathogen in swine. This study was performed to document the early cellular invasion of Salmonellaserovar Typhimurium in swine ileum. Ileal gut-loops were surgically prepared in ten 4- to 5-week-old mixed-breed pigs and inoculated for 0-60 minutes. Loops were harvested and prepared for both scanning and transmission electron microscopy (SEM and TEM, respectively). Preferential bacterial adherence to microfold cells (M cells) was seen within 5 minutes, and by 10 minutes bacterial invasion of the apical membrane was seen in M cells, goblet cells, and enterocytes. This multicellular invasion was observed throughout the course of infection. In addition, SEM revealed a specific affinity of Salmonella serovar Typhimurium to sites of cell extrusion. Using TEM, bacteria in these areas were focused in the crevices formed by the extruding cell and the adjacent cells and in the cytoplasm immediately beneath the extruding cell. Our results suggest that early cellular invasion by Salmonella serovar Typhimurium is nonspecific and rapid in swine. Furthermore, the combination of SEM and TEM data suggests that Salmonella serovar Typhimurium may use sites of cell extrusion as an additional mechanism for early invasion

Salmonella Typhimurium infections in pigs: a closer look at the pathogenesis

Salmonellose bij de mens wordt vaak veroorzaakt door Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium). De ziekte veroorzaakt door deze bacterie is voornamelijk geassocieerd met het eten van varkensvlees. Varkens die geïnfecteerd zijn met Salmonella Typhimurium zijn meestal symptoomloze dragers. De mechanismen die Salmonella Typhimurium gebruikt om deze dragerdieren blijvend te infecteren zijn niet gekend. Het doel van deze thesis was om inzicht te verkrijgen in de mechanismen die Salmonella Typhimurium gebruikt om varkens te kolonizeren en er in te persisteren. In het eerste hoofdstuk van deze thesis werd een Salmonella Typhimurium stam geselecteerd die een persisterende infectie bij varkens kan veroorzaken. Hiervoor werd een veldstam, geïsoleerd uit een persistent geïnfecteerd varken, vergeleken met een standaard laboratorium stam die vaak wordt gebruikt voor onderzoek in muizen. De laboratorium stam was virulenter in muizen, maar de varkensstam was efficiënter in het veroorzaken van een persisterende infectie bij varkens. Deze varkensstam werd gekozen om te gebruiken in verdere experimenten. In het tweede hoofdstuk werden verschillende in vitro en in vivo modellen op punt gesteld om de pathogenese van Salmonella Typhimurium infecties bij varkens te onderzoeken. In het derde hoofdstuk werd de rol van verschillende virulentiegenen van Salmonella in de pathogenese van Salmonella Typhimurium infecties bij het varken onderzocht. In een eerste reeks experimenten werd de rol nagegaan van de genen die gelegen zijn op het Salmonella Pathogeniciteitseiland 1 (SPI-1). Deze genen waren essentieel voor Salmonella Typhimurium om varkensdarmcellen en macrofagen te kunnen binnen dringen. Alle SPI-1 mutanten waren sterk verzwakt in hun vermogen om diarree te veroorzaken. Zowel vroege als late celdood werd gezien in de macrofagen, maar enkel de vroege celdood bleek SPI-1 afhankelijk. Wanneer varkens peroraal geïnfecteerd werden met een combinatie van de Salmonella Typhimurium veldstam enerzijds en een SPI-1 deletiemutant anderzijds, bleek dat de mutant stam sterk verzwakt was in het invaderen en kolonizeren van de darmen, maar niet in het kolonizeren van de tonsillen. In een tweede reeks experimenten werd de rol nagegaan van de genen die gelegen zijn op het Salmonella Pathogeniciteitseiland 2 (SPI-2). Hierbij werd gebruik gemaakt van een mutant in het ssrA gen. Deze mutant was minder goed in staat om in vitro in macrofagen te vermeerderen en om de organen te kolonizeren van varkens die intraveneus geïnfecteerd werden. Bij biggen die oraal geïnoculeerd werden met de ssrA mutant stam verliep de infectie evenwel gelijkaardig in vergelijking met biggen die geïnoculeerd waren met de wild type stam. In een derde reeks experimenten, werd het belang van het fibronectine bindend eiwit ShdA in het ontstaan van een persisterende infectie onderzocht. Alhoewel dit gen bij muizen recent geïdentificeerd is als een belangrijke factor in de intestinale kolonizatie en persistentie in muizen, kon dit in deze experimenten niet worden bevestigd voor Salmonella Typhimurium infecties bij varkens. De resultaten van deze thesis tonen aan dat zowel de gastheermodellen als de Salmonella stammen met zorg gekozen moeten worden om relevant onderzoek te kunnen doen naar de pathogenese van Salmonella infecties. Tevens kan geconcludeerd worden dat SPI-1 afhankelijke invasie cruciaal is voor de kolonizatie van de darmen, maar niet van de tonsillen. De bijdrage van SPI-2 en shdA tot de persistentie van Salmonella Typhimurium in varkens is veel kleiner dan beschreven voor muizen

Host adapted serotypes of <i>Salmonella enterica</i>

Salmonella constitutes a genus of zoonotic bacteria of worldwide economic and health importance. The current view of salmonella taxonomy assigns the members of this genus to two species: S. enterica and S. bongori. S. enterica itself is divided into six subspecies, enterica, salamae, arizonae, diarizonae, indica, and houtenae, also known as subspecies I, II, IIIa, IIIb, IV, and VI, respectively. Members of Salmonella enterica subspecies enterica are mainly associated with warm-blooded vertebrates and are usually transmitted by ingestion of food or water contaminated by infected faeces. The pathogenicity of most of the distinct serotypes remains undefined, and even within the most common serotypes, many questions remain to be answered regarding the interactions between the organism and the infected host. Salmonellosis manifests itself in three major forms: enteritis, septicaemia, and abortion, each of which may be present singly or in combination, depending on both the serotype and the host involved. Although currently over 2300 serovars of Salmonella are recognized, only about 50 serotypes are isolated in any significant numbers as human or animal pathogens and they all belong to subspecies enterica. Of these, most cause acute gastroenteritis characterized by a short incubation period and a severe systemic disease in man or animals, characterized by septicaemia, fever and/or abortion, and such serotypes are often associated with one or few host species. It is the intention of this review to present a summary of current knowledge of these host-adapted serotypes of S. enterica. The taxonomic relationships between the serotypes will be discussed together with a comparison of the pathology and pathogenesis of the disease that they cause in their natural host(s). Since much of our knowledge on salmonellosis is based on the results of work on Typhimurium, this serotype will often be used as the baseline in discussion. It is hoped that an appreciation of the differences that exist in the way these serotypes interact with the host will lead to a greater understanding of the complex host–parasite relationship that characterizes salmonella infections

Tissue-specific Salmonella Typhimurium gene expression during persistence in pigs

Salmonellosis caused by Salmonella Typhimurium is one of the most important bacterial zoonotic diseases. The bacterium persists in pigs resulting in asymptomatic 'carrier pigs', generating a major source for Salmonella contamination of pork. Until now, very little is known concerning the mechanisms used by Salmonella Typhimurium during persistence in pigs. Using in vivo expression technology (IVET), a promoter-trap method based on Delta purA attenuation of the parent strain, we identified 37 Salmonella Typhimurium genes that were expressed 3 weeks post oral inoculation in the tonsils, ileum and ileocaecal lymph nodes of pigs. Several genes were expressed in all three analyzed organs, while other genes were only expressed in one or two organs. Subsequently, the identified IVET transformants were pooled and reintroduced in pigs to detect tissue-specific gene expression patterns. We found that efp and rpoZ were specifically expressed in the ileocaecal lymph nodes during Salmonella peristence in pigs. Furthermore, we compared the persistence ability of substitution mutants for the IVET-identified genes sifB and STM4067 to that of the wild type in a mixed infection model. The Delta STM4067::kanR was significantly attenuated in the ileum contents, caecum and caecum contents and faeces of pigs 3 weeks post inoculation, while deletion of the SPI-2 effector gene sifB did not affect Salmonella Typhimurium persistence. Although our list of identified genes is not exhaustive, we found that efp and rpoZ were specifically expressed in the ileocaecal lymph nodes of pigs and we identified STM4067 as a factor involved in Salmonella persistence in pigs. To our knowledge, our study is the first to identify Salmonella Typhimurium genes expressed during persistence in pigs

Quantitative analysis of the immune response upon Salmonella typhimurium infection along the porcine intestinal gut

Salmonella enterica serovar Typhimurium causes enteric disease and compromises food safety. In pigs, the molecular response of the intestine to S. typhimurium has been traditionally characterized by in vitro models that do not reflect the actual immunological competence of the intestinal mucosa. In this work, we performed an oral S. typhimurium infection study to obtain insight into the in vitro response in three different sections (jejunum, ileum and colon) of the porcine intestine. For this, samples from one-month-old infected piglets were collected during a time course comprising 1, 2, and 6 days post inoculation to evaluate the intestinal response by quantifying the mRNA expression of gene coding for 28 innate immune system molecules using quantitative real-time PCR assays. In addition, samples from non-infected control animals were also employed to establish differences in the steady state gene expression between intestinal sections. The panel of quantified molecules included an assortment of cytokines, chemokines, pattern-recognition receptors, intracellular signaling molecules, transcription factors and antimicrobial molecules. Changes in gene expression occurred in the three different parts of the intestine and during the course of the S. typhimurium infection. Moreover, the high variation observed in expression patterns of genes coding for inflammatory mediators could indicate that each intestinal section responds differently to the infection. Thus, on the contrary to findings in the jejunum and colon, a down-regulation and lack of induction of some proinflammatory cytokine transcripts was observed in the ileum. Nevertheless, all chemoattractant cytokines assayed were up-regulated in the ileum and jejunum whereas only interleukin-8 and MIP-1α mRNA were over expressed in the colon. In conclusion, our results reveal regional differences in gene expression profiles along the porcine intestinal gut as well as regional differences in the inflammatory response to S. typhimurium infection. Taken together, these data should provide a basis for a complete understanding of the porcine intestinal response to bacterial infection

An update on pathogenesis and control of Salmonella infections in pigs

C

Pathophysiological effects of oral in[n]oculation of growing pigs with Salmonella enterica serovars Typhimurium or Choleraesuis

Master of ScienceDepartment of Animal Sciences and IndustryJ. Ernest MintonEnteric pathogens are responsible for major economic losses in the swine industry. In the U.S., Salmonella enterica subspecies enterica serovar Typhimurium (ST) and serovar Choleraesuis (SC) account for essentially all cases of salmonellosis in swine. Previous studies documented that oral ST eroded growth and produced unmistakable changes in the endocrine stress and somatotropic axis of young growing pigs. However, these effects occurred in the absence of elevated systemic inflammatory cytokines that were previously thought to accompany disease-associated growth retardation. In the current study, it was hypothesized that SC would produce very different systemic inflammatory cytokine responses compared to ST given the likelihood of SC to produce systemic disease in pigs. Weaned pigs were housed two per pen with free access to feed and water during a 14 d experiment. On d 0, pigs were fed either 108 CFU SC or 108 ST, and bacteria were re-fed twice weekly through the course of the experiment. Control pigs were fed dough without bacteria. Serum was collected on d 0, 7, and 14 for determination of tumor necrosis factor alpha (TNFα), interleukin-1beta (IL-1β), and insulin-like growth factor-I (IGF-I) were determined. Rectal temperatures (RT) were monitored daily beginning 2 d prior to challenge with bacteria and until 7 d following the first bacterial feeding. Pigs were weighed initially, and at the conclusion of the study. Daily body weight gain was reduced by 25.4% in pigs fed SC (P<.0001) compared to control, while growth was similar between control pigs and those fed ST. Pigs fed SC had increased RT beginning on d 2 and continuing though d 7 (P < 0.05) with the greatest elevation spike on d 3 (P < 0.001) when compared to controls. On d 7, pigs fed SC had reduced IGF-I when compared to both control (P < 0.01) and ST pigs (P = 0.01). Despite the obvious febrile response, and the reductions in body weight gain and serum IGF-I, circulating TNFα and IL-1β were not affected by treatment. It was concluded that elevated TNFα and IL-1β are not obligatory correlates of SC-induced pathology and growth retardation in weaned pigs

Transcriptional analysis of porcine intestinal mucosa infected with Salmonella Typhimurium revealed a massive inflammatory response and disruption of bile acid absorption in ileum

Infected pork meat is an important source of non-typhoidal human salmonellosis. Understanding of molecular mechanisms involved in disease pathogenesis is important for the development of therapeutic and preventive strategies. Thus, hereby we study the transcriptional profiles along the porcine intestine during infection with Salmonella Typhimurium, as well as post-transcriptional gene modulation by microRNAs (miRNA). Sixteen piglets were orally challenged with S. Typhimurium. Samples from jejunum, ileum and colon, collected 1, 2 and 6 days post infection (dpi) were hybridized to mRNA and miRNA expression microarrays and analyzed. Jejunum showed a reduced transcriptional response indicating mild inflammation only at 2 dpi. In ileum inflammatory genes were overexpressed (e.g., IL-1B, IL-6, IL-8, IL1RAP, TNFα), indicating a strong immune response at all times of infection. Infection also down-regulated genes of the FXR pathway (e.g., NR1H4, FABP6, APOA1, SLC10A2), indicating disruption of the bile acid absorption in ileum. This result was confirmed by decreased high-density lipoprotein cholesterol in serum of infected pigs. Ileal inflammatory gene expression changes peaked at 2 dpi and tended to resolve at 6 dpi. Furthermore, miRNA analysis of ileum at 2 dpi revealed 62 miRNAs potentially regulating target genes involved in this inflammatory process (e.g., miR-374 and miR-451). In colon, genes involved in epithelial adherence, proliferation and cellular reorganization were down-regulated at 2 and 6 dpi. In summary, here we show the transcriptional changes occurring at the intestine at different time points of the infection, which are mainly related to inflammation and disruption of the bile acid metabolism. © 2016 Uribe et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated