Ileocaecal Nodes Research Articles

Diagnostic performance of faecal and tissue multiplex qPCR IS900/F57 for the detection of Mycobacterium avium subspecies paratuberculosis in cattle

Mycobacterium avium subspecies paratuberculosis (MAP) is responsible for bovine-paratuberculosis (bPTB), which causes high production losses in cattle. A cross-sectional study was conducted in 228 cattle to evaluate the validity and diagnostic utility of a multiplex real-time PCR (qPCR) on faecal and intestinal samples [ileocaecal valve (ICV) and ileocaecal lymph nodes (ICLN)], using intestinal tissue culture as a reference test.Based on the sensitivity, specificity, and likelihood ratios (LR) obtained, the diagnostic value of faecal qPCR for confirming MAP infection was moderate (sensitivity 50.3%, specificity 93.5%, positive LR 7.8), and low to rule it out (negative LR 0.5). In areas with a prevalence of >23% the credibility of positive results was higher than 70%. In the case of negative results, their credibility was higher than 90% in herds with an infection rate below 19%, so faecal qPCR would be very useful in these areas to certify the absence of infection. For post-mortem diagnosis, qPCR on ICV samples showed good diagnostic accuracy to confirm the disease (sensitivity 71.7%, specificity 93.3%, positive LR 10.8), with a credibility higher than 70% in animals from areas or herds with a prevalence of infection greater than or equal to 18%. The best strategy to rule out the disease was the parallel combination of both tissues (ICV + ICLN) (sensitivity 81.3%, specificity 89.5%, negative LR 0.2) with a credibility of over 95% in animals from areas with an infection prevalence of 0–20%. Faecal and tissues qPCR techniques can be used to monitor bPTB, the interpretation of results, according to epidemiological situation of the herd or area, are shown.

Prevalence of Salmonella spp. in slaughter pigs and carcasses in Irish abattoirs and their antimicrobial resistance

BackgroundSalmonella is an important zoonotic pathogen and is one of the main causes of foodborne outbreaks and infections in the European Union. Pigs are a significant reservoir and are frequently subclinical carriers of this organism. Salmonella can be shed in the faeces allowing infection to spread to other pigs, the environment, transport vehicles, lairages and other areas. Inadvertent spillage of gut contents during the slaughter process also leads to contamination. A pig Salmonella control programme has operated in Ireland since 2002 but many local surveys and an EUMS baseline survey in 2008 continued to indicate high levels of the organism in the pig sector. The objectives of this study were to generate updated information on the prevalence of Salmonella spp, in slaughter pigs and carcasses in Irish abattoirs. Five pigs from each of 164 herds were randomly sampled over a 14-week period during 2016. One sample from each of the five pigs of; caecal content, ileo-caecal lymph nodes and carcass swabs (pre-chill) were collected. The five caeca and lymph node samples from each herd were processed as one pool of caecal samples and one pool of lymph node samples, respectively, while the five carcass swabs were tested as individual samples. All isolates were characterised by serotyping and antimicrobial susceptibility.ResultsIn total, 235 Salmonella spp. were isolated from 820 individual carcass swabs, 164 pooled lymph nodes and 164 caecal contents. Salmonella spp. were isolated from 54.3% of the caecal contents and from 31.7% of the ileo-caecal lymph node sample pools. A total of 11.5% of carcass-swab samples yielded Salmonella spp. S. Typhimurium 4,[5],12:i:1,2 or its monophasic variant 4,[5],12:i:-: predominated among isolates from all positive samples; accounting for 73% of lymph nodes, 68% of caecal contents and 56% of carcass swab isolates. S. London and S. Derby were the next most common isolated serotypes.ConclusionsThese results confirm continuing high levels of Salmonella in fattening pigs in Ireland although reductions in carcass contamination compared to previous surveys were noted. A high prevalence of Salmonella in lymph nodes suggests that it remains a significant problem pre slaughter and a challenge to abattoirs in adhering to process hygiene requirements. The high prevalence of monophasic S. Typhimurim 4,[5],12:i:-: is of serious concern.Therefore, it is important to identify contributing factors in the dissemination of this pathogen in the pork industry in order to minimise the risk of human salmonellosis cases.

Parasite-Probiotic Interactions in the Gut: Bacillus sp. and Enterococcus faecium Regulate Type-2 Inflammatory Responses and Modify the Gut Microbiota of Pigs During Helminth Infection.

Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and fecal microbiota, notably including an enrichment of fecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.

Fasciola hepatica products can alter the response of bovine immune cells to Mycobacterium avium subsp. paratuberculosis.

BackgroundFasciola hepatica causes economically important disease in livestock worldwide. The relevance of this parasitic infection extends beyond its direct consequences due to its immunoregulatory properties.ObjectivesGiven the importance of the T helper 1 (Th1) immune response in controlling infections with Mycobacterium avium subspeciesparatuberculosis (MAP) in cattle, we aimed to establish the immunological consequences that co‐infection with F. hepatica might have on the course of Johne’s disease (JD).MethodsThis study compared the in vitro response of bovine immune cells to infection with MAP or exposure to MAP antigens following F. hepatica infection or stimulation with F. hepatica products.ResultsWe found a decreased proliferation of peripheral blood mononuclear cells (PBMCs) after infection with F. hepatica. This reduction was inversely correlated with fluke burden. Pre‐stimulation with F. hepatica molecules produced a significant reduction of ileocaecal lymph node leucocyte proliferation in response to MAP antigens. Additionally,F. hepatica products reduced expression of the CD14 receptor by macrophages and increased levels of apoptosis and bacterial (MAP) uptake.ConclusionsOverall, F. hepatica infection had little impact on the in vitro response of immune cells to MAP, whereas in vitro co‐stimulation with F. hepatica molecules had a measurable effect. Whether this is likely to affect JD progression during in vivo chronic conditions remains unclear.

A DIVA vaccine strain lacking RpoS and the secondary messenger c-di-GMP for protection against salmonellosis in pigs

Salmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA).

Comparative evaluation of different diagnostic techniques for detection of naturally occurring paratuberculosis in Gaddi goats

The present study was aimed to evaluate the diagnostic efficacy of different techniques for detection of naturally occurring caprine paratuberculosis, considering histopathology as a parameter of infection. A total of 68 Gaddi goats slaughtered during September 2013 to March 2014 were investigated. Samples were screened for paratuberculosis by gross, histopathology, Ziehl-Nielsen (ZN) staining of impression smear and tissues sections (terminal ileum, ileo-caecal lymph node and mesenteric lymph node), ELISA, AGID and paraffin embedded tissue PCR. On gross, histopathology, ZN staining of impression smear as well as tissue sections from 68 goats revealed typical lesions, acid-fast bacilli and granulomatous inflammatory changes suggestive of paratuberculosis in 46.15% (18/39), 57.35% (39/68), 43.58% (17/39) and 35.89% (14/39) respectively. The sensitivity of ELISA, AGID and IS900/IS1311 paraffin tissue PCR was 74.35% (29/39), 61.53% (24/39) and 43.58% (17/39) respectively. All tests viz. histopathology, ZN staining, ELISA, AGID and paraffin embedded tissue PCR showed relatively higher sensitivity in animals with grade III and IV lesions when compared to grade I and II. Agreement among different tests was statistically analyzed by kappa statistics and chi-square test. The findings of the present study indicate that ELISA was most sensitive amongst the serological assays and histopathology was most sensitive of the post-mortem tests. However, combination of one of the serological tests along with histopathology and paraffin tissue PCR was found to be a better indicator for making confirmatory diagnosis of paratuberculosis in Gaddi goats.

Spread of an Experimental Salmonella Derby Infection in Antibiotic-Treated or Lawsonia intracellularis Vaccinated Piglets.

Simple SummaryToday, pigs repeatedly suffer from diarrhoea which requires treatment with antibiotics. Infections with Lawsonia intracellularis are one of the most common diseases, which, although usually subclinical, have a negative impact on performance. The alternative to antibiotic treatment is vaccinations against Lawsonia intracellularis, not least because antibiotic treatments are suspected of promoting the spread of certain zoonotic pathogens. A study was carried out with piglets from a farm that had a problem with Lawsonia intracellularis infections. In half of the animals, antibiotic treatments with tylosin were carried out in piglet rearing. In the other group, the piglets had been vaccinated against Lawsonia intracellularis as suckling piglets. Individual animals from both groups were subsequently artificially infected with Salmonella as piglets. A total of 72 animals were included in the study, 12 of which were primarily infected. The other animals had the possibility of becoming infected via direct animal contact or the faeces of infected animals. The detection of Salmonella in stool and intestinal lymph nodes was significantly higher in animals previously treated with antibiotics. Treatment with tylosin may significantly increase the spread of the Salmonella infection not observed after early Lawsonia intracellularis vaccination.Lawsonia intracellularis infections are a common reason for antibiotic treatment in pig production. Experimental studies in animals naturally infected with Lawsonia intracellularis comparing the course of an experimental Salmonella infection in piglets previously treated with tylosin or vaccinated against Lawsonia intracellularis are scarce. A total of 72 seven-week-old Salmonella-free pigs were taken from a herd with a Lawsonia intracellularis history in piglet rearing. The pigs were divided into two groups with three replicates each. Animals had either been previously treated with tylosin (10 mg/kg body weight) for seven days (AB+VAC−) or had been vaccinated as suckling pigs by drenching (Enterisol®Ileitis; AB−VAC+). Two animals per replicate were primarily infected with Salmonella Derby (1.04 × 108 colony-forming units per animal). The detection of Salmonella in faeces (p < 0.0001, odds ratio: 3.8364) and in the ileocaecal lymph nodes (p = 0.0295, odds ratio: 3.5043) was significantly more frequent in AB+VAC− animals. Overall, the odds ratio for detecting Salmonella in any substrate or organ was significantly higher in the AB+VAC− group animals (p = 0.0004, odds ratio: 5.9091). Treatment with tylosin can significantly increase the spread of a Salmonella infection, which is not observed after early Lawsonia intracellularis vaccination.

Pathology of subclinical paratuberculosis in goats of Mahakausal region

The present study was carried out to determine the prevalence and the associated enteric pathology of subclinical paratuberculosis in goats of Mahakausal region. Tissue samples including intestine, mesenteric and ileocaecal lymph nodes were collected from 70 goats and examined for presence of acid-fast bacilli in the smears, MAP genome by IS 900 PCR and gross/histopathological changes. The postmortem prevalence of subclinical paratuberculosis was recorded in 7.14% (5/70) goats by Ziehl Neelsen staining of the intestine and lymph node smears and MAP genome was detected in intestine tissue of 4.28% (3/70) goats. Characteristics gross lesions of paratuberculosis were observed as mild to moderate thickening of intestinal wall and focal to generalized mucosal corrugation. Mesenteric lymph nodes were pale, oedematous and enlarged. The microscopic lesions in intestine and mesenteric lymph nodes were characterized by infiltration of macrophages, lymphocytes and epithelioid cells. Histopathologically lesions were classified as paucibacillary and multibacillary based on inflammatory cell infiltration and distribution of acid fast bacilli(AFB) laden epithelioid cells.

Immunogenic potential of a Salmonella Typhimurium live vaccine for pigs against monophasic Salmonella Typhimurium DT 193

BackgroundMonophasic Salmonella Typhimurium (mSTM) strains account for up to 8.6% of all human Salmonellosis cases. They have an increasing prevalence during recent years and several human cases with hospitalisation were reported. These strains are often isolated from pigs and pork - one primary source of human infection. A Salmonella Typhimurium (STM) live vaccine has been proven successful in controlling of STM infections in pigs for many years. The aim of this study was to test the immunogenicity of the vaccine in weaners during oral challenge with a virulent mSTM strain and to examine the kinetics of STM-specific IgA, IgM and IgG antibodies induced by vaccination and infection.ResultsDespite clinical signs being present in both groups, the vaccination led to a significant reduction of diarrhoea, overall clinical symptoms and a milder elevation of the body temperature. Necropsy revealed fewer pathological lesions in the gastrointestinal tract of vaccinated compared to control animals. Moreover, in the ileal and caecal mucosa and in the ileocaecal lymph nodes the challenge strain burden was significantly reduced by vaccination. Significant differences in the antibody responses of both groups were present during the vaccination period and after infection. In vaccinated animals Salmonella-specific IgA and IgG antibody levels increased significantly after vaccination and were even more pronounced in response to challenge. In contrast, similarly low levels of IgM antibodies were detected during the vaccination period in both vaccinated and non-vaccinated animals. However, after challenge IgM antibody levels increased significantly in control pigs while neither IgA nor IgG antibodies were detectable.ConclusionThe data demonstrate that mSTM can evoke clinical signs in weaners. Due to the vaccination their incidence and magnitude were significantly milder. Vaccination also led to a significantly reduced challenge strain burden in the intestine and the lymph nodes which is comparable to previous studies using the same vaccine in a challenge with biphasic STM. Therefore, it is concluded that this vaccine induces immunity against monophasic and biphasic STM strains. Furthermore, the results of antibody profiles in response to vaccination and infection provide additional evidence for humoral immune mechanisms triggered during Salmonella infection or vaccination.

Pathways and Genes Associated with Immune Dysfunction in Sheep Paratuberculosis

Multibacillary and paucibacillary paratuberculosis are both caused by Mycobacterium avium subspecies paratuberculosis. Multibacillary lesions are composed largely of infected epithelioid macrophages and paucibacillary lesions contain T cells but few bacteria. Multibacillary disease is similar to human lepromatous leprosy, with variable/high levels of antibody and a dysfunctional immune response. Animals with paucibacillary disease have high cell-mediated immunity and variable levels of antibody. This study aims to characterize the immunological dysfunction using TruSeq analysis of the ileocaecal lymph node that drains disease lesions. Immune dysfunction is highlighted by repression of TCR/CD3 genes, T cell co-receptors/co-stimulators, T cell activation and signal-transduction genes. Inflammation was an acute phase response and chronic inflammation, with little evidence of acute inflammation. The high levels of immunoglobulin and plasma cell transcripts is consistent with the anti-MAP antibody responses in paratuberculosis sheep. Also notable was the overwhelming reduction in mast cell transcripts, potentially affecting DC activation of the immune response. This study also shows that there were no fundamental differences in the gene expression patterns in multibacillary and paucibacillary disease, no shift in T cell genes from Th1 to Th2 pattern but rather an incremental decline into immune dysfunction leading to multibacillary pathology.

Investigation of in-feed organic acids as a low cost strategy to combat Salmonella in grower pigs

Salmonella carriage in pigs is a significant food safety issue. Dietary supplementation with organic acids has previously been shown to reduce shedding and transmission of Salmonella. Therefore, this study aimed to examine the effect of three commercially available organic acid-based products on Salmonella levels in grower pigs, using a model of experimental infection that closely mimics natural exposure to the organism. Seven week old trial pigs (n=40) with a mean weight of 14.7kg were placed in one of four pens with 10 pigs/pen. Pens had previously been contaminated with Salmonella Typhimurium 4,[5],12;i;- via seeder pigs. Trial pigs received one of four diets for 28days: 1, control diet; 2, sodium butyrate supplemented diet; 3, benzoic acid supplemented diet and 4, formic-citric acid supplemented diet. A further 10 pigs were placed in a Salmonella-free pen receiving the control diet. Pigs were weighed and blood sampled on days 0 and 28. Faeces was collected on day 0, 2, 3, 5, 7, 14, 21 and 28 and examined for Salmonella. On day 28, 5 pigs/group were euthanised and ileocaecal lymph nodes (ILN) and caecal contents sampled for culture. The remaining 5 pigs/pen were then fed the control diet and faeces were collected on days 35 and 42. On day 42 pigs were euthanised and ILN and caecal contents tested for Salmonella levels. The trial was repeated once.Within the first two days of exposure to the contaminated environment, 96% (77/80) of pigs became infected. Most pigs shed Salmonella at levels of between 100–103 CFU/g faeces for at least 7days post-exposure. A significant reduction in Salmonella faecal concentration was observed after supplementation with sodium butyrate (p=0.001) and a formic citric acid blend (p<0.0001). Average daily weight gain (ADWG) was significantly increased in all groups fed the supplemented feed when compared to the positive control group. The use of sodium butyrate or a blend of formic and citric acid in feed could be considered a cost-effective control measure to reduce Salmonella faecal shedding and improve ADWG in Salmonella infected herds.

Detection of Paratuberculosis in Migratory Gaddi Sheep of Himachal Pradesh using Pathological Techniques

The present study was planned to investigate the pathology of terminal ileum and associated lymph nodes in Gaddi sheep in Himachal Pradesh (HP) with particular emphasis on paratuberculosis. The tissue samples were collected from 39 sheep slaughtered for table purpose around Palampur and from Government slaughter house Shimla, Himachal Pradesh, during September, 2013 to March, 2014. The terminal ileum, along with ileo-caecal junction (ICJ) and associated lymph nodes were critically examined at gross and histopathology for lesions suggestive of paratuberculosis. The scrapping smear cytology of ileal mucosa and ileo-caecal lymph node (ICLN) was stained by Ziehl-Neelsen’s (ZN) staining. Based on gross pathology, a total of 25(64.10%), 3(7.69%) and 1(2.56%) cases showed lesions suggestive of mild, moderate and severe paratuberculosis, respectively, with an overall 74.36% cases. ZN stained cytology demonstrated acid-fast bacilli (AFB) in 9 (23.08%) cases. On routine histopathological examination, the tissue samples were graded from 0 to IV and revealed only one (2.56%), 21 (53.84%), 13 (33.33%) and 3 (7.69%) Grade I, II, III and IV cases respectively, with an overall 97.43% cases. Only one histopathological Grade III case showed AFB (paucibacillary type) in ZN stained tissue sections from ileum and ileo-caecal lymph node (ICLN). Irrespective of the grade of paratuberculosis lesion the lymph nodes revealed mild to severe fibrosis in most of the cases suggesting replacement of granulomatous areas. In conclusion, histopathological examination was found to be the most effective method for detection of paratuberculosis in sheep and present study suggests alarming level of paratuberculosis among Gaddi sheep in HP.

Variations in T cell transcription factor gene structure and expression associated with the two disease forms of sheep paratuberculosis

Two different forms of clinical paratuberculosis in sheep are recognised, related to the level of bacterial colonization. Paucibacillary lesions are largely composed of lymphocytes with few bacteria, and multibacillary pathology is characterized by heavily-infected macrophages. Analysis of cytokine transcripts has shown that inflammatory Th1/Th17 T cells are associated with development of paucibacillary pathology and Th2 cytokines are correlated with multibacillary disease. The master regulator T cell transcription factors TBX21, GATA3, RORC2 and RORA are critical for the development of these T cell subsets. Sequence variations of the transcription factors have also been implicated in the distinct disease forms of human mycobacterial and gastrointestinal inflammatory diseases. Relative RT-qPCR was used to compare expression levels of each transcript variant of the master regulators in the ileo-caecal lymph nodes of uninfected controls and sheep with defined paucibacillary and multibacillary pathology. Low levels of GATA3 in multibacillary sheep failed to confirm that multibacillary paratuberculosis is caused simply by a Th2 immune response. However, high levels of TBX21, RORC2 and RORC2v1 highlights the role of Th1 and Th17 activation in paucibacillary disease. Increased RORAv1 levels in paucibacillary tissue suggests a role for RORα in Th17 development in sheep; while elevated levels of RORAv4 hints that this variant might inhibit RORα function and depress Th17 development in multibacillary sheep.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0368-3) contains supplementary material, which is available to authorized users.

Variations in IL-23 and IL-25 receptor gene structure, sequence and expression associated with the two disease forms of sheep paratuberculosis.

The immunopathology of paucibacillary and multibacillary sheep paratuberculosis is characterized by inflammatory T cell and macrophage responses respectively. IL-23 and IL-25 are key to the development of these responses by interaction with their complex receptors, IL-23R/IL-12RB1 and IL-17RA/IL-17RB. In humans, variations in structure, sequence and/or expression of these genes have been implicated in the different pathological forms of tuberculosis and leprosy, and in gastrointestinal inflammatory disorders such as Crohn’s disease. Sequencing has identified multiple transcript variants of sheep IL23R, IL12RB1 and IL17RB and a single IL17RA transcript. RT-qPCR assays were developed for all the identified variants and used to compare expression in the ileo-caecal lymph node of sheep with paucibacillary or multibacillary paratuberculosis and uninfected animals. With IL-23 receptor, only the IL12RB1v3 variant, which lacks the receptor activation motif was differentially expressed and was significantly increased in multibacillary disease; this may contribute to high Th2 responses. Of the IL17RB variants only full length IL17RB was differentially expressed and was significantly increased in multibacillary pathology; which may also contribute to Th2 polarization. IL17RA expression was significantly increased in paucibacillary disease. The contrast between the IL17RA and IL17RB results may indicate that, in addition to Th1 cells, Th17 T cells are also involved in paucibacillary pathology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0314-4) contains supplementary material, which is available to authorized users.

Maternal immunity induced by inactivated S. Typhimurium vaccine is less protective to S. Derby challenge than to S. Typhimurium challenge in suckling piglets

Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Derby are the most common serovars of Salmonella enterica ssp. enterica found in pigs in Europe. We previously observed that suckling piglets of sows vaccinated with an S. Typhimurium-based inactivated vaccine are protected against homologous strain challenge. To develop this vaccine for commercial use, potential crossprotectivity of this vaccine to challenge with S. Derby was tested. Two sows were vaccinated with an S. Typhimurium-based inactivated vaccine while two other sows remained serologically negative. Four-day-old suckling piglets from both groups were orally challenged with S. Derby or S. Typhimurium. Maternally-derived immunity against S. Typhimurium protected piglets against S. Typhimurium challenge, when a significant (P < 0.05) decrease in S. Typhimurium count was found in ileocaecal and submandibular lymph node, tonsil, ileum and ileum content. On the other hand, after S. Derby challenge, significant (P < 0.05) decrease in S. Derby count was detected only in ileum content. Although both serovars belong to the same O:4 serogroup, other antigenic structures, for example the flagellin, are different. In a subsequent in-vitro experiment, we found that serum from vaccinated sows inhibited the motility of S. Typhimurium but not the motility of S. Derby. Our results indicate that protectivity of S. Typhimurium vaccine against S. Derby infection is limited.

High diversity of viable bacteria isolated from lymph nodes of slaughter pigs and its possible impacts for food safety.

Ileocaecal lymph nodes (ICLNs) of pigs, the key immune inductive site for bacterial systemic invasion, were examined in this study with emphasis on viable and cultivable bacteria. Asymptomatic and pathologically altered ICLNs of slaughter pigs (n = 16) with hyperplasia, purulence or granulomatous formations were aerobically and anaerobically cultivated. In total, 209 isolates were collected and the near full-length 16S rRNA gene from each isolate was sequenced. Taxonomic classification revealed that 68% of the isolates belonged to Proteobacteria, 27% to Firmicutes and 5% to Actinobacteria. Purulent and granulomatous ICLNs generally tended to contain more Proteobacteria than asymptomatic and enlarged ICLNs (P = 0·061). The isolates could be assigned to 25 species belonging to 17 genera including Escherichia, Carnobacterium, Lactobacillus, Staphylococcus and Acinetobacter. Furthermore, pathogens such as Streptococcus suis and Salmonella enterica were detected. The most abundant isolate (57%) was most similar (>99%) to Escherichia coli. Random amplified polymorphic DNA (RAPD) profiling revealed a high genetic diversity among E. coli isolates and 24% of these isolates were positive for at least one gene associated with enterohemorrhagic disease (eae, fliC, stx1 or hlyA). Compared with a recently published DNA-based high-throughput sequencing data set including the same ICLNs, 4% of species detected were cultivable. The presence of viable, commensal and pathogenic bacterial phylotypes could be proven in ICLNs with Proteobacteria being dominant. This is the first study that broadly characterizes viable bacteria from ICLNs of pigs. The presence of bacteria in lymph nodes of farm animals has practical relevance for host colonization and possible chronic infection. It is also of great interest for basic research investigating translocation of bacteria from the gastrointestinal tract to ICLNs.

Relative abundance of Mycobacterium in ovine Johne's disease

No study has determined what proportion of the total microbiota comprises the genus Mycobacterium in ovine Johne's disease (OJD) tissues. We aimed to assess the relative abundance of Mycobacterium in the ileocaecal lymph node, involved and uninvolved ileal mucosa from sheep with and without OJD, using three extraction methods. Eight sheep, four with and four without OJD, were recruited. Pyrosequencing of the 16Sr RNA gene amplicons for all samples revealed that Mycobacterium represented between 0-92% (average 38%) of the total microbiota of samples from sheep with OJD, and 0-85% (average 13%) of sheep without OJD. Only sheep with OJD had samples that were positive for the IS900 (MAP) element. Mycobacterial strains other than MAP may provide competitive exclusion of MAP and should be further investigated.

Abattoir based survey of Salmonella in finishing pigs in the United Kingdom 2006–2007

All European Union (EU) Member States (MSs) were required to conduct a baseline survey from October 2006 to September 2007 to estimate the prevalence of Salmonella infection in finisher pigs at slaughter. In the United Kingdom (UK), samples for microbiological culture were collected from the ileo-caecal lymph nodes, from carcass swabs and from caecal contents. Meat juice samples were also collected for testing in an enzyme-linked immunosorbent assay (MJE) to detect a serological response. Salmonella was isolated from 22% (CI95% 19–25%) of ileo-caecal lymph nodes, 15% (CI95% 12–18%) of carcass swabs and 22% (CI95% 19–26%) of caecal contents. A quarter (25%, CI95% 22–29%) of MJE samples were positive for Salmonella antibodies (cut-off, S/P≥0.25). The most frequently identified serovars were Salmonella typhimurium (57%) and Salmonella derby (26%). The results were used to investigate some factors associated with Salmonella infection in slaughter pigs. The weight of the carcass was significant, with a lower probability of obtaining a positive meat juice result from pigs with a weight greater than 75kg (p=0.03). The weight of the lymph node sample was significantly associated with Salmonella status, with a heavier sample of lymph nodes being more likely to be positive (OR=2.16 CI95% 1.07–4.39). ‘Carcass weight’ in two of the classes – 75–79kg and 80–84kg – (OR=0.44 CI95% 0.28–0.70; OR=0.64 CI95% 0.49–0.85) and ‘fewer-than-1500-pigs’ scheduled for slaughter on the day of sampling’ (OR=0.41 CI95% 0.24–0.71) were also associated with a reduced risk of isolating Salmonella from lymph node. The model for carcass swabs showed a positive association with ‘time-elapsed-since-the-start-of-the-line’ indicating a higher risk of contamination as the day progressed (p<0.01). This model also showed positive association between isolation of Salmonella from a carcass swab and the occurrence of Salmonella in caecal contents from the same pig (OR=2.22 CI95% 1.38–3.59) and a negative association with time in singeing units (OR=0.88 CI95% 0.79–0.98).

Use of a live attenuated Salmonella enterica serovar Typhimurium vaccine on farrow-to-finish pig farms

Salmonella enterica infection in pigs is economically important and poses a zoonotic risk. In this study, the efficacy of an attenuated S. enterica serovar Typhimurium strain was evaluated in three farrow-to-finish pig herds. In each herd, 120 piglets were vaccinated orally at 3 and 24 days of age, while 120 piglets served as unvaccinated controls. Faeces, ileocaecal lymph nodes and caecal contents were examined for S. Typhimurium by isolation and serum was analysed for antibodies against S. Typhimurium by ELISA. All pigs were weighed at pre-weaning and slaughter to determine daily weight gain. In vaccinated pigs prior to slaughter, significantly fewer animals excreted S. enterica, there was a significantly lower S. enterica-specific mean antibody titre and there was a significantly higher mean daily weight gain compared to unvaccinated controls. In two herds, there were significantly lower proportions of S. enterica positive ileocaecal lymph nodes and caecal contents at slaughter between the vaccinated and control groups, but this difference was not significant across all three herds. S. enterica with the same auxotrophic characteristics and genotype as the vaccine strain was isolated from several samples of faeces, ileocaecal lymph nodes and caecal contents from vaccinated pigs. These findings indicate that vaccination with an attenuated S. Typhimurium strain reduces S. enterica shedding, but the reduction is not consistent and the vaccine strain may persist in tissues.

Rapid testing and quantification of Salmonella in ileocaecal lymph nodes of Austrian pigs slaughtered for consumption

Traditionally, quantitative microbial risk assessment (QMRA) is based on culture-dependent technologies. However, molecular quantification could forge additional, detailed information. A prerequisite of quantitative real-time PCR in animal science is a tissue preparation method where large volumes of tissue material can be reduced and particularly target cells can be concentrated. An easy-to-use sample preparation method for food (Matrix-Lysis) was recently adapted to tissues and now permits quantification of target cells from up to 5 g of organic matrix. The aim of this study was to examine the suitability of Matrix-Lysis for quantification of Salmonella in porcine ileocaecal lymph nodes (ICLNs). After demonstrating constant recovery rates, ICLNs from 540 pigs were examined for Salmonella spp. with Matrix-Lysis. Samples were also analysed using ISO 6579:2002, a combined enrichment/qPCR method and a lateral flow test. It could be shown that qPCR coupled with Matrix-Lysis can contribute to QMRA in food safety by enabling reproducible quantitative data, even at low contamination rates.