Investigation of an emergent, tetracycline-resistant, abortifacient Campylobacter jejuni clone in a pregnant guinea pig model

Campylobacter spp. have been recognized as an important cause of sheep abortion for decades; however, recent analyses have revealed that C. jejuni has replaced C. fetus subsp. fetus as the predominant species isolated, and that a single clone of tetracycline-resistant C. jejuni, named clone SA for Sheep Abortion, has emerged as the predominant isolate across multiple states and multiple lambing seasons. The emergence of this genetic clone reveals a dramatic shift in the epidemiology of ovine campylobacteriosis and warrants further investigation. Herein we describe oral inoculation of the pregnant guinea pig as an appropriate model to study the pathogenesis of septic abortion due to clone SA with 60% (6/10) of animals aborting within 21 days of inoculation with a clinical isolate (C. jejuni IA3902) of this highly pathogenic clone. In placentas obtained from these aborting guinea pigs, we observed significantly increased TLR2 protein in subplacental syncytiotrophoblast cells while TLR4 expression was relatively unchanged, suggesting a role for TLR2 in the pathogenesis of Campylobacter-associated abortion. We describe resolution of infection after intraperitoneal challenge in guinea pigs as a means to compare efficacy of two commercially available Campylobacter vaccines and an experimental bacterin. In this challenge study, one commercial vaccine was entirely ineffective and may suggest a means of positive selection for the emergence of clone SA. Lastly, we describe the spatial localization of mucin, iron and L-fucose within the guinea pig placental unit and compare the effects of these chemicals, placental tissue extracts, blood, and bile from pregnant guinea pigs on the growth and chemotaxis of C. jejuni. Our findings reveal the presence of growth promoting and chemoattractive substances for C. jejuni within the guinea pig placental unit and suggest a role for these factors in the fetoplacental tropism observed with this strain. The results of these studies provide insight into potential mechanisms underlying the pathogenesis of septic abortion following infection with clone SA of C. jejuni, and to the emergence of this highly pathogenic strain

Improved Tissue-Based Analytical Test Methods for Orellanine, a Biomarker of Cortinarius Mushroom Intoxication.

Orellanine (OR) toxin is produced by mushrooms of the genus Cortinarius which grow in North America and in Europe. OR poisoning is characterized by severe oliguric acute renal failure, with a mortality rate of 10%-30%. Diagnosis of OR poisoning currently hinges on a history of ingestion of Cortinarius mushrooms and histopathology of renal biopsies. A key step in the diagnostic approach is analysis of tissues for OR. Currently, tissue-based analytical methods for OR are nonspecific and lack sensitivity. The objectives of this study were: (1) to develop definitive HPLC and LC-MS/MS tissue-based analytical methods for OR; and (2) to investigate toxicological effects of OR in mice. The HPLC limit of quantitation was 10 µg/g. For fortification levels of 15 µg/g to 50 µg/g OR in kidney, the relative standard deviation was between 1.3% and 9.8%, and accuracy was within 1.5% to 7.1%. A matrix-matched calibration curve was reproduced in this range with a correlation coefficient (r) of 0.97-0.99. The limit of detection was 20 ng/g for LC-MS/MS. In OR-injected mice, kidney OR concentrations were 97 ± 51 µg/g on Day 0 and 17 ± 1 µg/g on termination Day 3. Splenic and liver injuries were novel findings in this mouse model. The new tissue-based analytical tests will improve diagnosis of OR poisoning, while the mouse model has yielded new data advancing knowledge on OR-induced pathology. The new tissue-based analytical tests will improve diagnosis of OR poisoning, while the mouse model has yielded new data advancing knowledge on OR-induced pathology

Alterations in the Colonic Microbiota of Pigs Associated with Feeding Distillers Dried Grains with Solubles

In an effort to reduce feed costs, many pork producers have increased their use of coproducts of biofuel production in commercial pig diets, including increased feeding of distiller’s dried grains with solubles (DDGS). The inclusion of DDGS increases the insoluble fiber content in the ration, which has the potential to impact the colonic microbiota considerably as the large intestine contains a dynamic microenvironment with tremendous interplay between microorganisms. Any alteration to the physical or chemical properties of the colonic contents has the potential to impact the resident bacterial population and potentially favor or inhibit the establishment of pathogenic species. In the present study, colonic contents collected at necropsy from pigs fed either 30% or no DDGS were analyzed to examine the relative abundance of bacterial taxa associated with feeding this ingredient. No difference in alpha diversity (richness) was detected between diet groups. However, the beta diversity was significantly different between groups with feeding of DDGS being associated with a decreased Firmicutes:Bacteriodetes ratio (P = .004) and a significantly lower abundance of Lactobacillus spp. (P = .016). Predictive functional profiling of the microbiota revealed more predicted genes associated with carbohydrate metabolism, protein digestion, and degradation of glycans in the microbiota of pigs fed DDGS. Taken together, these findings confirm that alterations in dietary insoluble fiber significantly alter the colonic microbial profile of pigs and suggest the resultant microbiome may predispose to the development of colitis

Pathogenicity of an emergent, ovine abortifacient Campylobacter jejuni clone orally inoculated into pregnant guinea pigs

Objective—To compare pathogenicity of an emergent abortifacient Campylobacter jejuni (IA 3902) with that of reference strains after oral inoculation in pregnant guinea pigs. Animals—58 pregnant guinea pigs. Procedures—12 animals were challenged IP with C jejuni IA 3902 along with 5 sham-inoculated control animals to confirm abortifacient potential. Once pathogenicity was confirmed, challenge via oral inoculation was performed whereby 12 guinea pigs received IA 3902, 12 received C jejuni isolated from ovine feces (OF48), 12 received a fully sequenced human C jejuni isolate (NCTC 11168), and 5 were sham-inoculated control animals. After abortions, guinea pigs were euthanized; samples were collected for microbial culture, histologic examination, and immunohistochemical analysis. Results—C jejuni IA 3902 induced abortion in all 12 animals following IP inoculation and 6 of 10 animals challenged orally. All 3 isolates colonized the intestines after oral inoculation, but only IA 3902 induced abortion. Evidence of infection existed for both IA 3902 and NCTC 11168; however, C jejuni was only recovered from fetoplacental units of animals inoculated with IA 3902. Immunohistochemical analysis localized C jejuni IA 3902 infection to subplacental trophoblasts, perivascular tissues, and phagocytes in the placental transitional zone. Conclusions and Clinical Relevance—This study revealed that C jejuni IA 3902 was a unique, highly abortifacient strain with the ability to colonize the intestines, induce systemic infection, and cause abortion because of its affinity for the fetoplacental unit. Guinea pigs could be effectively used in the study of septic abortion after oral inoculation with this Campylobacter strain

Comparison of two commercial ovine Campylobacter vaccines and an experimental bacterin in guinea pigs inoculated with Campylobacter jejuni

Objective—To compare efficacy of 2 commercial ovine Campylobacter vaccines and an experimental bacterin in guinea pigs following IP inoculation with Campylobacter jejuni IA3902. Animals—51 female guinea pigs. Procedures—Pregnant and nonpregnant animals were randomly assigned to 1 of 4 treatment groups and administered a commercial Campylobacter vaccine labeled for prevention of campylobacteriosis in sheep via two 5-mL doses 14 days apart (vaccine A; n = 13), another labeled for prevention of campylobacteriosis via two 2-mL doses (vaccine B; 12), an experimental bacterin prepared from the challenge strain (12), or a sham vaccine (14). Ten days later, animals were challenged IP with C jejuni IA3902; 48 hours later, animals were euthanized, complete necropsy was performed, and blood and tissue samples were obtained for bacteriologic culture. Results—Administration of vaccine B or the experimental bacterin, but not vaccine A, significantly reduced 48-hour infection rates versus administration of the sham vaccine. A significantly reduced 48-hour infection rate was associated with administration of vaccine B independent of pregnancy status. Conclusions and Clinical Relevance—Administration of vaccine B significantly reduced infection in guinea pigs challenged with C jejuni IA3902, similar to a homologous bacterin. Results suggested that vaccine B or an autogenous product may be effective in controlling ovine campylobacteriosis caused by this emergent abortifacient strain. Bacteriologic culture of blood, liver, bile, and uterus in nonpregnant guinea pigs 48 hours after inoculation may be a useful screening tool for comparing efficacy of C jejuni vaccines

Dietary fiber affects pigs’ response to E. coli diarrhea

Pigs are subject to numerous health threats as they undergo various stresses at the time of weaning. At the same time, their immune system is not fully developed. Diarrhea is one of the common problems in a newly weaned group, causing substantial economic losses for producers due to the increased mortality and morbidity. Certainly, many factors can lead to post-weaning diarrhea, such as poor environmental hygiene, inappropriate feeding and infectious pathogens. Piglet diarrhea caused by Escherichia coli is one of the most common cases, also called colibacillosis

Critical Role of LuxS in the Virulence of Campylobacter jejuni in a Guinea Pig Model of Abortion

Previous studies on Campylobacter jejuni have demonstrated the role of LuxS in motility, cytolethal distending toxin production, agglutination, and intestinal colonization; however, its direct involvement in virulence has not been reported. In this study, we demonstrate a direct role of luxS in the virulence of C. jejuni in two different animal hosts. The IA3902 strain, a highly virulent sheep abortion strain recently described by our laboratory, along with its isogenic luxS mutant and luxScomplement strains, was inoculated by the oral route into both a pregnant guinea pig virulence model and a chicken colonization model. In both cases, the IA3902luxS mutant demonstrated a complete loss of ability to colonize the intestinal tract. In the pregnant model, the mutant also failed to induce abortion, while the wild-type strain was highly abortifacient. Genetic complementation of the luxSgene fully restored the virulent phenotype in both models. Interestingly, when the organism was inoculated into guinea pigs by the intraperitoneal route, no difference in virulence (abortion induction) was observed between the luxS mutant and the wild-type strain, suggesting that the defect in virulence following oral inoculation is likely associated with a defect in colonization and/or translocation of the organism out of the intestine. These studies provide the first direct evidence that LuxS plays an important role in the virulence of C. jejuni using an in vivomodel of natural disease

Impact of Brachyspira hyodysenteriae on intestinal amino acid digestibility and endogenous amino acid losses in pigs

Brachyspira hyodysenteriae (Bhyo) induces mucohemorrhagic diarrhea in pigs and is an economically significant disease worldwide. Our objectives were to determine the impact of Bhyo on apparent total tract digestibility (ATTD), ileal digestibility (AID), and ileal basal endogenous losses (BEL) in grower pigs. In addition, we assessed the effect of Bhyo on hindgut disappearance of DM, N, and GE. Thirty-two Bhyo negative gilts (38.6 ± 0.70 kg BW) were fitted with a T-cannula in the distal ileum and individually penned. In replicates 1 and 2, pigs were fed a complete diet (7 Bhyo−, 10 Bhyo+ pigs) or nitrogen-free diet (NFD; 4 Bhyo−, 11 Bhyo+ pigs), respectively. Across multiple rooms, the 21 Bhyo+ pigs (62.6 ± 1.39 kg BW) were inoculated with Bhyo on day post inoculation (dpi) 0, and the 11 Bhyo− pigs were sham inoculated. Feces were collected from 9 to 11 dpi and ileal digesta collected from 12 to 13 dpi. All pigs were euthanized at 14 to 15 dpi and intestinal tract pathology assessed. Within the complete diet and NFD treatments, data were analyzed to determine pathogen effects. All Bhyo− pigs remained Bhyo negative, and 5 Bhyo+ pigs in each replicate were confirmed Bhyo positive within 9 dpi. Infection with Bhyo reduced ATTD of DM, N, and GE and increased AID of Gly (P \u3c 0.05). No other AA AID differences were observed. Only BEL of Pro was reduced (P \u3c 0.05) while Arg, Trp, and Gly tended (P \u3c 0.10) to be reduced in Bhyo+ pigs. When calculated from AID and BEL, Bhyo infection reduced standardized ileal digestibility (SID) of N, Arg, Lys, Ala, Gly, Pro, and Ser (P \u3c 0.05) and tended to reduce Thr SID (P = 0.09). In the hindgut of Bhyo+ pigs, there was generally an appearance of nutrients rather than disappearance. In Bhyo+ pigs fed a complete diet, hindgut appearance of N and GE were increased (P \u3c 0.05) by 58 and nine-fold, respectively, and DM tended to be increased two-fold (P = 0.06). Similarly, in NFD fed pigs, hindgut appearance of N and GE was increased by 172% and 162%, respectively, although high variability led to no significance. Altogether, Bhyo infection decreases ATTD but has minimal impact on AID of AA, when corrected for BEL, SID of N, Arg, Lys and some nonessential AA are specifically reduced. Unexpectedly, BEL of several AA involved in mucin production were unaffected by Bhyo infection. This may suggest an increased need for specific AA and energy during a Bhyo challenge

Impact of PRRSV infection and dietary soybean meal on ileal amino acid digestibility and endogenous amino acid losses in growing pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) is a significant disease in the swine industry, and increasing soybean meal (SBM) consumption during this disease challenge may improve performance. Our objectives were to determine the impact of SBM level on apparent total tract (ATTD) and ileal (AID) digestibility during PRRSV infection and to determine ileal basal endogenous losses (BEL) during PRRSV infection. Forty PRRSV negative gilts were fitted with a T-cannula in the distal ileum. Treatments were arranged in a 2 × 2 factorial with high and low SBM (HSBM, 29% vs. LSBM, 10%), with and without PRRSV (n = 6/treatment). The remaining pigs (n = 8/challenge status) were fed a N-free diet. Chromic oxide was used as an indigestible marker. On day post inoculation (dpi) 0, at 47.7 ± 0.57 kg BW, 20 pigs were inoculated with live PRRSV; 20 control pigs were sham inoculated. Infection was confirmed by serum PCR. Feces were collected at dpi 5 to 6 and dpi 16 to 17, and ileal digesta collected at dpi 7 to 8 and dpi 18 to 19. Feed, feces, and digesta were analyzed for DM, N, and GE. Digesta and feed were analyzed for AA. Data were analyzed in a 2 × 2 + 2 factorial design to determine main effects of diet and PRRSV and their interaction. Data from N-free fed pigs were analyzed separately to determine BEL and hindgut disappearance due to PRRSV infection. All control pigs remained PRRSV negative. There were no interactions for AID of AA; however, HSBM reduced DM, GE, Lys, and Met AID and increased Arg and Gly AID during both collection periods (P \u3c 0.05). At dpi 7 to 8 only, PRRSV reduced DM and GE AID (P \u3c 0.05). At 7 to 8 dpi, BEL of Arg, Ala, and Pro were reduced (P \u3c 0.05) due to PRRSV by 64%, 39%, and 94%, respectively. At dpi 18 to 19, BEL of Thr tended (P = 0.06) to be increased in PRRSV-infected pigs; however, no other differences were observed. Pigs fed LSBM had increased Lys, Met, Thr, Trp, and Pro standardized ileal digestibility (SID), primarily at 7 to 8 dpi. At 7 to 8 dpi, PRRSV reduced Arg, Gly, and Pro SID (P \u3c 0.01), and SID Pro continued to be reduced by 17% at dpi 18 to 19. Compared with HSBM pigs, LSBM reduced hindgut disappearance of DM and GE at dpi 5 to 8 and dpi 16 to 19, while N disappearance was reduced at dpi 5 to 8. There were no differences between control and PRRSV N-free fed pigs. Altogether, SBM inclusion impacts SID of AA and hindgut disappearance of nutrients, regardless of PRRSV. In contrast, there is minimal impact of PRRSV on BEL, and therefore, SID of most AA are not different

Acute infection with Brachyspira hyodysenteriae affects mucin expression, glycosylation, and fecal MUC5AC

IntroductionInfection with strongly β-hemolytic strains of Brachyspira hyodysenteriae leads to swine dysentery (SD), a production-limiting disease that causes mucohemorrhagic diarrhea and typhlocolitis in pigs. This pathogen has strong chemotactic activity toward mucin, and infected pigs often have a disorganized mucus layer and marked de novo expression of MUC5AC, which is not constitutively expressed in the colon. It has been shown that fucose is chemoattractant for B. hyodysenteriae, and a highly fermentable fiber diet can mitigate and delay the onset of SD.MethodsWe used lectins targeting sialic acids in α-2,6 or α-2,3 linkages, N-acetylglucosamine (GlcNAc), α-linked L-fucose, and an immunohistochemical stain targeting N-glycolylneuraminic acid (NeuGc) to investigate the local expression of these mucin glycans in colonic tissues of pigs with acute SD. We used a commercial enzyme-linked immunosorbent assay (ELISA) to quantify fecal MUC5AC in infected pigs and assess its potential as a diagnostic monitoring tool and RNA in situ hybridization to detect IL-17A in the colonic mucosa.ResultsColonic mucin glycosylation during SD has an overall increase in fucose, a spatially different distribution of GlcNAc with more expression within the crypt lumens of the upper colonic mucosa, and decreased expression or a decreased trend of sialic acids in α-2,6 or α-2,3 linkages, and NeuGc compared to the controls. The degree of increased fucosylation was less in the colonic mucosa of pigs with SD and fed the highly fermentable fiber diet. There was a significant increase in MUC5AC in fecal and colonic samples of pigs with SD at the endpoint compared to the controls, but the predictive value for disease progression was limited.DiscussionFucosylation and the impact of dietary fiber may play important roles in the pathogenesis of SD. The lack of predictive value for fecal MUC5AC quantification by ELISA is possibly due to the presence of other non-colonic sources of MUC5AC in the feces. The moderate correlation between IL-17A, neutrophils and MUC5AC confirms its immunoregulatory and mucin stimulatory role. Our study characterizes local alteration of mucin glycosylation in the colonic mucosa of pigs with SD after B. hyodysenteriae infection and may provide insight into host-pathogen interaction