A preliminary study investigating effects of oral monensin sodium in an enteric Mycobacterium avium ssp. paratuberculosis infection model of calves.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, an enteric infection of ruminants that causes significant economic burden for dairy and beef producers. Efforts to control MAP in endemic herds typically focus on herd management practices such as limiting exposure or early culling of infected animals and, occasionally, vaccination. The ionophore monensin sodium may have protective effects against MAP both in vivo and in vitro; however, this has not been thoroughly evaluated experimentally. Using a direct intestinal MAP challenge model, we have observed similarities regarding persistence of MAP in tissues and apparent resilience to infection compared with experimental oral infection or natural disease. Here we sought to investigate the effects of oral monensin supplementation in experimentally MAP-infected calves. We examined the persistence of MAP in the intestinal tissues, MAP-induced intestinal inflammation, fecal MAP shedding, and seroconversion using a commercial serologic assay. Monensin-supplemented MAP-infected calves demonstrated evidence for resilience to MAP infection earlier in this study compared with monensin-free MAP-infected calves. However, statistical modeling did not identify a significant effect of monensin on outcomes of infection, and more work is required to understand how monensin affects early tissue colonization of MAP in calves.

Gamma-delta T-cell responses during subcutaneous Mycobacterium avium subspecies paratuberculosis challenge in sensitized or naive calves using matrix biopolymers.

We have developed a model to explore the early immune response against Mycobacterium avium subspecies paratuberculosis (Map) infection in the bovine calf using subcutaneously placed liquid gel matrix biopolymer (matrigel) containing live Map. Matrigel rapidly polymerizes in vivo, retains recruited cellular infiltrates and soluble immune mediators, and can be rapidly removed 48 hours later and depolymerized for analysis. In this study, we examined early host immune events at matrigel/Map sites; recruited cells were evaluated by histopathology and flow cytometry, and cytokines were measured by flow cytometry, enzyme-linked immunosorbent assay, and Luminex bead immunoassay. Our results demonstrate earlier recruitment of gamma-delta (γδ) T cells to matrigel/Map challenge sites compared to CD4+ T cells. We also show that significantly more γδ T cells were recruited to matrigel/Map sites postinfection day 7 compared to postinfection day 30 and that these cells produced significant amounts of the cytokine interferon gamma. We also provide evidence that peripheral blood-derived γδ T-cell subsets in cattle differentially generate interferon gamma, suggesting distinct roles for these cells. These data provide unique insight into initial antimycobacterial host cellular immune responses following Map infection in calves.

Direct inoculation of Mycobacterium avium subspecies Paratuberculosis into ileocecal Peyer's patches results in colonization of the intestine in a calf model.

The objective of this study was to develop an intestinal model of Mycobacterium avium subspecies paratuberculosis (Map) infection in the calf for evaluation of mucosal pathology and local and systemic immunologic responses. Map was inoculated into Peyer's patches of young calves using a right flank surgical approach in standing calves to exteriorize the ileocecal junction. Inoculum doses ranging from 10(3) to 10(9) colony-forming units of strain K10 Map were injected through the serosal surface into Peyer's patches of the distal ileum near the ileocecal valve. Fecal samples were collected for culture from each calf weekly until termination of the study. Calves were necropsied at 7, 30, 60, and 90 days after infection, when inoculation sites, lymph nodes, spleen, and peripheral blood were collected for evaluation. Ileocecal lymph nodes were consistently colonized by Map in the 10(5) to 10(9) groups. The ileocecal valve was also colonized in 10(7) and 10(9) groups. This correlated with fecal culture results as infected calves intermittently shed Map in their feces throughout the study. Granulomatous lesions with giant cells and acid-fast bacilli at the ileocecal junction, ileocecal lymph nodes, and lamina propria of high-dose animals (10(7) and 10(9)) were identified from each time point. Flow cytometry was used to detect antigen-specific production of interferon-γ and interleukin-4 locally (ileocecal lymph node) and systemically (peripheral blood mononuclear cells), which defined distinct immunologic profiles in low-dose and high-dose calves. This study demonstrates intestinal Map infection via Peyer's patch inoculation, a novel model with many shared features of natural Map infection.

Duodenitis-Proximal Jejunitis in Horses After Experimental Administration of Clostridium difficile Toxins.

BACKGROUND: Duodenitis-proximal jejunitis (DPJ) is an acute sporadic gastrointestinal disorder of horses of unknown cause. HYPOTHESIS/OBJECTIVES: We hypothesize that Clostridium difficile toxins are involved in the pathogenesis of DPJ in horses. The objective of this study was to determine whether experimentally delivered C. difficile toxins cause clinical signs and histologic lesions similar to those of naturally occurring DPJ. ANIMALS: Six healthy mature mixed breed horses. METHODS: Experimental study: animal model of animal disease. Fasted horses were administered crude C. difficile toxins via gastroscopy and monitored for up to 48 hour. Blood was collected for complete blood cell count, biochemistry profile, and plasma fibrinogen assay, and abdominal fluid was collected for cytologic analysis and total solids before and after toxin administration. Physical examination and abdominal ultrasonography were performed throughout the study period. Tissues were collected from the gastrointestinal tract and processed for routine histologic analysis, and lesions were scored. RESULTS: Clinical signs were observed in 2 of 6 horses that are typical although not specific for horses with naturally occurring DPJ. Histopathologic lesions were observed in 6 of 6 horses and were similar to those reported in horses with naturally occurring DPJ. Two horses were severely affected. CONCLUSIONS AND CLINICAL IMPORTANCE: Duodenitis-proximal jejunitis is likely a syndrome with multiple causes that result in the same clinical and pathologic findings, and our data suggest that the toxins of C. difficile represent one cause of this syndrome. Toxin dose and variation in individual animal susceptibility might affect the clinical signs and lesions after administration of C. difficile toxins.

Presence of the Streptococcus suis suilysin gene and expression of MRP and EF correlates with high virulence in Streptococcus suis type 2 isolates.

Nineteen Streptococccus suis type 2 isolates that had been analyzed previously for hemolysin production, ribotype, and virulence in pigs were examined for presence of the gene coding for suilysin by PCR amplification, and southern blot and hybridization techniques. Based on southern blot and hybridization analysis, all isolates tested contained at least a portion of the suilysin gene. PCR amplification of the entire gene resulted in gene fragments from five of the seven highly virulent isolates and none of the moderately virulent or avirulent isolates. Additional PCR analysis showed that mutation or deletions at the 5' end of the suilysin gene in the less virulent isolates prevented amplification of the sly gene fragment from those isolates. The MRP+ (muramidase-released protein) EF+ (extracellular protein) phenotype was also expressed by the same five highly virulent/sly+ isolates.

Use of ribotyping and hemolysin activity to identify highly virulent Streptococcus suis type 2 isolates.

Nineteen Streptococcus suis type 2 isolates were evaluated for their virulence in pigs and mice. Of these, seven were determined to be highly virulent in pigs on the basis of clinical sign scores and gross pathology and histopathology results. Clinical sign scores correlated with gross pathology and histopathology scores at P equal to 0.004 and P equal to 0.009, respectively. The virulence of highly virulent isolates in pigs compared somewhat with virulence in mice, but the correlation was not significant. No correlation of virulence was noted among the moderately virulent and avirulent isolates in pigs and mice. Chromosomal DNAs from all S. suis isolates were evaluated by PstI, PvuII, EcoRI, and HaeIII restriction enzyme digestion followed by hybridization with a digoxigenin-11-dUTP-labeled cDNA probe transcribed from 16S and 23S rRNAs from Escherichia coli. The hybridization patterns (ribotypes) varied depending upon the enzyme used, but a significant number of isolates determined to be highly virulent in pigs had unique hybridization patterns compared with those of the moderately virulent and avirulent isolates (P = 0.002). In addition, hemolysin activity showed a high correlation to virulence (P = 0.00008) and ribotype (P = 0.002).