Evaluation of different Campylobacter jejuni isolates to colonize the intestinal tract of commercial turkey poults and selective media for enumeration.

Consumption of contaminated poultry products is the main source of human campylobacteriosis, for which Campylobacter jejuni is responsible for 90% of human cases. Although chickens are believed to be a main source of human exposure to C. jejuni, turkeys also contribute to cases of human infection. Little is known about the kinetics of C. jejuni intestinal colonization in turkeys, or best selective media for their recovery. Enumeration of C. jejuni from intestinal samples can be challenging because most selective Campylobacter media support the growth of non-Campylobacter organisms. In this study, we sought to compare a) C. jejuni isolates that persistently colonize different compartments of the poult intestinal tract, and b) selective media to enumerate C. jejuni from turkey intestinal samples. Three-week-old poults were orally colonized with C. jejuni isolates NCTC 11168 or NADC 20827 (isolated from a turkey flock). Mock-colonized poults were orally gavaged with uninoculated media. Poults were euthanized at d 3, 7, and 21 post colonization and direct plated on different selective Campylobacter media [Campy Line agar with sulfamethoxazole (CLA-S), CHROMagar Campylobacter (CAC) and Campy Cefex] for enumeration. Isolates NCTC 11168 and NADC 20827 poorly colonized the distal ileum. Both isolates colonized the colon, but the number of NADC 20827 significantly decreased at d 21. Isolates NCTC 11168 and NADC 20827 persistently colonized the cecum for up to 21 days. There was no significant difference in the Campylobacter amount recovered on CLA-S and CAC. Campy Cefex failed to prevent growth of background microbes to enumerate C. jejuni from turkey samples. Two independent PCR assays (multiplex PCR and qPCR) confirmed that colonies grown on CLA-S or CAC were C. jejuni. Data from this study demonstrated that isolates NCTC 11168 and NADC 20827 persistently colonized the cecum, and CLA-S or CAC were successful to enumerate Campylobacter from intestinal samples. These findings will be useful to evaluate the host response by C. jejuni in turkeys, and test pre-harvest strategies to reduce its colonization and promote food safety.

Evaluation of disinfectants and antiseptics to eliminate bacteria from the surface of turkey eggs and hatch gnotobiotic poults.

Bird eggs are in contact with intestinal microbiota at or after oviposition, but are protected from bacterial translocation by a glycoprotein cuticle layer, the shell, and internal membranes. In a preliminary study, turkey eggs were hatched in a germ-free environment. Firmicutes 16S rRNA gene was detected in the cecal microbiota of hatched poults, suggesting that poults may acquire spore-formers by exposure to shell contents during hatching. Generating gnotobiotic poults for research requires elimination of bacteria from the egg's surface without damaging the developing embryo. The ability of different disinfectants and antiseptics to eliminate eggshell bacteria without harming the developing embryo was tested. Different classes of disinfectants and antiseptics (halogens, biguanidines, and oxidants) were selected to target spores and vegetative bacteria likely present on the egg's surface. Eggs were treated by fully immersing in heated antiseptic (betadine or chlorhexidine) or disinfectant (alkaline bleach, acidified bleach, chlorine dioxide, Oxysept-333, or Virkon S) solutions for up to 15 minutes. Shells were aseptically harvested for aerobic and anaerobic culturing of bacteria. Toxicity to the developing embryo was assessed by gross evaluation of developmental changes in treated eggs incubated up to 27 d of embryonation. Halogen disinfectants acidified bleach and chlorine dioxide, and oxidants Oxysept-333 and Virkon-S eliminated viable bacteria from eggshells. However, addition of oxidants, alone or in combination with other treatments, produced significant (P < 0.05) embryotoxicity. The combination treatment of acidified bleach, chlorine dioxide, and betadine produced minimal embryotoxicity and eliminated viable bacteria from whole turkey eggs, and produced hatched poults in a gnotobiotic isolator. As a control, eggs were treated with PBS, incubated, and hatched under germ-replete conditions. After hatching, poults were euthanized and treated poults had no detectable bacterial growth or 16S rRNA gene qPCR amplification, demonstrating that acidified sodium hypochlorite, chlorine dioxide, and betadine safely hatched gnotobiotic poults. Generation of germ-free poults is an important tool and will be used to evaluate the host-pathogen interaction by foodborne pathogens such as Campylobacter spp.

Inflammatory cytokines enhance the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood neutrophils in vitro.

Mannheimia (Pasteurella) haemolytica A1 produces several virulence factors that play an important role in the pathogenesis of bovine pneumonic pasteurellosis. Foremost among these is a leukotoxin (LKT) that specifically kills ruminant leukocytes. Recent evidence suggests that M. haemolytica LKT binding to bovine leukocytes is mediated by the beta(2)-integrin CD11a/CD18 (lymphocyte function-associated antigen 1 [LFA-1]), which subsequently induces activation and cytolysis of these cells. Inflammatory cytokines, which are released during viral and bacterial infection, are reported to increase LFA-1 expression and conformational activation. We investigated the effects of the inflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) on the interaction of M. haemolytica LKT with bovine peripheral blood neutrophils (PMNs). In this study we demonstrated, by flow cytometry, that bovine PMNs increased their binding to an anti-bovine LFA-1 monoclonal antibody (BAT75A) following in vitro incubation with IL-1beta, TNF-alpha, or IFN-gamma. Incubation with cytokines also increased CD18 expression, as assessed by real-time PCR and by Western blotting. Increased LFA-1 expression by PMNs exposed to cytokines was associated with increased LKT binding and cytotoxicity. The latter represented, at least in part, enhanced PMN apoptosis, as assessed by propidium iodine staining and caspase-3 activation. The results of this study suggest that inflammatory cytokines may play an important role in enhancing the biological response of bovine PMNs to M. haemolytica LKT.

Effect of experimental infection of cattle with bovine herpesvirus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with Mannheimia (Pasteurella) haemolytica leukotoxin.

Mannheimia (Pasteurella) haemolytica A1 produces an extracellular leukotoxin (LKT) that is reported to bind the beta(2)-integrin CD11a/CD18 (LEA-1) on ruminant leukocytes. LKT binding induces activation, and subsequent cytolysis, of these cells. It is well known that active viral infection greatly increases the susceptibility of cattle to pasteurellosis. To better understand the mechanism by which this occurs, we investigated the effects of experimental in vivo infection of cattle with bovine herpes virus-1 (BHV-1) on the ex vivo interaction of bovine leukocytes with the M. haemolytica LKT. In this study, we demonstrated that active BHV-1 infection increased the expression of the beta(2)-integrin CD11a/CD18 (as defined by the mAb BAT75) on bovine peripheral blood neutrophils, enhanced the binding of LKT to bronchoalveolar lavage (BAL) leukocytes and peripheral blood neutrophils, and increased the killing of BAL leukocytes and peripheral blood leukocytes by LKT. In addition, BHV-1 greatly increased the number of BAL, resulting in many more LKT-responsive cells being present in the lungs. These findings might explain in part the increased susceptibility of BHV-1 infected cattle to pneumonic pasteurellosis.

Use of Hoechst 33342 staining to detect apoptotic changes in bovine mononuclear phagocytes infected with Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis is an intracellular pathogen of macrophages that causes a chronic enteritis (Johne's disease) in ruminants. The purpose of this study was to determine whether M. avium subsp. paratuberculosis infection causes apoptosis in bovine monocytes. Using Hoechst 33342 staining, we observed increased numbers of apoptotic monocytes within 6 h of infection with M. avium subsp. paratuberculosis, and these numbers increased further at 24 and 48 h. This effect appeared to require viable bacilli, because monocytes infected with heat-killed M. avium subsp. paratuberculosis did not exhibit a significant increase in apoptosis. Preincubation of monocytes with bovine growth hormone prior to infection with M. avium subsp. paratuberculosis did not significantly alter the number of apoptotic cells.

Haemophilus somnus induces apoptosis in bovine endothelial cells in vitro.

Haemophilus somnus causes pneumonia, reproductive failure, infectious myocarditis, thrombotic meningoencephalitis, and other diseases in cattle. Although vasculitis is commonly seen as a result of systemic H. somnus infections, the pathogenesis of vascular damage is poorly characterized. In this study, we demonstrated that H. somnus (pathogenic isolates 649, 2336, and 8025 and asymptomatic carrier isolates 127P and 129Pt) induce apoptosis of bovine endothelial cells in a time- and dose-dependent manner, as determined by Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end labeling, DNA fragmentation, and transmission electron microscopy. H. somnus induced endothelial cell apoptosis in as little as 1 h of incubation and did not require extracellular growth of the bacteria. Viable H. somnus organisms induced greater endothelial cell apoptosis than heat-killed organisms. Since viable H. somnus cells release membrane fibrils and blebs, which contain lipooligosaccharide (LOS) and immunoglobulin binding proteins, we examined culture filtrates for their ability to induce endothelial cell apoptosis. Culture filtrates induced similar levels of endothelial cell apoptosis, as did viable H. somnus organisms. Heat inactivation of H. somnus culture filtrates partially reduced the apoptotic effect on endothelial cells, which suggested the presence of both heat-labile and heat-stable factors. We found that H. somnus LOS, which is heat stable, induced endothelial cell apoptosis in a time- and dose-dependent manner and was inhibited by the addition of polymyxin B. These data demonstrate that H. somnus and its LOS induce endothelial cell apoptosis, which may play a role in producing vasculitis in vivo.

Recombinant bovine interleukin-1beta amplifies the effects of partially purified Pasteurella haemolytica leukotoxin on bovine neutrophils in a beta(2)-integrin-dependent manner.

The influx and death of polymorphonuclear leukocytes within the infected lung are hallmarks of bovine pasteurellosis. Recent reports have shown that the Pasteurella haemolytica leukotoxin (LKT) and other RTX toxins bind beta(2)-integrins on target cells. In this study we demonstrate that exposure of bovine neutrophils to recombinant bovine interleukin-1beta upregulates beta(2)-integrins (CD11a/CD18), which in turn enhance the binding and amplify the biological effects of partially purified LKT on these cells. LKT binding and cytotoxicity were inhibited by addition of an anti-integrin antibody (CD11a/CD18). These findings help to clarify the early events that occur in bovine pasteurellosis and support the hypothesis that inflammatory mediators might increase the severity of pasteurellosis by causing upregulation of beta(2)-integrins that serve as an LKT receptor on bovine neutrophils.

Apoptosis: a possible tactic of Haemophilus somnus for evasion of killing by bovine neutrophils?

Haemophilus somnus is an important veterinary pathogen that causes respiratory disease, arthritis, septicaemia and abortion in cattle and sheep. In the present study we investigated the possibility that H. somnus resists killing by bovine neutrophils, by causing the latter to undergo morphological changes consistent with apoptosis. Both serum-sensitive and serum-resistant strains of H. somnus enhanced bovine neutrophil chromatin condensation and shape change (i.e. zeiosis) in vitro, suggesting that the cells were undergoing apoptosis. Heat-killed or formalin-killed H. somnus had less effect than viable H. somnus. Chromatin margination of neutrophils was greater whenH. somnus was opsonized with adult bovine serum, which facilitates phagocytosis of the bacteria. H. somnus culture filtrates did not cause bovine neutrophil chromatin condensation. These findings suggest that direct contact with H. somnus is required for the maximal effect on bovine neutrophils. Apoptosis was confirmed by flow cytometry, using propidium iodide staining to detect DNA fragmentation. These findings suggest that H. somnus can evade killing by bovine neutrophils, in part, by inducing these cells to undergo apoptosis.

Antibacterial activity of a synthetic peptide (PR-26) derived from PR-39, a proline-arginine-rich neutrophil antimicrobial peptide.

PR-39 is a proline-arginine-rich (PR) neutrophil antibacterial peptide originally identified and purified from the porcine small intestine. We report on the synthesis of a functional antibacterial domain of PR-39, the first 26 amino acid residues of the NH2 terminus. PR-26 was as potent as or more potent than PR-39 against enteric gram-negative bacteria. This truncated form of PR-39 potentiated neutrophil phagocytosis of Salmonella choleraesuis and decreased the level of S. typhimurium invasion into intestinal epithelial cells. Scanning electron microscopy confirmed that these peptides did not lyse cells by pore-forming mechanisms; however, they potentiated the antibacterial capabilities of a pore-forming peptide, magainin A. In addition, PR-26 was not toxic to epithelial cells at concentrations several times greater than its bactericidal concentration. These data suggest that PR-39 and its functional domain, PR-26, may potentiate the host's defense capabilities against gram-negative infections.