Reducing Campylobacter jejuni colonization in broiler chickens by in-feed supplementation with hyperimmune egg yolk antibodies.

Campylobacter infections sourced mainly to poultry products, are the most important bacterial foodborne zoonoses worldwide. No effective measures to control these infections in broiler production exist to date. Here, we used passive immunization with hyperimmune egg yolks to confer broad protection of broilers against Campylobacter infection. Two novel vaccines, a bacterin of thirteen Campylobacter jejuni (C. jejuni) and C. coli strains and a subunit vaccine of six immunodominant Campylobacter antigens, were used for the immunization of layers, resulting in high and prolonged levels of specific immunoglobulin Y (IgY) in the hens' yolks. In the first in vivo trial, yolks (sham, bacterin or subunit vaccine derived) were administered prophylactically in the broiler feed. Both the bacterin- and subunit vaccine-induced IgY significantly reduced the number of Campylobacter-colonized broilers. In the second in vivo trial, the yolks were administered therapeutically during three days before euthanasia. The bacterin IgY resulted in a significant decrease in C. jejuni counts per infected bird. The hyperimmune yolks showed strong reactivity to a broad representation of C. jejuni and C. coli clonal complexes. These results indicate that passive immunization with hyperimmune yolks, especially bacterin derived, offers possibilities to control Campylobacter colonization in poultry.

Oral glutathione supplementation drastically reduces Helicobacter-induced gastric pathologies.

Helicobacter (H.) suis causes gastric pathologies in both pigs and humans. Very little is known on the metabolism of this bacterium and its impact on the host. In this study, we have revealed the importance of the glutamate-generating metabolism, as shown by a complete depletion of glutamine (Gln) in the medium during H. suis culture. Besides Gln, H. suis can also convert glutathione (GSH) to glutamate, and both reactions are catalyzed by the H. suis γ-glutamyltranspeptidase (GGT). Both for H. pylori and H. suis, it has been hypothesized that the degradation of Gln and GSH may lead to a deficiency for the host, possibly initiating or promoting several pathologies. Therefore the in vivo effect of oral supplementation with Gln and GSH was assessed. Oral supplementation with Gln was shown to temper H. suis induced gastritis and epithelial (hyper)proliferation in Mongolian gerbils. Astonishingly, supplementation of the feed with GSH, another GGT substrate, resulted in inflammation and epithelial proliferation levels returning to baseline levels of uninfected controls. This indicates that Gln and GSH supplementation may help reducing tissue damage caused by Helicobacter infection in both humans and pigs, highlighting their potential as a supportive therapy during and after Helicobacter eradication therapy.

Effect of a DIVA vaccine with and without in-feed use of coated calcium-butyrate on transmission of Salmonella Typhimurium in pigs.

BACKGROUND: For satisfactory Salmonella control, good biosecurity along the pork production chain is crucial, although additional control measures on-farm need to be considered. This study evaluated the effect of two potential control measures against the spread of Salmonella Typhimurium via a transmission experiment with 56 piglets (3-15 weeks of age): two groups were orally vaccinated with 107 - 108 Colony Forming Units (CFU)/2 mL of a new attenuated Salmonella Typhimurium vaccine 'Salmoporc-∆rfaJ' with DIVA capacities (Differentiation between Infected and Vaccinated Animals) (n = 2x16); the feed of one group was additionally supplemented with coated calcium-butyrate salt. Two weeks post vaccination, four pigs per group were orally challenged with 107 CFU/2 mL of a Salmonella Typhimurium strain 112910a. Both groups were compared with a positive (challenged/untreated; n = 16) and negative (unchallenged/untreated; n = 8) control group. Until six weeks post challenge, blood, individual faecal and finally tissue samples were examined. Adjusted transmission ratios 'Ra' were estimated, based on the challenge strain isolation from faecal and/or tissue samples. RESULTS: In both intervention groups, Ra values were lower compared to the positive control group, although these differences were not significant. In the combination group DIVA vaccine + coated butyrate, less non-challenged contact animals excreted Salmonella and less tissue samples were found Salmonella-positive in all pigs, when compared to the positive control group (P < 0.01). Seroconversion was detected in none of the vaccinated animals before challenge, when using a commercial lipopolysaccharide (LPS) ELISA targeting only Salmonella O-antigens, deleted in this vaccine. This was in contrast with an in-house whole-cell ELISA testing for various Salmonella antigens, in which Salmonella-specific antibodies were found pre-challenge in the serum of the vaccinated pigs. CONCLUSIONS: Both interventions showed a limited, non-significant reduction of Salmonella transmission between piglets. They may have applications towards Salmonella control and surveillance. Firstly, the number of Salmonella excreting contact pigs was significantly lower in the group where vaccination was combined with coated calcium-butyrate salt in the feed; secondly, the new vaccine confirmed its DIVA capacity. Therefore, these interventions merit further research with larger sample sizes, to optimize their use for Salmonella programmes.

Effects of Helicobacter suis γ-glutamyl transpeptidase on lymphocytes: modulation by glutamine and glutathione supplementation and outer membrane vesicles as a putative delivery route of the enzyme.

Helicobacter (H.) suis colonizes the stomach of the majority of pigs as well as a minority of humans worldwide. Infection causes chronic inflammation in the stomach of the host, however without an effective clearance of the bacteria. Currently, no information is available about possible mechanisms H. suis utilizes to interfere with the host immune response. This study describes the effect on various lymphocytes of the γ-glutamyl transpeptidase (GGT) from H. suis. Compared to whole cell lysate from wild-type H. suis, lysate from a H. suis ggt mutant strain showed a decrease of the capacity to inhibit Jurkat T cell proliferation. Incubation of Jurkat T cells with recombinantly expressed H. suis GGT resulted in an impaired proliferation, and cell death was shown to be involved. A similar but more pronounced inhibitory effect was also seen on primary murine CD4(+) T cells, CD8(+) T cells, and CD19(+) B cells. Supplementation with known GGT substrates was able to modulate the observed effects. Glutamine restored normal proliferation of the cells, whereas supplementation with reduced glutathione strengthened the H. suis GGT-mediated inhibition of proliferation. H. suis GGT treatment abolished secretion of IL-4 and IL-17 by CD4(+) T cells, without affecting secretion of IFN-γ. Finally, H. suis outer membrane vesicles (OMV) were identified as a possible delivery route of H. suis GGT to lymphocytes residing in the deeper mucosal layers. Thus far, this study is the first to report that the effects on lymphocytes of this enzyme, not only important for H. suis metabolism but also for that of other Helicobacter species, depend on the degradation of two specific substrates: glutamine and reduced glutatione. This will provide new insights into the pathogenic mechanisms of H. suis infection in particular and infection with gastric helicobacters in general.

Antibacterial therapeutics for the treatment of chytrid infection in amphibians: Columbus's egg?

BACKGROUND: The establishment of safe and effective protocols to treat chytridiomycosis in amphibians is urgently required. In this study, the usefulness of antibacterial agents to clear chytridiomycosis from infected amphibians was evaluated. RESULTS: Florfenicol, sulfamethoxazole, sulfadiazine and the combination of trimethoprim and sulfonamides were active in vitro against cultures of five Batrachochytrium dendrobatidis strains containing sporangia and zoospores, with minimum inhibitory concentrations (MIC) of 0.5-1.0 µg/ml for florfenicol and 8.0 µg/ml for the sulfonamides. Trimethoprim was not capable of inhibiting growth but, combined with sulfonamides, reduced the time to visible growth inhibition by the sulfonamides. Growth inhibition of B. dendrobatidis was not observed after exposure to clindamycin, doxycycline, enrofloxacin, paromomycin, polymyxin E and tylosin. Cultures of sporangia and zoospores of B. dendrobatidis strains JEL423 and IA042 were killed completely after 14 days of exposure to 100 µg/ml florfenicol or 16 µg/ml trimethoprim combined with 80 µg/ml sulfadiazine. These concentrations were, however, not capable of efficiently killing zoospores within 4 days after exposure as assessed using flow cytometry. Florfenicol concentrations remained stable in a bathing solution during a ten day period. Exposure of Discoglossus scovazzi tadpoles for ten days to 100 µg/ml but not to 10 µg florfenicol /ml water resulted in toxicity. In an in vivo trial, post metamorphic Alytes muletensis, experimentally inoculated with B. dendrobatidis, were treated topically with a solution containing 10 µg/ml of florfenicol during 14 days. Although a significant reduction of the B. dendrobatidis load was obtained, none of the treated animals cleared the infection. CONCLUSIONS: We thus conclude that, despite marked anti B. dendrobatidis activity in vitro, the florfenicol treatment used is not capable of eliminating B. dendrobatidis infections from amphibians.

Influence of mycotoxins and a mycotoxin adsorbing agent on the oral bioavailability of commonly used antibiotics in pigs.

It is recognized that mycotoxins can cause a variety of adverse health effects in animals, including altered gastrointestinal barrier function. It is the aim of the present study to determine whether mycotoxin-contaminated diets can alter the oral bioavailability of the antibiotics doxycycline and paromomycin in pigs, and whether a mycotoxin adsorbing agent included into diets interacts with those antibiotics. Experiments were conducted with pigs utilizing diets that contained blank feed, mycotoxin-contaminated feed (T-2 toxin or deoxynivalenol), mycotoxin-contaminated feed supplemented with a glucomannan mycotoxin binder, or blank feed supplemented with mycotoxin binder. Diets with T-2 toxin and binder or deoxynivalenol and binder induced increased plasma concentrations of doxycycline administered as single bolus in pigs compared to diets containing blank feed. These results suggest that complex interactions may occur between mycotoxins, mycotoxin binders, and antibiotics which could alter antibiotic bioavailability. This could have consequences for animal toxicity, withdrawal time for oral antibiotics, or public health.

Efficacy of four enrofloxacin treatment regimens against experimental infection in turkey poults with avian pneumovirus and Ornithobacterium rhinotracheale.

Drinking-water treatment with enrofloxacin is widely used to cure respiratory infections in turkeys. The current treatment regimen advises a 5-day treatment at 10 mg/kg body weight. Since enrofloxacin exerts a concentration-dependent activity it might be useful to provide the total treatment dose of 50 mg/kg total dose in a single-day treatment regimen. We therefore assessed whether single-day treatment regimens with 50 mg/kg body weight were clinically equivalent to the advised multiple-day treatment regimen with 10 mg/kg body weight for 5 days. For this purpose, five groups of 16 turkeys, 22 days old, were experimentally inoculated with avian metapneumovirus (APV) and Ornithobacterium rhinotracheale and subsequently treated in the drinking water with enrofloxacin, using either a single-day treatment regimen at 50 mg/kg body weight during a 5-h, 10-h or 20-h period or a standard 5-day treatment regimen at 10 mg/kg body weight/ day for 20 h. Although initially all dosage regimens cleared O. rhinotracheale from the trachea, 4 days after onset of treatment O. rhinotracheale bacteria were re-excreted in the single-day regimens but without worsening of the clinical symptoms. The 5-day treatment with 10 mg enrofloxacin/kg in turkeys provided the best results for the treatment of an O. rhinotracheale infection in turkeys by shortening the course and reducing the severity of clinical disease and by eliminating O. rhinotracheale from the respiratory tract without re-emergence. None of the used treatment regimens promoted the selection of bacterial clones with reduced susceptibility or resistance.

The cereal type in feed influences gut wall morphology and intestinal immune cell infiltration in broiler chickens.

In broiler chickens, a diet where the major cereal types are wheat, rye and/or barley has a lower digestibility compared with a diet in which maize is the major cereal type. In the present study, the effects of two different dietary cereal types, maize v. wheat/rye, on host factors (inflammation and gut integrity) and gut microbiota composition were studied. In addition, the effects of low-dose Zn-bacitracin supplementation were examined. Broilers given a wheat/rye-based diet showed more villus fusion, a thinner tunica muscularis, more T-lymphocyte infiltration, higher amount of immune cell aggregates in the mucosa, more and larger goblet cells and more apoptosis of epithelial cells in the mucosa than those given a maize-based diet. Adding Zn-bacitracin generally reversed these alterations. The microbiota composition was analysed by the use of terminal-restriction fragment length polymorphism, showing changes in the microbiota composition depending on the cereal type used in the diets. The effect of the change of cereal type on the gut microbiota composition was larger than that of Zn-bacitracin supplementation. In conclusion, a wheat/rye-based diet evoked mucosal damage, an alteration in the composition of the microbiota and an inflammatory bowel type of condition.

Designing a successful antimicrobial treatment against Devriesea agamarum infections in lizards.

Dermatitis caused by Devriesea agamarum poses a major health problem for the captive maintenance of several desert lizard species. This study was conducted to determine the optimal antimicrobial treatment to eliminate D. agamarum infections from lizards. First, the in vitro susceptibility of 42 D. agamarum isolates was determined for 10 different antimicrobial agents using an agar dilution method. In none of the isolates acquired antimicrobial resistance was demonstrated. Then, two intramuscular treatment protocols using either enrofloxacin or ceftiofur were tested in bearded dragons (Pogona vitticeps) experimentally infected with a D. agamarum strain showing a MIC of 2 microg/ml for enrofloxacin and 0.12 microg/ml for ceftiofur. While D. agamarum could no longer be isolated after 17-18 days of ceftiofur administration, enrofloxacin administration and sham treatment failed in clearing the infection after 27 days of treatment. Based on these results, intramuscular injection of ceftiofur at 5 mg/kg BW q24h was used to treat naturally and clinically infected Uromastyx lizards. This resulted in marked clinical improvement and clearance of infection after 12 days on average. In conclusion, intramuscular administration of ceftiofur at 5 mg/kg BW q24h eliminates D. agamarum in lizards, resulting in clinical cure.

Short-chain fatty acids and L-lactate as feed additives to control Campylobacter jejuni infections in broilers.

The usefulness of butyrate, acetate, propionate and l-lactate for the control of Campylobacter jejuni infections in broilers was assessed. For this purpose, the effect of these acids on the growth of C. jejuni in broth and intestinal mucous was determined, as well as their influence on the invasiveness of C. jejuni in intestinal epithelial cells. From these in vitro obtained results, one acid was retained for use as a feed additive in an in vivo trial. Butyrate was the most successful of the short-chain fatty acids, with 12.5 mM being bactericidal for C. jejuni at pH 6.0. Propionate and acetate had a bacteriostatic effect at 50 mM. None of the short-chain fatty acids had a bactericidal effect at pH 7.5 at a maximum concentration of 50 mM. Mucous increased the minimum bactericidal concentration of butyrate, but not the bacteriostatic concentrations of propionate or acetate. When C. jejuni was incubated in growth subinhibitory concentrations of butyrate, acetate or propionate or 25 mM L-lactate, no alteration in the invasive capabilities of C. jejuni in Caco-2 cells was observed. The addition of butyrate-coated micro-beads to the feed was unsuccessful to reduce C. jejuni caecal colonization in a seeder model using 2-week-old broilers. In conclusion, despite the marked bactericidal effect of butyrate towards C. jejuni in vitro, butyrate-coated micro-beads do not protect broilers from caecal colonization with C. jejuni in the applied test conditions. This might be partially ascribed to the protective effect of mucous and the rapid absorption of butyrate by the enterocytes.

Induction of the carrier state in pigeons infected with Salmonella enterica subspecies enterica serovar typhimurium PT99 by treatment with florfenicol: a matter of pharmacokinetics.

Paratyphoid caused by Salmonella enterica subsp. enterica serovar Typhimurium is the main bacterial disease in pigeons. The ability of Salmonella serovar Typhimurium to persist intracellularly inside pigeon macrophages results in the development of chronic carriers, which maintain the infection in the flock. In this study, the effect of drinking-water medication with florfenicol on Salmonella infection in pigeons was examined. The pharmacokinetics of florfenicol in pigeons revealed a relatively high volume of distribution of 2.02 liters/kg of body weight and maximum concentrations in plasma higher than the MICs for the Salmonella strain used (4 microg/ml) but quick clearance of florfenicol due to a short half-life of 1.73 h. Together with highly variable bioavailability and erratic drinking-water uptake, these parameters resulted in the inability to reach a steady-state concentration through the continuous administration of florfenicol in the drinking water. Florfenicol was capable of reducing only moderately the number of intracellular salmonellae in infected pigeon macrophages in vitro. Only at high extracellular concentrations (>16 microg/ml) was a more-than-10-fold reduction of the number of intracellular bacteria noticed. Florfenicol treatment of pigeons via the drinking water from 2 days after experimental inoculation with Salmonella serovar Typhimurium until euthanasia at 16 days postinoculation resulted in a reduction of Salmonella shedding and an improvement in the fecal consistency. However, internal organs in florfenicol-treated pigeons were significantly more heavily colonized than those in untreated pigeons. In conclusion, the oral application of florfenicol for the treatment of pigeon paratyphoid contributes to the development of carrier animals through sub-MIC concentrations in plasma that do not inhibit intracellular persistency.