Research Note: Effect of a phlorotannin extract of the brown seaweed Ascophyllum nodosum as a potential control strategy against Campylobacter in broilers.

Poultry is generally recognized as the main source of human campylobacteriosis and Campylobacter is highly prevalent at the farm level. To reduce the relative risk of human campylobacteriosis attributable to broiler meat, it is necessary to reduce Campylobacter loads in broiler ceca but to date, no effective, reliable and practical strategy is available. The marine environment is a rich source of original natural compounds exhibiting different biological activities. The objective of this study was to test a phlorotannin extract of the brown seaweed Ascophyllum nodosum as a potential control strategy against Campylobacter in broilers. Bactericidal activity has been demonstrated in vitro, on several Campylobacter spp. strains at a range of 0.06 to 0.47 mg/mL. Therefore, an in vivo trial in experimental facilities was performed to evaluate addition of 0.2% (w/w) of an A. nodosum extract to feed distributed at the end of rearing from day 31 to day 35, and to assess the effect on artificial Campylobacter jejuni colonization. No statistical differences in Campylobacter enumeration were observed between the treated and control groups. Another trial was performed in a commercial broiler flock. Feed containing the extract at 0.2% (w/w) (2 kg/t) was distributed during the last 5 days of rearing (day 33-day 38). No significant effects on Campylobacter colonization and on growth parameters were observed compared to the control group. Additional studies are needed to assess whether active polyphenols are found in the cecum.

Randomized control trial to test the effect of a feed additive on Campylobacter contamination in commercial broiler flocks up to slaughter.

A randomized controlled trial (RCT) was carried to evaluate the effect of a feed additive on Campylobacter contamination of broilers reared in commercial conditions. Twenty-four broiler flocks naturally contaminated with Campylobacter were enrolled in the RCT: 12 were assigned to a control group (C) fed with a conventional finishing feed from 4 weeks of age to slaughter (around 35 days), and the other group of 12 flocks (S) was fed with a finishing feed supplemented with 250 ppm of a patented feed additive (an ion-exchanged clay compound) previously proven to reduce Campylobacter contamination in broiler caeca under experimental conditions. Enumeration of Campylobacter colonies in caeca (8 per flock) was carried out following ISO standards before feed distribution and at slaughter. Before treatment, the caecal Campylobacter load tended to be lower in C flocks (7.1 ± 1.9 log CFU/g, CI95% [6.6-7.5]) than in S flocks (7.7 ± 1.0 log UFC/g, CI95% [7.5-7.9]) (p = .05). At slaughter, the bacterial load was similar in the S (7.7 ± 1.0 log CFU/g, CI95% [7.5-7.9]) and C groups (7.5 ± 1.2 log CFU/g, CI95% [7.2-7.8]) (p = .73). Therefore, the feed additive had no significant effect on the caecal Campylobacter load at slaughter under the tested conditions. The logistical constraints inherent in field trials and the natural variability of Campylobacter contamination in naturally infected broiler flocks make it difficult to reproduce experimental results in in situ farm conditions. RCT testing of an intervention strategy in commercial situation is therefore a key step in evaluating pre-harvest interventions against food-borne pathogens.

Promising new vaccine candidates against Campylobacter in broilers.

Campylobacter is the leading cause of human bacterial gastroenteritis in the European Union. Birds represent the main reservoir of the bacteria, and human campylobacteriosis mainly occurs after consuming and/or handling poultry meat. Reducing avian intestinal Campylobacter loads should impact the incidence of human diseases. At the primary production level, several measures have been identified to reach this goal, including vaccination of poultry. Despite many studies, however, no efficient vaccine is currently available. We have recently identified new vaccine candidates using the reverse vaccinology strategy. This study assessed the in vivo immune and protective potential of six newly-identified vaccine antigens. Among the candidates tested on Ross broiler chickens, four (YP_001000437.1, YP_001000562.1, YP_999817.1, and YP_999838.1) significantly reduced cecal Campylobacter loads by between 2 and 4.2 log10 CFU/g, with the concomitant development of a specific humoral immune response. In a second trial, cecal load reductions results were not statistically confirmed despite the induction of a strong immune response. These vaccine candidates need to be further investigated since they present promising features.

Influence of enrichment and isolation media on the detection of Campylobacter spp. in naturally contaminated chicken samples.

Investigating Campylobacter epidemiology requires adequate technique and media to ensure optimal culturing and accurate detection and isolation of Campylobacter strains. In the present study, we investigated the performances of three enrichment durations in Bolton broth (0, 24 and 48h) and compared four isolation media (mCCDA, Karmali, Butzler no. 2 and CampyFood agar (CFA)) for the detection of Campylobacter positive samples and the identification of Campylobacter species, from naturally contaminated broiler chicken samples (caeca, neck skin from carcasses, and skin from thighs). We compared our local results to those we obtained with samples from a European survey (caeca and neck skin) and a national survey (neck skin, thigh skin, and breast). Direct plating favored the detection of positive samples highly contaminated by Campylobacter (caeca and neck skin from carcasses) whatever the media. A longer enrichment reduced the rates of Campylobacter recovery except when using Butzler no. 2, more particularly for neck skin which background microflora was less important than in caeca. As a matter of fact, enrichment allowed a higher detection rate of positive samples with low Campylobacter contamination levels (breast, thigh skin), this detection being enhanced when using Butzler no. 2. When comparing the 3 other selective media, CFA was the 2nd most efficient media prior to mCCDA and Karmali. Interestingly, enrichment promoted the growth of Campylobacter coli but this promotion was least with Butzler no. 2 agar. Our study has confirmed the need to adapt the method to the types of samples for improving the detection of Campylobacter and that the method may affect the prevalence of the species.

Efficacy of feed additives against Campylobacter in live broilers during the entire rearing period.

Poultry meat is the major source of human campylobacteriosis, the most frequently reported zoonosis in the EU. The prevalence of Campylobacter colonization in European broiler flocks is 71%. Despite considerable efforts, there is still no effective strategy available to prevent or reduce Campylobacter colonization in broilers. This study tested a wide variety of feed additives to reduce Campylobacter shedding in primary poultry production. Twelve additives containing organic or fatty acids, monoglycerides, plant extracts, prebiotics, or probiotics were tested. For each additive, broilers contaminated with Campylobacter jejuni were fed with an additive free diet (control group) or with a supplemented diet (treated group) and Campylobacter loads compared at three sampling times. No treatment was able to prevent broiler colonization by Campylobacter, and there was a high degree of variation in contamination among the birds. At 14 d of age, eight treatments significantly decreased the colonization level compared to the control group by a maximum of 2 log10 CFU/g. At 35 d of age, three of these treatments still had a significant effect with a maximum reduction of 1.88 log10 CFU/g for a probiotic. At 42 d of age, only one short-chain fatty acid was still significantly efficient with a mean reduction over 2 log10 CFU/g. In addition, a probiotic and a prebiotic-like compound significantly decreased the contamination by a maximum of 3 log10 CFU/g, only at the 42-d sampling period. This study gives promising results regarding the use of feed additives to reduce Campylobacter infection in flocks. Nevertheless, a global approach, combining intervention measures at the different steps of the broiler meat production chain could have a greater impact on the reduction of public health risk.

Prevalence of and risk factors for Campylobacter colonisation in broiler flocks at the end of the rearing period in France.

1. A study was conducted to estimate the prevalence and quantification by species of Campylobacter infection in broiler flocks at the end of the rearing period and to identify associated risk factors. 2. A questionnaire about the rearing period was completed and caecal samples were collected from 121 broiler flocks in Brittany, France, during 2008. 3. Campylobacter was isolated in 87 out of 121 flocks--a prevalence of 71.9% (95% CI, 63.7-80.1%), including 40.5% of Campylobacter jejuni and 29.8% of Campylobacter coli. 4. The average concentration, in positive flocks, was 7.96 log10 cfu/g and ranged from 3.15 to 10.32 log10 cfu/g. 5. The average concentration by species was: 7.57 log10 cfu/g for C. jejuni and 8.44 log10 cfu/g for C. coli. 6. There was a seasonal effect, with increased risk of Campylobacter colonisation in June, July and August (odds ratio (OR) = 9.59, 95% CI 1.15-79.75). 7. The other factors, associated with lower risk of Campylobacter colonisation, were the acidification of drinking water (OR = 0.33, 95% CI 0.13-0.86), antibiotic treatment at the beginning of the rearing period (OR = 0.20, 95% CI 0.07-0.55) and rodent control around the house (OR = 0.18, 95% CI 0.03-0.95). 8. The results show that hygiene practices and biosecurity measures could lead to a reduction in Campylobacter colonisation.

Regulation of a high-affinity diamine transport system in Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi epimastigotes take up exogenous [3H]putrescine and [3H]cadaverine by a rapid, high-affinity, transport system that exhibits saturable kinetics (putrescine K(m) 2.0 microM, V(max) 3.3 nmol/min per 10(8) cells; cadaverine K(m) 13.4 microM, V(max) 3.9 nmol/min per 10(8) cells). Putrescine transport is temperature dependent and requires the presence of a membrane potential and thiol groups for activity. Its activity is altered in response to extracellular putrescine levels and as the cells proceed through the growth cycle. This transporter shows high specificity for the diamines putrescine and cadaverine, but low specificity for the polyamines spermidine and spermine. The existence of rapid diamine/polyamine transport systems whose activity can be adjusted in response to the growth conditions is of particular importance, as they seem unable to synthesize their own putrescine [Hunter, Le Quesne and Fairlamb (1994) Eur. J. Biochem. 226, 1019-1027].

Identification and biosynthesis of N1,N9-bis(glutathionyl)aminopropylcadaverine (homotrypanothione) in Trypanosoma cruzi.

Radiolabelling studies using tritiated ornithine, arginine and lysine, together with the relevant amino acid decarboxylase enzyme assays, indicate that the epimastigote stage of Trypanosoma cruzi is unable to synthesise significant amounts of putrescine and cadaverine de novo, compared to the amounts of these diamines scavenged from the growth medium. Radiolabelled putrescine is readily incorporated into spermidine, spermine and the trypanosomatid-specific polyamine-glutathione conjugate trypanothione (N1,N8-bis(glutathionyl)spermidine). Likewise, radiolabelled cadaverine is incorporated into the analogous polyamines aminopropylcadaverine, bis(aminopropyl)cadaverine and another major unidentified component. Subsequent studies showed this major component to be a novel polyamine-thiol conjugate whose structure was confirmed by chemical synthesis to be N1,N9-bis(glutathionyl)aminopropylcadaverine (homotrypanothione). Kinetic analyses using recombinant T. cruzi trypanothione reductase demonstrated that homotrypanothione disulphide is readily reduced by this enzyme with kinetic parameters similar to trypanothione disulphide, suggesting that it is a physiological substrate in vivo. Thus the epimastigote form of T. cruzi differs significantly from the African trypanosomes and Leishmania in (a) being unable to synthesise significant amounts of diamines de novo, (b) converting significant amounts of putrescine and cadaverine to spermine and bis(aminopropyl)cadaverine, respectively and (c) the ability to synthesise homotrypanothione as well as trypanothione. The implications of these findings with respect to the prospective chemotherapy of Chagas' disease are discussed.