Final Report Summary - CAMPYBRO (Control of Campylobacter infection in broiler flocks through two-steps strategy: nutrition and vaccination)
Executive Summary:
The objective of CAMPYBRO project, under the 7th FRAME PROGRAM of UE (Capacities- Research for SME Associations FP7-SME GA 605385) is to develop practical strategies to decrease the population of Campylobacter jejuni in broilers through two strategies, nutrition and vaccination. The project started in September 2013 and will finish in August 2016, is coordinated by IMASDE AGROALIMENTARIA S.L. (IMASDE), and is formed by 10 partners: 5 poultry producers National Associations (FIA and CIDEF from France, PROPOLLO from Spain, NEPLUVI from The Netherlands, BTT from Hungary), a vaccine laboratory (CZV, Spain), a poultry producer (EXPLOTACIONES AVÍCOLAS JOSÉ LUIS REDONDO S.A. Spain) and a lab (MIKROLAB, Hungary). The Research centers involved are ANSES (France) and IMASDE (Spain).
Into the nutrition strategy the steps were: i) identify single additives that decreased in vivo cecal Campylobacter infection, ii) identify synergisms between the most efficient additives, iii) look for changes in the feed form that also decreased in vivo cecal Campylobacter infection, iv) look for synergisms between feed form and combination of additives, v) optimize the nutrition program (time of administration, dose), vi) test the additives strategy in field (experimental and commercial) conditions with natural contamination, vi) demonstration field trials, (Spain, France, The Netherlands, Hungary). Single additives: A total of 24 single products belonging to the next categories of products: ORGANIC ACIDS (OA, 42%), PLANT EXTRACTS (PE, 33%), PREBIOTICS (PRE, 4%) or PROBIOTICS (PRO, 21%) were tested in vivo through challenge trials. In total, 46% of the products showed a significant reduction in Campylobacter counts anytime. The best results were obtained with a yeast extract, with a probiotic based on B. subtilis, and with monoglycerides of medium chain fatty acids (MCFA). Group of additives: A total of 10 combinations between families of groups of additives (PE, PRO, PRE and OA) were designed and tested. One of the combinations tested (mixture of a combination of organic acids and monoglycerides of MCFA [MGOA] and a B. subtilis-based probiotic [CALS]) dramatically decreased the Campylobacter population. Feed form studies: A total of 4 experiments were done, testing: the diet main cereal (corn, wheat or barley), the inclusion of oat hulls (OH), grinding screen size (Ø2 vs 5mm), feed presentation (mash vs pellets) or whole wheat (ww) inlusion. Only the inclusion of ww and 5% of OH decreased the Campylobacter contamination at 42d. Synergisms between additives and feed form: No synergisms were found, and the additives strategy was selected to continue. Additive program optimization: The combination of MGOA and CALS had a synergistic effect, although this effect was variable: in one trial there was an effect at 35d but not at 42d, in two trials the effect was maintained at 35 and 42d, and in one trial there was not any effect. The yeast extract did not show any effect on Campylobacter cecal population. Field trials: In field experimental conditions, the natural infection did not occur in 3 of 5 experiments, and in the other 2 experiments the combination of MGOA+CALS had not any effect on Campylobacter infection. In Spain, the pattern of natural contamination was very different and there were not any effect of the additives. In Hungary, for unknown reasons, the additive addition not only did not decrease the Campylobacter population, but it increased it. Demonstration activities: In France.... In Spain, the treatment delay the infection in two trials, and the MGOA by water decreased the infection in other trial. In The Netherlands, no natural infection was achieved. In Hungary, there were no effect of treatment on final contamination level.
Vaccine development: a list of 12 potential highly immunogenic vaccine antigens against C. jejuni was obtained through reverse vaccinology. A vaccine protocol was studied using flagellin as a model. Once the model was developed, the new potential antigens were tested, with promising results on Campylobacter cecal reduction of at least five antigens.
Final conclusions: i) a combination of additives (MGOA+CALS) decreased the Campylobacter cecal population in challenge trials, ii) the effect of this combination was not homogenous, iii) the interaction between in vitro trials and in vivo trials was avoided, but there is an interaction between challenge trials and natural contamination infection trials, iv) much more knowledge is needed in Campylobacter epidemiology, since the natural infection did not follow classical patrons of infection, v) the demonstration trials will define if the strategies developed are effective in field conditions in some cases, vi) a potential vaccine could be developed, although its commercial development could be compromised by the price and the vaccination program.
Project Context and Objectives:
Campylobacteriosis is the most frequently reported zoonotic illness at the EU, with 238,851 declared cases in 2014, and a total annual cost of 2.4 billion € per year in the EU27.
However, In order to appraise more realistically the impact of Campylobacteriosis in EU/EEA MS we compiled data from three different sources: an ECDC-funded sero-epidemiological study, reported cases in The European Surveillance System (TESSy) database, and data stemming from literature reviews. ECDC commissioned sero-epidemiological study aimed at estimating the exposure to Campylobacter spp. in selected EU/EEA MS by measuring antibody responses in human serum samples and back-calculating the estimated time of infection. Results from this serological study were anchored to published community studies in order to derive the actual incidence of Campylobacteriosis disease. In the EU/EEA MS, the annual rate of exposure to Campylobacter spp. is estimated to be around 0.83 per person-year, translating in more than 420 million yearly infections. The vast majority of exposed cases does not develop the clinical disease and remain asymptomatic. Based on community studies, the related incidence of Campylobacteriosis disease is 475 per 100 000 (CI 95%: 423-524 per 100 000) or 2.4 million cases per year amongst European citizens. Underestimation of the disease, therefore, is considered to be 11 times the notification rate. Moreover, in a recent burden of disease study (BCoDE 2015), ECDC estimated that about 600 deaths are related to Campylobacteriosis every year, largely among elderly people. Results from BCoDE 2015 also found that Campylobacteriosis is the food and water-borne disease producing the highest number of DALYs (Cassini, 2015 ).
We are therefore faced with a major problem of public health, and the EC has been discussing for a long time the possible measures to be taken to decrease the risk of campilobacteriosis infection. After a stakeholder meeting in Brussels , and the consultation of all the EU countries, very recently has been launched a Regulation proposal amending Regulation (EC) No 2073/2005 as regards Campylobacter in broiler carcasses. This Regulation will be published in short time, and in theory it will be enter into force the 01/09/2016. Based on the EFSA opinions of 2010 and 2011, the Commission commissioned an analysis of setting certain control measures for reduction of Campylobacter in broiler meat at different stages of the food chain. The main conclusion of this analysis is that setting a process hygiene criterion to Campylobacter in broiler carcasses would provide one of the best balances between reducing human Campylobacteriosis attributed to the consumption of poultry meat and economic consequences from the application of the criterion. In view to reduce administrative burden for food business operators, the sampling plan for the criterion on Campylobacter should follow the same testing approach as for the process hygiene criterion set out in Row 2.1.5 of Chapter 2 of the Regulation (EC) No 2073/2005 for Salmonella in poultry carcasses. The same samples used for testing the compliance with criterion 2.1.5 may therefore be used for the Campylobacter analyses. The international standard ISO/TS 10272-2 will be the horizontal method for the enumeration of Campylobacter spp in food and feed stuffs.
When testing against the process hygiene criterion in poultry carcasses in slaughterhouses, neck skins from a minimum of 15 poultry carcasses shall be sampled at random after chilling during each sampling session. A piece of approximately 10 g from neck skin shall be obtained from each poultry carcass. On each occasion the neck skin samples from three poultry carcasses from the same flock of origin shall be pooled before examination in order to form 5 x 25 g final samples. The food business operators of slaughterhouses or establishments producing minced meat, meat preparations, mechanically separated meat or fresh poultry meat shall take samples for microbiological analysis at least once a week. The day of sampling shall be changed each week to ensure that each day of the week is covered.
Campylobacteriosis is produced by Campylobacter. These rods are negative, curved or spiral shaped, gram movable by flagellum are not sporulate and microaerophilic. There are at least 23 species, but the most important are thermophilic, growing at 32-47 º C , and are those that cause human gastroenteritis. The most important are C. jejuni and C. coli, with other minor (C. lari, C. upsaliensis, C. helveticus). Of the reported cases of Campylobacteriosis in the EU, 81.1 % were C. jejuni, C. coli 6.2%, 0.2% C. lari, C. upsaliensis 0.06%, and 0,01% C. fetus . The rest were doubts among C. jejuni and C. coli. Another feature of Campylobacter is their sensitivity to desiccation and extreme temperatures, both heat and cold. The natural reservoir of Campylobacter is the gastrointestinal tract of domestic and wild animals, most notably birds ( chickens, ducks , turkeys, etc. ), pigs, and cattle, and rodents , dogs , cats and insects (mainly flies). Poultry meat is the major source of human infection, and there is a direct correlation between the contaminations of the herds with the carcasses. However, also other foods such as raw milk, are reporting a growing number of cases.
Campylobacter is commensal bacteria that do not produce illness or morbidity in broilers (apparently, it does not produce any immune response), and its capacity of (horizontal) infection is very effective, with the whole farm infected within one week form a few animals infected. Basically Campylobacter lives in the caeca of broilers, and for that reason it is complicated to design nutritional strategies to decrease the colonization, since most of the products have been absorbed or changed during its pass through the gastrointestinal tract. Moreover, this is the main reason of the lack of coincidence between in vitro and in vivo trials. Therefore, there is still no effective, reliable and practical strategy available to prevent or to reduce Campylobacter colonization in broilers.
One of the most important problems to deal with is the lack of knowledge on Campylobacter epidemiology. It is not known the source of Campylobacter in the continuous contamination process within a farm. There are a lot of possible sources, such us the rest or organic matter in drinkers or feeders from the previous flock, the drinking water, the farmers tools, several pests, wild animals, etc. However, no conclusive studies have been done, and in fact, multiple strains has been found in several studies in consecutive contaminated batches, which suggest that there are several sources of contamination, and that the origin is not necessarily the same source between consecutive flocks. Also, there is a lack of knowledge on the transmission of Campylobacter along the food chain. In theory, the strains that appear in the live broilers should be the same than in the slaughterhouse and in the carcasses. But recent research has demonstrated that this is not always true, and the strains appears and disappears along the food chain with a logical explanation.
Starting from the premise that there is no vertical transmission reduction strategies fall into two groups: pre-slaughter and post sacrifice. Within the pre-slaughter strategies, there are different alternatives: Biosecurity, Vaccine or Nutritional strategies. The two last are being developed by the project CAMPYBRO "Control of Campylobacter infection in broiler flocks through two-steps strategy: nutrition and vaccination”.
The biosecurity measures to control Campylobacter have been extensively analyzed by the CAMCON European project (www.camcon-eu.net). This project has generated a good manufacturing practices manual, in which a lot of biosecurity measures are recommended. However, many of them are difficultly applicable in most of the real farms within the EU, mainly because of the farm design and age. In addition, the thinning practice (in which part of the animals are slaughtered at 32-36d and the rest are maintained until the final slaughtering), is a clear break of the biosecurity, and most of the measures lack their efficacy after this process. To avoid the thinning is not possible in practice, because of the huge economic impact for the producers in a very complex context, with very low margins and the threat of imported chickens for third countries.
Also, the most efficient biosecurity measures such as the fly control through nets, have demonstrated their efficacy only after other very strict measures have been implemented, which is not always possible. On the other hand, the measures proposed by CAMPYBRO project (nutrition and vaccination) can be implemented immediately and horizontally to the entire poultry sector in the EU and worldwide. However, in practice, the most logical procedure should be to combine all the available techniques in order to decrease the possibility of contamination.
Regarding the vaccine development, there are different initiatives. The University of Arizona has recently presented Anivax, and they are searching investors to initiate the registration in the USDA. Nothaft et al. (2015 ) engineered the C. jejuni N-glycan heptasaccharide to be expressed on the surface of E. coli cells as a heptasaccharide-LPS fusion. Vaccination of chickens with live E. coli expressing the heptasaccharide fusions resulted in >8 log(10) reduction of C. jejuni colonization compared to the control. However for the development and approval of this potential vaccine there is a long period, and since it is a GMO vaccine, its approval in Europe should be complicated.
The objective of the present project is to develop a two-steps strategy in order to fight against Campylobacter in primary poultry production. The first step, through a nutritional approach, and in a second step, a more medium-term strategy through the development of a vaccine. The nutritional approach consisted in a combination of two strategies: i) nutritional additives, such as plant extracts, organic acids, prebiotics, probiotics, trying to find synergisms between them, and ii) feed presentation (type of cereal, mash vs pellets, particle size, whole wheat or oat hulls inclusion). This nutritional strategy was tested in trials with (challenged) infected broilers first, and then with natural contamination in real conditions (either experimental or commercial). In a second step, the vaccination approach was based on a novel development of a Campylobacter vaccine through the use of reverse vaccinology. First, 12 novel antigens were detected based on their theoretical antigenicity. After, a vaccination model was developed by using falgellin protein. Finally, the new candidates were tested for their capacity of generating immunity and their capacity to reduce the Campylobacter numbers in vivo.
The objectives of the project are to develop i) a short term strategy to decrease the Campylobacter infection in vivo through new nutritional strategies based on a synergic combination of feed additives and feed presentation, ii) a medium term strategy through a novel vaccine to reduce the prevalence of Campylobacter in broiler flocks.
Project Results:
WP1: Efficacy of several compounds against Campylobacter in broilers orally infected looking for synergies
The WP1 “Efficacy of several compounds against Campylobacter in broilers orally infected looking for synergies” has developed the additives strategy through three tasks: T1.1: “In vivo effectiveness of products based on plant extracts, organic acids, prebiotics, and probiotics against Campylobacter”; T1.2: “In vitro effectiveness of mixtures of products: Synergistic effect”; and T1.3 “In vivo effectiveness of product mixtures based on plant extracts, organic acids, prebiotics, and probiotics against Campylobacter”.
The objectives of this WP were to design combinations of additives that effectively decrease the Campylobacter population of infected broilers in vivo. To fulfill this objective, in the T1.1. a total of 24 additives were tested. The 24 products involved belong to the next categories of products: ORGANIC ACIDS ([OA], 42%), PLANT EXTRACTS ([PE], 33%), PREBIOTICS ([PRE], 4%) or PROBIOTICS ([PRO], 21%). The 24 additives were tested in vivo through challenge trials (50% each RTD: ANSES and IMASDE AGROALIMENTARIA, S.L.) in which all the animals were infected with C. jejuny ST-45. After inoculation, Campylobacter jejuny was analyzed at 14/21, 35 or 42d of age, reflecting the commercial practice in different countries across the EU. Campylobacter counts were analyzed by ISO/TS 10272-2:2006. At 14d, the products that decrease the Campylobacter counts were BRC, Biotronic, PoultryStar, Alliin, Adimix, Antaphy and Campylostat. At 21d, only the caprilic+capric as acids or monoglycerides (MG), and the combination of organic acids (OA) plus MG of Medium Chain Fatty Acids (MCFA) had a significant effect. At 35 d, BRC, PoultryStar, Adimix, caprilic+capric as acids or MG, and the combination of OA plus MG of MCFA also reduced the contamination. At 42d, the probiotic Calsporin, the prebiotic XPC, the acid Adimix, MG of caprilic+capric acids, and the combination of OA plus MG of SMCFA decreases the caecal Campylobacter counts. The usual level of contamination in control animals were between 7-9 Log10UFC/g, and the reductions were between 1 and 3 Log10UFC/g. Therefore, in total, 46% of the products (11 of 24) showed a significant reduction in Campylobacter counts in any of the periods tested.
although all of them had a significant effect in vivo, only three products had a significant bacteriostatic effect in vitro: monoglycerides (MG) of Medium Chain Fatty Acids (MCFA), MG-MCFA in combination with organic acids (OA), and the PE Alliin. Interestingly, results did not reveal important synergistic effects between the products tested. However, potential antagonistic effects were also observed.
These results were used to define the 10 treatments of Task 1.3 based on the mixture of five products (MG-MCFA, PE, coated OA, OA+MG-MCFA, and the yeast-wall based PRE) and the two PRO (based on multi-species and B. subtilis). A total of 10 combinations between families of groups of additives (PE, PRO, PRE and OA) were designed and tested in ANSES (6 combinations) and IMASDE (4 combinations). This mixtures combine between 1 and 2 products from three families of products (PE, PRE and OA), plus one PRO.
In the ANSES trial, five combinations decreased the Campylobacter population at 21d (mixture of Lactobutyrin BRC and Calsporin; mixture of Excential Alliin Plus and Calsporin; mixture of Monoglycerides and Calsporin; mixture of Adimix 30 coated, Excential Alliin Plus and Calsporin; and the mixture of Lactobutyrin BRC, XPC and PoultryStar), but only one mixture, (Lactobutyrin BRC, XPC and PoultryStar) significantly decreased Campylobacter counts at 35 days of age and none of them at 42d.
In the IMASDE trial, one of the combinations tested (mixture of Campylostat and Calsporin) clearly decreased the Campylobacter population, mainly avoiding the contamination of birds, with reductions of 3.52 4.09 and 3.98 log10 CFU/g; or 75, 58 and 75% of inoculated birds negative at 21, 35 and 42d, respectively. The other treatments showed numerical but not significant reductions, although the other mixture which contained Campylostat (plus Alliin and PoultryStar) also tended to show a significant reduction at 21d, with 66.7% of birds not infected.
It can be concluded that 7, 2 and 1 combinations of the products tested in T1.1 gave significant reductions at 21, 35 and 42d. Therefore, the combination that showed the best results at 42d (mixture of Campylostat and Calsporin), and some other individual products that showed the biggest reductions in T1.1 alone, will be used in WP3.
Therefore, the objectives of the WP1 were achieved, since several individual compounds and combinations have demonstrated its ability to decrease the Campylobacter population. However, it was expected a higher level of synergisms between individual additives. This effect is not well understood, and it is related with the ecology in the caeca of birds, and the mode of action of each group of additives: acting directly against Campylobacter (such as OA, MCFA, MG-MCFA or PE), o modifying the microflora of caeca for displace the Campylobacter populations (PRO, PRE). Also, although for the moment is only a theory, it was discovered that the best way to decrease the Campylobacter infection is to avoid the contamination, rather than decrease the cecal populations (once the bird is infected, it is very difficult to decrease the populations). This is the case of the combination of Campylostat and Calsporin, which showed that 75, 58 and 75% of inoculated birds were negative at 21, 35 and 42d, respectively.
There were not deviations in connection with the expected works. However, there was a delay, due to two reasons: i) There were some works in the Animal Biosafety Level 2 (ABL2) facilities in the RTD ANSES, which lasted from September to December of 2013. Therefore, there was a delay of three months, since the trials started in January 2014, ii) In IMASDE, the experiment EXP.EU02 has to be repeated. The reason was that the chickens suffer an E. coli infection, which origin is unknown, which was not controlled by the selective medium mCCDA. This causes that most of the plates presented a huge growth of E. coli, which was invasive, that make impossible to count the C. jejuny counts. This make impossible to reach any conclusion after the trial, and the products tested were re-tested in trials EXP.EU04 and EXP.EU05. This caused a parallel delay of three months approximately, from which two has been recovered (last experimental work finished in December). Therefore, all the works have the same delay, but at this time, all the work expected for WP1 has been achieved. The consortium will try to recover the rest of this delay in WP3.
WP2. Feed presentation strategies against Campylobacter
The WP2 “Feed presentation strategies against Campylobacter” has developed the feed form strategy through two tasks: T2.1: “Effect of feed composition, particle size and feed presentation on the prevalence of Campylobacter in broilers orally infected”; and T2.2: “Effect of whole grain feeding on the prevalence of Campylobacter in broilers orally infected”.
The objectives of this WP were to design feed forms that effectively decrease the Campylobacter population of infected broilers in vivo. A total of 4 experiments (2 experiments in each Task) were done. In the first experiment (EXP.03) there were five treatments, based on the main cereal of the diet (corn, wheat or barley), and the inclusion of 5% of oat hulls (OH) in the corn and wheat-based diets. Oat hulls were added by dilution. The second experiment (EXP.06) was done with wheat-based diets, and there were four treatments factorially arranged with two screen sizes of wheat (Ø2 vs 5mm) and two feed presentations (mash vs pelleted). In the third experiment (EXP.07) there were three treatments, a mash wheat-based diet (Control, T1), T1+ whole wheat (ww) at 7.5% from 0-21d and 15% from 21 to 42d (T2), and T2+5% of OH. The fourth experiment (EXP.08) compromised six wheat-based treatments factorially arranged, with two feed forms (mash vs pellet), and three levels of ww: zero, 7.5% from 0-21d and 15% from 21 to 42d, and 15% from 0-21d and 30% from 21 to 42d. All the experiments were performed by IMASDE AGROALIMENTARIA, S.L. and were done in vivo through challenge trials, in which all the animals were infected at 14d with C. jejuny ST-45. After inoculation, Campylobacter jejuny was analyzed in the ceca of animals at 21, 35 or 42d of age, reflecting the commercial practice in different countries across the EU. Experimental feeds were given to animals along the whole experimental period. Campylobacter counts were analyzed by ISO/TS 10272-2:2006 from direct sampling from the ceca of animals. In EXP.03 EXP.06 and EXP.08 several intestinal physiology measures were recorded: the weight of gastrointestinal tract, empty body, proventriculus (whole or empty), gizzard (whole or empty), and caeca; and the pH at proventriculus, gizzard and caeca.
In the first experiment (EXP.03) corn-based diets numerically showed less contamination, but neither cereal type nor oat hulls inclusion significantly decreased the level of contamination.In the second experiment (EXP.06) there were no significant differences in C. jejuny contamination between experimental group. Birds fed pelleted diets showed higher feed consumption and growth. In the third experiment (EXP.07) the inclusion of ww (7.5% between 0-21d, and 15% from 21 to 42d) and 5% of oat hulls significantly decreased the C. jejuny contamination at 42d with respect to a mash diet or a mash diet plus ww (8.10 vs 9.48 and 9.00 log10UFC/g, respectively, P<0.05). In the last experiment (EXP.08) neither the feed form (mash vs pellet) nor ww inclusion affected the Campylobacter level at the ceca of animals.
The intestinal physiology was clearly affected by treatments, with bigger gizzards in animals fed with OH, ww or higher particle size. However, there was no clear relationship with this physiological changes and the level of C. jejuny counts at ceca level. It is concluded that despite of the clear physiological changes derived of the feed form (mash or pellets), particle size, type of cereal, and OH or ww inclusion, no clear reductions in C. jejuny populations in the ceca were observed, although the inclusion of ww and OH to a mash diet decreased significantly the contamination at 42d in EXP.07.
Therefore, the objectives of the WP2 were achieved, since one effective combination of feed form was obtained: mash diets in overall tended to show lower Campylobacter counts, and the addition of ww and OH to mash diets also decreased the Campylobacter cecal population.
WP3: Interactions between products and feed presentation against Campylobacter. Synergies.
In the WP3 “Interactions between products and feed presentation against Campylobacter. Synergies”. the experiments were done with the best combination of products as a result of WP1, combined with the best Functional diet as a result of WP2. The products tested in this WP were: a combination of monoglycerides of medium chain fatty acids and organic acids (Campylostat, MGOA); three probiotics, two based on Bacillus subtilis: C-3102 DSM 15544 [Calsporin®, CALS] and DSM 17299 [GalliPro®, GAL], and a multispecies probiotic [PoultryStar®, PS]; and a yeast extract (XPC). The Functional diet consisted in the addition of 5% oat hulls [OH] and 7.5/12/15% Whole Wheat [WW].
A total of 7 experiments (6 in IMASDE AGROALIMENTARIA S.L. –IMASDE- and 1 in ANSES) were done. Two of six experiments in IMASDE came from repetitions for several causes, and this causes a delay of 4 months in the deliverable. In the first experiment (EXP. 10) there were four treatments in a factorial design with the addition of MGOA (1.5% form 0-10d and 2.5% thereafter), CALS (0.01%), or a combination of both. All diets were Functional. Due to unknown reasons, animals were not infected neither at 21 nor at 35d after inoculation at 14d, and they were re-inoculated at 39d. At 42d, animals fed MGOA either alone or in combination with CALS, decreased the number of infected animals to middle (P<0.05). However, this trial was repeated because the first inoculation process was unsuccessful. In the second experiment (EXP.11) there were four treatments factorially arranged based on two types of diet: a Control diet (C) and a functional (F) diet (with 7.5 12 and 15% of whole wheat form 1-10, 10-21 and 21-42d, respectively); with or without the addition of MGOA (1.5% from 1-10d and 2.5% thereafter) + CALS (0.01%). At 35d, feed supplemented with MGOA+CALS was able to reduce the colonization of Campylobacter both in control and functional diets (6.573 3.705 6.464 4.915 log10 CFU/g for Control, Control+MGOA+CALS, Functional and Functional+MGOA+CALS, respectively, P=0.036). This reduction was mainly because of the number of not infected birds in animals fed the diets supplemented with MGOA and CALS (14.30 71.40 7.10 and 35.70% of not infected birds for Control, Control+MGOA+CALS, Functional and Functional+MGOA+CALS, respectively, P=0.001). However, this effect of MGOA was lost at 42d of age (7.444 7.188 6.882 7.082 log10 CFU/g for Control, Control+MGOA+CALS, Functional and Functional+MGOA+CALS, respectively, P=0.351). In this experiment an additional treatment wqas included to evaluate the product GAL. The combination of MGOA +GAL showed the same effect (or even better) that MGOA+CALS at 21 and 35d, but its effect was also lost at 42d. In the third experiment (EXP.12) there were five treatments depending on the time of administration of the combination of MGOA (1.5% from 0-10d and 2.5% thereafter) + CALS (0.01%): a Control diet (C), and C plus MGOA+CALS in four periods: <21d, >21d, >10d, 0-42d for treatments 2 to 5, respectively. In this experiment the environmental conditions were very poor and the trial was repeated (EXP.15). In the fourth experiment (EXP.13) there were four treatments factorially arranged: T1, a Control diet (C); T2, C plus MGOA at 0.5% from 0-10d and 2.5% thereafter; T3, C plus CALS at 0.01%, and T4, C plus the addition of MGOA (1.5% from 0-10d and 2.5% thereafter)+CALS (0.01%). At 21d of age, broilers fed MGOA (alone or with CALS) showed less infection than broilers fed control diet or the control diet supplemented with CALS (4.463 2.374 4.656 2.266 log10 CFU/g for T1 to T4, respectively, P<0.001). Those differences came from the proportion of negative animals (43.8 93.8 37.5 and 93.8% for T1 to T4, respectively, P<0.001). At 35d of age (21d after challenge), broilers fed MGOA+CALS showed less infection than broilers fed the rest of treatments (6.816 6.557 6.394 4.451 log10 CFU/g for T1 to T4, respectively, P=0.024). Those differences came from the proportion of negative animals (6.30 6.30 12.50 43.80% for T1 to T4, respectively, P=0.012). Finally, at 42d, broilers fed MGOA+CALS showed less infection than control animals, showing the rest of treatments an intermediate Campylobacter population (8.514 7.299 8.276 and 6.887 log10 CFU/g for T1 to T4, respectively, P=0.001).
In the fifth experiment (EXP.14) there were four treatments depending on the MGOA dose: T1, a Control diet (C); T2, C plus MGOA at 0.5% from 0-10d and 1% thereafter and CALS at 0.01%; T3, C plus MGOA at 0.5% from 0-10d and 2% thereafter and CALS at 0.01%; T4, C plus MGOA at 0.5% from 0-10d and 3% thereafter and CALS at 0.01%. At 21d of age, the level of infection was very poor and there were not effect of treatment on Campylobacter. At 35d of age, birds fed the diets with the higher levels of MGOA from 10 to 42 d (2 or 3%) showed a clear reduction of Campylobacter infection (6.450 5.678 3.780 3.670 log10 CFU/g for T1 to T4, respectively, P=0.009). Those differences came from the proportion of negative animals (12.5 25.0 62.5 and 68.8% for T1 to T4, respectively, P=0.002). At 42d of age, again birds fed the diets with the higher levels of MGOA from 10 to 42 d (2 or 3%) showed a clear reduction of Campylobacter infection (6.441 5.741 3.979 and 4.156 log10 CFU/g for T1 to T4, respectively, P=0.009) and again, those differences came from the proportion of negative animals (7.1 25.0 62.5 56.3% for T1 to T4, respectively, P=0.005). In the sixth experiment (EXP.15) there were four treatments depending on the time of administration of the combination of MGOA (0.5% from 0-10d and 2.5% thereafter)+CALS (0.01%): a Control diet (C), and C plus MGOA + CALS in four periods: 0-42d, <21d, >21d, for treatments 2 to 4, respectively. At 21d of age, birds fed diets containing MGOA and CALS (T2 and T3) showed less contamination than the animals fed control diet (T1 and T4) (7.852 6.596 6.799 and 7.214 log10 CFU/g for T1 to T4, respectively, P=0.026). However, surprisingly this effect was lost at 35 and 42d.
It can be concluded that the combination of at least 2% of MGOA plus a probiotic based on B. subtilis (either CALS or GAL) administrated during the whole fattening period decrease the Campylobacter populations at 35 and 42d, but for unknown reasons, this effect is inconsistent. The next step will be testing this combination of products in field conditions with natural infected animals.
WP4. Application of different nutritional strategies against Campylobacter in experimental farm and field trials.
In the WP4 “Application of different nutritional strategies against Campylobacter in experimental farm and field trials”, the experiments were done with the best combination of products as a result of WP1 and WP3, since the feed form tested in WP2 or the combination of the most effective feed form and additives did not shown any synergistic effect. The products or combinations tested were: i) a combination of monoglycerides of medium chain fatty acids and organic acids (Campylostat, MGOA); ii) two probiotics based on Bacillus subtilis: C-3102 DSM 15544 [Calsporin®, CALS] and DSM 17299 [GalliPro®, GAL]; and iii) a yeast extract (XPCTM). There were three tasks: T4.1. Effect of different strategies against Campylobacter on performance parameters and level of infection of broilers chickens in experimental farm; T4.2. Effect of different strategies against Campylobacter on performance parameters and level of infection of broilers chickens in commercial farms; and T4.3. Effect of different strategies against Campylobacter on performance parameters and level of infection of turkeys in commercial farms.
In Task 4.1 a total of five experiments were done (EXP.16 to EXP.20) in the experimental facilities (two different farms) of IMASDE AGROALIMENTARIA S.L. –IMASDE- and one in ANSES. Those experiments differ with the field conditions in the experimental unit: in the experimental farm the experimental unit is a box within a single barn (all treatments together), and in the field trials the experimental unit is the barn (treatments separated in different barns). In EXP.16 the experimental design was a completely randomized design with 6 different experimental treatments: T1, Control (C); T2, T3 and T4, C + MGOA at different dosages + CALS; T5, C + MGOA+ GAL and T6, C + XPC (yeast extract). Each treatment was applied to 12 pens of 20 birds. The natural Campylobacter infection only appeared in some boxes at 35d, and was forced to the rest through the dissemination of litter, but the additives supplementation had not effect at 41d (6.68 6.92 6.84 6.80 6.23 and 6.64 log10 cfu/g for T1 to T6, respectively, P=0.306). In EXP.17 the experimental design was a completely randomized design with 6 different experimental treatments: T1, Control (C); T2, T3 and T4, C + MGOA at different dosages + CALS; T5, T6 and T7, C + MGOA at different dosages + GAL and T8, C + XPC. Each treatment was applied to 12 pens of 20 birds. Unexpectedly, all analyzed broiler caeca at 35 and 42 days of age were Campylobacter spp. free. In EXP.18 the experimental design was a completely randomized design with 3 different experimental treatments: T1, Control (C); T2, C + MGOA + CALS, and T3, C + [(MGOA + CALS) only 0-10d] + XPC (1-42d). Each treatment was applied to 8 pens of 22 birds. Although the hygienic barriers were very low and the trial was done in summer time, the natural Campylobacter infection was not implemented (additionally to the caeca samples, a fecal swab of all the animals was taken and were all negative). In EXP.19 the experimental design was a completely randomized design with 4 different experimental treatments (supplied by drinking water at 46-47d/48-49d): T1, Control (C); T2, C + 0.1/0.2% of MGOA; T3, C + 0.3/0.6% MGOA and T4, C + 0.5/1.0% MGOA. Each treatment was applied to 2 pens of 22 birds. The MGOA supplementation decreased the water consumption. Campylobacter infection was implemented, but water treatment did not show any effect on Campylobacter counts (9.04 9.08 9.15 and 9.27 log10 cfu/g for T1 to T4, respectively, P=0.756). In EXP.20 the experimental design was a completely randomized design with 3 different experimental treatments: T1, Control (C); T2, C + MGOA (1.5%) + CALS (0.01%), and T3, C + CALS (0.1% all periods) + MGOA (3%, only in the finisher diet). Each treatment was applied to 8 pens of 22 birds. Although the infection was detected in only nine replicates at 35d control (T1: 2.63 3.61 4.17 and 6.95; T2: 2.75 and 3.16; T3: 2.43 3.30 and 4.79 log10 cfu/g), when the analysis was repeated at 42 days of age (78 samples in total), all the samples except 3, were Campylobacter spp. free (T2: 0.47 and 3.31 T3: 3.29 log10 cfu/g).
Unfortunately, although we detected repeatedly Campylobacter in the two experimental farms previously to the project, the natural infection was not achieved in most of the cases. Some factor related with the structural disposition of the boxes could be the cause of the lack of infection.
In the first trial of ANSES, the additives increased the Campylobacter population both at 35 and 42d. On the contrary in the second trial, the additives avoid the contamination in most of the birds, but unfortunately the effect was lost either at 35 or 42d.
In Task 4.2 the trials were carried out in Spain (three different farms with twin buildings, one Control and the other Treated with MGOA+CALS), and Hungary (one single farm with three pairs of twin buildings with the same design). In Spain, previously to the feeding trial, a survey was carried out between 23 EXPLOTACIONES AVÍCOLAS JOSÉ LUIS REDONDO S.A. (REDONDO) farms at 42/29d. Most farms (21; 91.3%) were positive with more than 107 cfu/g. In the first farm, broilers were Campylobacter free until the last week, in which they reached 7log10 cfu/g, but no differences among treatments were found. However, carcasses form birds fed the supplemented diets showed more contamination than the controls birds (3.37 vs 3.92 log10 cfu/g for control and treated animals, respectively, P=0.002). In the second farm, the infection was very high at 26d, and it naturally decreased until 47d. Unexpectedly, the decrease in the control barn was higher than in the treated one, so the final infection was higher for treated animals than for control animals (5.31 vs 6.46 log10 cfu/g for control and treated animals, respectively, P=0.009). However, the treated animals presented lower contamination in the neck skin (1.67 vs 0.58 log10 cfu/g for control and treated animals, respectively, P=0.020). In the third farm, both barns were positive at 32d, and the infection was maintained high until the end of the fattening period without any difference between treatments, neither in live animals nor in the neck skin. So, from the Spanish trials we can conclude that the infection patron is completely different among farms, and that the additive addition had not a positive effect on Campylobacter infection neither in live animals nor in the carcasses.
In the Hungarian trials, the infection was only achieved at the end of the trial, and unexpectedly, diet supplementation with additives significantly increased the Campylobacter infection, since Control animals clearly showed lower contamination (4.433 vs 8.912 log10 cfu/g for Control and Treated animals, respectively, P<0.001).
In Task 4.3 two strategies were followed: MGOA+CALS in the feed and the MGOA in the water during the withdrawal period. In females (12-13 weeks of age), the addition of MGOA+CALS in feed surprisingly increased the Campylobacter population in two of the three farms tested, but in males (18/19 weeks) the addition of the additives did not modify the Campylobacter cecal population. On the other hand, the addition of MGOA through the water in females decreased the Campylobacter population in one farm but no in other, and in males it increased the Campylobacter population in one farm but no in other.
It can be concluded that i) for unknown reasons, the natural infection was only implanted in part of the trials, which could not be explained with the actual state of art on the Campylobacter epidemiology, ii) the combination of at least 1.5% of MGOA plus a probiotic based on B. subtilis (either CALS or GAL) or the yeast extract XPC administrated during the whole fattening period, did not decrease the Campylobacter populations at 35 and 42d under natural conditions.
WP5 Development of a novel vaccine against Campylobacter based on reserve vaccinology
In order to evaluate the protective potential of the selected antigens, we had to develop an experimental vaccine model. Since we would like to test several antigens, the way of producing the vaccine for each potential vaccine antigen should be as easy as possible. We have a huge experience in DNA vaccination at ANSES. Therefore we decided to start with DNA vaccination, which is an easy way to produce vaccine prototypes. Flagellin A was used as a vaccine antigen model since it was described in the literature to induce some protective immune response in chickens.
In order to analyze the immune responses induced by the vaccination and infection, we developed and optimized ELISAs to quantify the levels of systemic antibodies in the blood (IgY type antibodies) and mucosal antibodies in the bile (IgA-type antibodies).
In a first assay we evaluated a vaccine protocol based only on DNA vaccination. In total 155 Ross broilers divided into 5 groups were used. 2 adjuvants were compared: CpG-ODN and interleukin 2. These adjuvants were selected because they were massively used in DNA vaccination in chickens. The DNA vaccine was injected through the sub-cutaneous route, a route also largely used in chicken vaccination. A prime injection was performed in one-day old chicks. A boost injection was performed in 10-days old chicks. The challenge with Campylobacter jejuni 81-176 strain was performed on day 21. Cecal loads of Campylobacter were measured on days 28 and 42. This DNA vaccination protocol did not induce detectable levels of antibodies at any time-point of the assay. No reduction of Campylobacter loads were observed at any of the time-points tested.
Nevertheless this assay was useful to design the next assay. First we observed a negative effect of interleukin 2 on Campylobacter load; only the CpG-ODN will be used as DNA vaccine adjuvant afterward. Second, the route of injection will be modified (intramuscular). Third, the vaccine regimen will be strengthened by including a boost-injection with the protein form of the vaccine. Fourth, we observed massive presence of maternal derived antibodies in the chickens; this should be taken into account for the next assays.
In the second assay, the vaccine was injected via the intramuscular route. We compared the DNA-DNA regimen to the DNA-protein regimen. CpG-ODN was used as adjuvant for the DNA vaccine and montanide as adjuvant for the protein vaccine. We included also groups of specific pathogen free (SPF) Leghorn hens which are from a Campylobacter-free breeding in order to study the impact of the maternal derived antibodies on the vaccine efficacy. In total 104 chickens divided in 6 groups were used. The prime and the boost injections were performed on 1-day and 7-day old chicks, respectively. The challenge with the same strain of Campylobacter as assay 1 was performed on day 15. For the Ross broilers, the DNA / protein regimen induced the production of specific antibodies against Campylobacter, but no reduction of the Campylobacter load on day 42. The DNA/DNA regimen was inefficient for all the parameters tested. For the SPF Leghorn hens, the DNA / protein vaccine regimen did not only induce the production of specific antibodies, but also permitted to eliminate entirely Campylobacter in the caecum. These results are showing that the DNA/protein vaccine regimen is highly powerful to induce a protective immune response, but the SPF Leghorn hens are not the targeted chickens. The vaccine has to be efficient in Ross broilers.
Therefore we performed a third assay with only Ross broilers where the prime-injection of the vaccine was done on day 5 (less maternal antibodies are present at this time point) and the boost on day 12. We also compared the DNA/protein regimen to the protein/protein one. In total, 128 chickens divided into 6 groups were used. None of these regimens induced the reduction of Campylobacter loads. The levels of antibodies were higher in the DNA/protein boost regimen performed on days 5 and 12.
In an additional assay we performed the DNA prime injection of day 14 and the boost injection on day 21. Unfortunately the challenge was inefficient; therefore we could not measure the protective effect of this vaccine regimen. Nevertheless, the level of antibodies against Campylobacter was equivalent on day 42 to the ones of the chickens injected on days 5 and 12.
Therefore the vaccine regimen that will be used in the assays to evaluate the protective potentials of the potential vaccine antigens will be the prime-boost regimen injected on days 5 and 12. The challenge with Campylobacter is performed on day 19.
The in vitro test to evaluate if the proteins are recognized or not by antibodies against Campylobacter has been developed by using the flagellin A as a protein model and a commercial polyclonal vaccine against Campylobacter jejuni.
We applied this method to the 12 potential vaccine antigens identified in task 5.1 (D5.1). Unfortunately, no staining was observed when the cells were transfected with all these antigens. Afterward, we produced a rabbit polyclonal antibody specifically directed against Campylobacter jejuni 81-176, the strain used in the project. Also no staining was obtained with any of the newly identified potential vaccine antigens. We do not know at this stage why this test was not successful for the newly identified potential vaccine antigens.
In conclusion, we were able under certain conditions to stain cells transfected with plasmids encoding Flagellin A. But this was not the case for other potential vaccine antigens. The in vitro test to select the antigens for the vaccine efficacy tests was therefore not useful. But this was not a barrier for the rest of the project since we used the alternative way to select the proteins to be tested for their protective potentials: a part of the proteins that have the strongest probability to be an antigenic protein were selected for the in vivo assessment.
We added some additional criteria for the selection. In some cases (YP_001001371.1 YP_001000204.1 YP_001000248.1) the proteins were not selected even if they were highly immunogenic because we found recently that these proteins were not common to many Campylobacter strains. These results were obtained now because there are now much more sequences of genomes of Campylobacter available. It is in fact important for the vaccine development that the vaccine proteins are shared among different Campylobacter strains in order to achieve a protection against several strains. In other cases (YP_001000153.1 YP_001000945.1) we were not able to produce all the components of the vaccine (protein form of the vaccine). In total 6 proteins were selected to test their protective potentials.
Two assays were performed for this part. We based the design of the assays on the results of task 5.3: DNA-prime / protein boost regimen injected on days 5 and 12, challenge with Campylobacter on day 19. Furthermore, we focused the determination of the Campylobacter loads on day 42, the slaughter date. In fact, the main goal of the vaccine is to get a reduction of Campylobacter load at this time point. The assays were designed to analyze at least 15 individual caeca from each batch. The chickens used are Ross broilers coming from a conventional hatchery.
The protocols of the in vivo experiments were designed in collaboration with Eugenia Puentos Colorado and Alberto Parra of CZV.
The first trial was performed with 3 potential vaccine antigens: YP_001000562.1 YP_001001115.1 and YP_999769.1. The vaccine potentials of these candidates were compared to a group injected with placebos (empty plasmid / PBS). 4 groups of chickens were used here. The vaccination protocol is initially the one of deliverable 5.2. The vaccine was injected through the intra-muscular route. On day 5, the antigens were injected in their DNA forms (300 µg); 50 µg CpG-ODN were added as adjuvants. On day 12, the boost was performed with the protein forms of the antigens (100 µg); montanide was used as adjuvant. On day 19, the chickens were challenged with the C. jejuni 81-176 strain (which was able to colonize the chickens in the previous assays – see deliverable 5.2). Unfortunately we observed no Campylobacter colonization of caecas 4 days later. Therefore we decided to modify the protocol in performing a second challenge with the same strain on day 28. Again, no colonization was observed. We finally decided to perform a challenge with the C. jejuni C97ANSES640 strain on day 42 and Campylobacter colonization was obtained. We stopped the assay on day 49 and analyzed the levels of Campylobacter colonization in each chicken. The induction of specific antibodies in the blood was analyzed weekly throughout the assay; mucosal specific antibodies were researched in the bile on day 49.
Our data showed that 2 of the 3 antigens of Campylobacter jejuni tested were able to induce the production of specific antibodies against Campylobacter (YP_001000562.1 - flagellin family protein and YP_001001115.1 - FlgK). No modifications of bile IgA were observed on day 49. Only YP_001001115.1 – FlgK induced under these particular experimental conditions used here a moderate but significant reduction of Campylobacter load in the caecum 1.4 log10). But the results should be interpreted with caution because the experimental conditions were particular following the failure of colonization observed after the first challenge. In total 3 challenges with 2 different Campylobacter strains were performed.
A second assay was performed with 6 vaccine antigens tested individually or mixed. Furthermore, we used as a challenge strain C. jejuni C97ANSES640 since it was able to induce high colonization levels in chickens in assay 1 (third challenge). Furthermore, this strain is representative of the strains isolated in chickens. So the modification of the strain does not modify the goal of the project.
Five out of the 6 antigens induced the production of antibodies against Campylobacter. For the last antigen (YP_999769.1 (FlgE-1)), we do not know now if significant levels of antibodies were produced because the ELISA used to measure the production of antibodies was not optimized for antibodies against this particular protein. More importantly, 4 out of 6 antigens induced significant reductions of Campylobacter loads in the caecum. The pool of the 6 antigens did not induce any reduction of Campylobacter loads. This may be due to the fact that low quantities of each antigen were used.
Two of these antigens were of particular efficacy and interest: YP_001000437.1 induced a reduction of Campylobacter loads of about 3.6 log10 and YP_999817.1 induced the highest reduction of Campylobacter loads of about 4.2 log10. In fact, a reduction of at least 3 log10 of Campylobacter in chickens may decrease by more than 90 % the risks of campylobacteriosis in humans (Romero-Barrios P. et al, Food Control 2013), indicating that the results we obtained are of main importance for future vaccine development to control Campylobacter in chickens.
Due to the delays of the beginning of the project, we were not able to perform a third assay to confirm the results we obtained. This assay has been postponed after the end of Campybro. It is expected that the assay starts the 16th of September 2016. Consequently the PhD student will finish her PhD training after the end of the project (probably during the first quarter of 2017).
WP6. Evaluation of the developed nutritional strategies in different geographical situations.
In the WP6 “Evaluation of the developed nutritional strategies in different geographical situations”, the experiments were done in practical farm conditions in the four countries that participate into the project: France, Hungary, Spain and The Netherlands. The products or combinations tested were: i) a combination of monoglycerides of medium chain fatty acids and organic acids (Campylostat, MGOA); and ii) a probiotic based on Bacillus subtilis: C-3102 DSM 15544 (Calsporin®, CALS). There was only a task: T6.1. Evaluation of developed nutritional strategies in South, Central, and East European conditions. In France, the trial was done in two farms with 2 twin buildings each (26,400 broilers per building, 96,900 in total) located at Sillé-le-Guillaume. The trial started the 23/06/2016 and lasted the 29th of July. The experimental feeds were done in the company LDC at Sillé-le-Guillaume, and there were two treatments: Control (C), and C + MGOA (0.5% 0-10d and 1.5% thereafter) + CALS (0.01% all the period). In The Netherlands, the trial was done in one farm with 3 twin buildings (63,400, 63,400 and 48,060 broilers per building, 174,860 in total) located at Grootschermer. The experimental feeds were done in the company Agrifirm at Mepple. The trial started the 01/07/2016 and lasted the 05/08/2016 and there were two treatments: Control (C), and C + MGOA (0.5% 0-7d and 1.5% thereafter) + CALS (0.01% all the period). In Spain there were three trials. The first trial was done in Cosuenda (Zaragoza) in one farm with twin buildings (4,500 birds each, 9,000 broilers in total). The experimental feed was done in Nanta (Casetas, Zaragoza). The trial started the 20/06/2016 and lasted 01/08/2016 and there were two treatments: Control, and C + MGOA (0.5% 0-10d and 1.5% thereafter) + CALS (0.01% all the period). The second trial was done in Fuente Álamo (Murcia) in one farm with three twin buildings (19,000 birds each, 57,000 broilers in total). The experimental feed was done in Avilesa (Torre Pacheco, Murcia). The trial started the 16/05/2016 and lasted the 04/07/2016 and there were three treatments: Control, C + MGOA (2.5% from 33 to 50d) + CALS (0.01% from 33 to 50d), C+ MGOA in drinking water at 0.06% from 43 to 50d. The third trial was done in Lominchar (Toledo) in one farm with two twin buildings (18,500 birds each, 37,000 broilers in total). The experimental feed was done in Explotaciones Avícolas José Luis Redondo S.A. (Pantoja, Toledo). The trial started the 17/05/2016 and lasted the 01/07/2016 and there were two treatments: Control, and C + MGOA (0.5% 0-10d and 1.5% thereafter) + CALS (0.01% all the period). In Hungary, the trial was done in one farm (Varjasi Farm Kft.) located at Újkígyós with 6 twin buildings (18,552 broilers per building, 110,760 in total) and there were two treatments: Control, and C + MGOA (0.5% 0-7d and 1.5% thereafter) + CALS (0.01% all the period). The experimental feeds were done in the company Agrifirm at Győr. The trial started the 23/06/2016 and lasted the 02/08/2016 and there were two treatments: Control, and C + MGOA (0.5% 0-10d and 1.5% thereafter) + CALS (0.01% all the period).
In France, in one of the farms the additives addition decreased the cecal Campylobacter counts (8.20 vs 7.18 Log10 cfu/g, P<0.001). In the other farm, the samples from control animals were incorrectly stored and showed difficulties in the analysis.
In The Netherlands, although the trial was done in the summer, unfortunately the natural infection started in one of the barns just in the final part of the trial, and the efficacy of the additives could not be tested.
In Spain, in the first trial the addition of MGOA (0.5% from 0 to 7d and 1.5% thereafter) + CALS (0.01%) in the feed decreased the Campylobacter infection at 24d (6.859 vs 6.006 Log10cfu/g for CONTROL and TREATED, respectively; P=0.019). However, the opposite effect was found at 35d, when birds fed the TREATED diet showed more Campylobacter counts than CONTROL birds (6.935 vs 7.387 Log10 cfu/g for CONTROL and TREATED, respectively; P=0.046). At 42d, there were no differences between treatments, although TREATED animals tended to show more Campylobacter counts than the CONTROL birds (6.502 vs 6.827 Log10cfu/g for CONTROL and TREATED, respectively; P=0.093). In the second trial, the MGOA application in water significantly decreased the Campylobacter contamination (8.25a 8.28a 8.14a 7.18b Log10 cfu/g for CONTROL, TREATED feed before thinning (37d), TREATED feed (50d), TREATED water, respectively, P=0.04). In the third trial, treatment decreased significantly the Campylobacter infection at 32d (7.25 vs 6.04 Log10 cfu/g for CONTROL and TREATED, respectively, P=0.046) and showed numerically lower values at 39 (6.21 vs 5.45 Log10 cfu/g for CONTROL and TREATED, respectively, P=0.131) and 42d (6.69 vs 5.71 Log10 cfu/g for CONTROL and TREATED, respectively, P=0.152).
In Hungary, There were not differences between treatments neither at 35d nor at the end of the experiment (7.708 vs 7.500 and 8.326 vs 8.805 Log10 cfu/g for CONTROL and TREATED, P=0.383 and 0.133 at 35 and 40d, respectively).
The addition of MGOA and CALS had a limited effect on Campylobacter infection and performance in all the countries studied. In France, in one of the farms the additives addition decreased the cecal Campylobacter counts (8.20 vs 7.18 Log10 cfu/g, P<0.001). In the other farm, the samples from control animals were incorrectly stored and showed difficulties in the analysis. In the Netherlands, natural infection did not occur during the experiment. In Spain, treatment delays the infection in two of the experiments, and decreases the final figures when MGOA was administrated in the water the last week before slaughtering in the other experiment. In Hungary, any effect was detected. Therefore, the solution developed by the project CAMPYBRO and tested in filed conditions in four countries in Europe, has a limited effect on campylobacter infection. Further investigations are needed to understand much better the epidemiology of Campylobacter for develop new strategies to decrease the contamination in broiler chickens.
Potential Impact:
The objective of CAMPYBRO project, under the 7th FRAME PROGRAM of UE (Capacities- Research for SME Associations FP7-SME GA 605835) is to develop practical strategies for EU poultry producers to decrease the population of Campylobacter jejuni in poultry, as a methodology to decrease the contamination of poultry carcasses. The expected results are practical strategies based on nutrition and vaccination which decrease the level of infection of Campylobacter jejuny in poultry batches.
The final conclusions are: i) the mixture of a combination of organic acids and monoglycerides of medium chain fatty acids [MGOA] and a B. subtilis-based probiotic [CALS]) decreased the Campylobacter cecal population in challenge trials, ii) the effect of this combination on Campylobacter reduction was not homogenous, iii) the interaction between in vitro trials and in vivo trials was avoided, but it look like there is an interaction between challenge trials and natural contamination infection trials, iv) much more knowledge is needed in Campylobacter epidemiology, since the natural infection did not follow classical patrons of infection, v) in the demonstration trials variable results were found, with a significant effect in some cases, a delay of infection in others and no effect in other cases, vi) a potential vaccine could be developed based on the antigens discovered.
The potential impact of the projects results are: i) Improved food safety and public health: the reduction of Campylobacter population in broilers may decrease the percentage of carcasses infected and the level of infection, which will cause an improvement of food safety, since campylobacteriosis is the most important zoonosis in the UE. The expected impact for a 1-2 Log10UFC/g is a reduction of 50-90%, and 90% for a 3 Log10UFC/g reduction. The expected costs are related to direct illness effects (laboratory diagnosis, consultations, medical cares, hospitalization, etc.) and indirect costs (work inefficacy, no-working days, etc.); ii) Meet future regulatory requirements: the European Commission launched a Proposal Regulation amending Regulation (EC) No 2073/2005, as regards Campylobacter in broiler carcasses. This proposal will limit the percentage of samples (sampling: once per week, 15 birds at random form one batch, pooled in 5 samples 3 birds) with more than 103 cfu Campylobacter/g of neck skin to 20% in 2017, 14% in 2018 and 2019, and 10% after 2020. These limits will be unachievable for many producers in most of the countries, especially in the summer. For that reason, the results of the project are also key for the EU Commission in order to know if it is feasible to decrease the Campylobacter infection in vivo through nutrition, vaccination or both; iii) To improve the competitiveness of EU poultry industry. Producing poultry meat with lower levels of Campylobacter will improve the sector competitiveness, with higher possibilities of exporting products, and avoiding possible future food safety barriers, iv) Improvement of health, welfare and productivity of chickens, and health and welfare of farmers, this project will have a significant effect on the quality of life of these workers due to a considerable lower risk of infection. v) Decrease in antimicrobial resistance: A decrease in Campylobacter population could decrease the risk of transmission of antimicrobial resistance both in broilers and humans (because of less cases of Campylobacteriosis treated with antibiotics).
However, the solution developed by the project CAMPYBRO and tested in filed conditions in four countries in Europe, has a limited and very variable effect on Campylobacter infection. Some relevant advances have been achieved on the vaccination area for the future development of a vaccine. Further investigations are needed to understand much better the epidemiology of Campylobacter for develop new strategies to decrease the contamination in broiler chickens.
Dissemination activities
The main communication activities within the first and second reporting period are: a webpage (www.campybro.eu) 8 newsletters, 5 scientific peer reviewed articles, 32 scientific communications, 6 invited conferences, 8 articles in the popular press, 23 press releases, a workshop per participant country (Spain, The Netherlands, Hungary and France), 10 presentation of the results to the industry of the participant countries and a scientific workshop were published regarding the Campylobacter issue and the project.
1. Website
The website www.campybro.eu has been created and updated with the last news about the project and the Campylobacter issue in poultry in general.
2. Newsletters of the project
There have been published 8 newsletters (3 per year) during the project.
3. Peer reviewed scientific publications (published)
It has been published 5 papers. Full articles appear in the Annex 2. The impact factor (2015) of the publications, according to research gate (www.researchgate.net) was: i) Poultry Science: 2.14 ii) Journal of Applied Microbiology: 2.38 iii) Clinical and Developmental Immunology: 3.87 iv) plos-one: 3.54. In the publications it is worth to be mentioned that the main authors were the researchers of the RTDs: ANSES (M. Guyard-Nicodème, D. Dory, M. Chemaly, M. Meunier, M., A. Keita, S. Quesne, M. Amelot, T. Poezevara, B. Le Berre, E. Hirchaud) and IMASDE (M. I. Gracia, C. Millán, J. Sánchez, Ó. Casabuena, P. Medel), but also other researchers from most of the partners: CIDEF (Y. Carre), BTT (A. Csorbai), FIA (J. Mayot), NEPLUVI (P. Vesseur), PROPOLLO (Á. Martín), CVZ (A. Parra). It is expected to send at least two more publications once the project will finish.
• Gracia, M. I., C. Millán, J. Sánchez, M. Guyard-Nicodème, J. Mayot, Y. Carre, A. Csorbai, M. Chemaly, P. Medel, 2016. Efficacy of feed additives against Campylobacter in live broilers during the entire rearing period: Part B. Poult. Sci. 95: 886-892. DOI: 10.3382/ps/pev346.
• Guyard-Nicodème, M., A. Keita, S. Quesne, M. Amelot, T. Poezevara, B. Le Berre, J. Sánchez, P. Vesseur, Á. Martín, P. Medel, M. Chemaly, 2016. Efficacy of feed additives against Campylobacter in live broilers during the entire rearing period. Poult. Sci. 95: 298-305. DOI: 10.3382/ps/pev303.
• Meunier, M., M. Guyard-Nicodème, D. Dory, M. Chemaly, 2016. Control Strategies against Campylobacter at the Poultry Production Level: Biosecurity Measures, Feed Additives and Vaccination. Journal of Applied Microbiology 120: 1139-1173. DOI: 10.1111/jam.12986.
• Meunier, M., M. Guyard-Nicodème , E. Hirchaud, A. Parra, M. Chemaly, D. Dory, 2016. Ientification of Novel Vaccine Candidates against Campylobacter through Reverse Vaccinology. Clinical and Developmental Immunology 2016 Article ID 5715790: 1-9. DOI: http://dx.doi.org/10.1155/2016/5715790
• Gracia, M. I., J. Sánchez, C. Millán, Ó. Casabuena, P. Vesseur, Á. Martín, F. J. García-Peña, P. Medel, 2016. Effect of Feed Form and Whole Grain Feeding on Gastrointestinal Weight and the Prevalence of Campylobacter jejuni in Broilers Orally Infected. PLoS ONE 11(8): e0160858. doi:10.1371/journal.pone.0160858.
4. Scientific publications (abstracts in Scientific Meetings or congress)
The results of the projects have been presented in the next scientific meetings covering the FIVE CONTINENTS:
• XIVth European POULTRY CONFERENCE. World's Poultry Science Association. 23-27 June, 2014, Stavanguer, Norway.
• 3rd International conference on Responsible use of Antibiotics in Animals. Amsterdam. 29th September-1st October, 2014. Amsterdam. The Netherlands.
• LI symposio científico de avicultura. 2-3 October, 2014. Alfara del Patriarca, Valencia, Spain.
• 11th Journées de la Recherche Avicole et palmipèdes a Foie Gras. 25-26 March 2015. Tours, France.
• European Symposium on Food Safety. 20-22 April 2015. Cardiff, Wales, UK.
• XXII European Symposium on the Quality of Poultry Meat » and the « XVI European Symposium on the Quality of Eggs and Egg Products. 10-13 May 2015. Nantes. France.
• Poultry Science Association 104th annual Meeting. July 27-30, 2015. Louisville, Kentucky, USA.
• 20th European Symposium on Poultry Nutrition. 24-27 August 2015. Prague, Chez Republic.
• 19th world Veterinary Poultry Association Congress. 7-11 September 2015. Cape Town, South Africa.
• 18th International Conference on Campylobacter, Helicobacter and related organisms. 1-5 November 2015. Rotoura, New Zealand.
• European Symposium on Food Safety. 11-13 may 2016. Athens, Greece.
• World Poultry Congress. 5-9 September 2016. Beijing, China.
All these publications are available on request to the Project Officer or Scientific evaluator.
A total of 32 communications were published in the Meetings listed above: The bibliographic references are:
• Millán, C., Casabuena, Ó., Porras, S., Sánchez, J., Gracia, M.I. 2014. Effect of feeding caproic and caprylic as acids or monoglycerides on Campylobacter counts in broilers. XIVth European POULTRY CONFERENCE. World's Poultry Science Associaction. Pp: 247
• Martín, Á., Mayot, J., den Hartog, M., Carre, Y., Molnar, G., Sánchez, F., Fernández, A., Tenk, M., Chemaly, M., Dory, D., Medel, P., 2014. CAMPYBRO PROJET: Strategies to fight the biggest zoonoses in the EU without antibiotics therapy. 3rd International conference on Responsible use of Antibiotics in Animals. Pp: 23
• Millán, C., Sánchez, J., Casabuena, O., Gracia, M.I. 2014. LI symposio científico de avicultura Efecto de la adición de un derivado de Allium sativum y de un probiótico sobre la población fecal de Campylobacter jenuni en broilers infectados experimentalmente. Asociación Española de Ciencia Avícola. Proceedings CD.
• Chemaly, M., Mayot, J., 2015. Les moyens de lute vis-à-vis de Campylobacter chez le poulet de chair: mythe ou realite?. 11th Journées de la Recherche Avicole et palmipèdes a Foie Gras. Tours, 25-26 March 2015.
• Guyard-Nicodème, M., Quesne, S., Poezevara, T., Le Berre, B., Amelot, M., Chemaly, M., 2015. A large scale study to test a wide variety of additives in broilers’ feed to decrease Campylobacter shedding. European Symposium on Food Safety. 20-22 April 2015. Cardiff, Wales, UK.
• Guyard-Nicodème, M., Houard, E., Poezevara, T., Chemaly, M., 2015. Combinations of broilers’ feed additives against Campylobacter. XXII European Symposium on the Quality of Poultry Meat » and the « XVI European Symposium on the Quality of Eggs and Egg Products. 10-13 May 2015. Nantes. France.
• Meunier, M., Guyard- Nicodème, M., Béven, V., Vigouroux, E., Chemaly, M., Dory, D., 2015. Optimization of a vaccination protocol against Campylobacter for poultry. XXII European Symposium on the Quality of Poultry Meat » and the « XVI European Symposium on the Quality of Eggs and Egg Products. 10-13 May 2015. Nantes. France.
• Chemaly M., 2015. Is this a fiction to tackle Campylobacter in the broiler food chain? XXII European Symposium on the Quality of Poultry Meat » and the « XVI European Symposium on the Quality of Eggs and Egg Products. 10-13 May 2015. Nantes. France.
• Gracia, M. I., O. Casabuena, F. Sánchez, J. Mayot, P. Medel, 2015. Effect of feeding a live yeast and a combination of organic acids and essential oils on Campylobacter colonization in broilers. Poult. Sci. 94 (E-Suppl. 1): 117.
• Sánchez, J., C. Millán, F. Sánchez, Y. Carre, P. Medel, 2015. Effect of type of cereal and oat hulls addition on cecal morphology and Campylobacter jejuni colonization of broilers orally infected. Poult. Sci. 94 (E-Suppl. 1): 119-120.
• Millán, C., A. Martín, M. G. Molnar, J. Sánchez, P. Medel, 2015. Effect of feed presentation and whole wheat addition on gastrointestinal morphology in orally infected broilers with C. jejuni. Poult. Sci. 94 (E-Suppl. 1): 82.
• Casabuena, O., V. Elgosi, M. Den Hartog, M.I. Gracia, P. Medel, 2015. Effect of particle size and feed presentation on gastrointestinal morphology in infected broilers with C. jejuni. Poult. Sci. 94 (E-Suppl. 1): 82.
• Casabuena, O., M. Chemaly, M. Den Hartog, P. Vesseur, M. I. Gracia. 2015. Effect of feeding different combinations of organic acids, mono-glycerides and probiotics on Campylobacter colonization in broilers. Pages 173-175 in Full Papers of the 20th European Symposium on Poultry Nutrition, Prague, Czech Republic.
• Gracia, M. I., F. J. García-Peña, M. Guyard, G. Le Pottier, M. Tenk. 2015. Effect of particle size and feed presentation (mash vs pellets) on cecal morphology and Campylobacter jejuni colonization of broilers orally infected. Pages 561-563 in Full Papers of the 20th European Symposium on Poultry Nutrition, Prague, Czech Republic.
• Millán, C., A. Martín, J. Mayot, J. Sánchez, M. I. Gracia. 2015. Effect of type of cereal and oat hulls addition on gastrointestinal morphology in broilers orally infected with Campylobacter jejuni. Pages 434-436 in Full Papers of the 20th European Symposium on Poultry Nutrition, Prague, Czech Republic.
• Sánchez, J., A. Parra, Y. Carre, A. Csorbai, M. I. Gracia. 2015. Effect of supplementing via drinking water a blend of mono-glycerides, alone or in combination with organic acids, on Campylobacter colonization in broilers. Pages 267-269 in Full Papers of the 20th European Symposium on Poultry Nutrition, Prague, Czech Republic.
• Millán, C., O. Casabuena, O. Benito, S. Porras, J. Sánchez, M. I. Gracia, 2015. Effect of feeding two different combinations of flavouring compounds on Campylobacter colonization in broilers. Proceedings of the 19th World Veterinary Poultry Association Congress, Cape Town, South Africa.
• Millán, C., O. Casabuena, I. Muñoz, S. Porras, J. Sánchez, M. I. Gracia, 2015. Effect of feeding two different combinations of essential oils on Campylobacter colonization in broilers. Proceedings of the 19th World Veterinary Poultry Association Congress, Cape Town, South Africa.
• Millán, C., O. Casabuena, M. Guyard, M. Tenk, P. Medel, 2015. Effect of feeding a combination of a yeast product and a probiotic, alone or in combination with a blend of mono-glycerides, on Campylobacter colonization in broilers. 18th International Workshop Campylobacter, Helicobacter & Related Organisms (CHRO) 1-5 November, 2015 Rotorua, New Zealand. O142. Pp: 113.
• Millán, C., P. Vesseur, E. Puentes, O. Casabuena, P. Medel, 2015. Effect of feed presentation (mash vs pellets) and whole wheat addition on cecal morphology and Campylobacter jejuni colonization of broilers orally infected. 18th International Workshop Campylobacter, Helicobacter & Related Organisms (CHRO) 1-5 November, 2015 Rotorua, New Zealand. O125. Pp: 102.
• Gracia, M.I. M.Chemally Á. Martín, A. Csorbai, P. Medel, 2015. Effect of oat hulls addition and whole wheat addition on cecal morphology and Campylobacter jejuni colonization of broilers orally infected. 18th International Workshop Campylobacter, Helicobacter & Related Organisms (CHRO) 1-5 November, 2015 Rotorua, New Zealand. P120. Pp: 178.
• Meunier, M., M. Guyard-Nicodème, E. Vigouroux, T. Poézëvara, V. Beven, S. Quesne, D. Dory, A. Parra, M. Chemaly, 2015. Sequential optimization of an avian vaccine protocol against Campylobacter. 18th International Workshop Campylobacter, Helicobacter & Related Organisms (CHRO) 1-5 November, 2015 Rotorua, New Zealand. O139. Pp: 111.
• Medel, 2016. Effect of feeding a combination of organic acids, mono-glycerides and a probiotic on Campylobacter colonization in broilers. European Symposium on Food Safety. 11-13 may 2016. Athens, Greece. P2-54. Pp 128.
• Millán, C., M. Chemaly, J. Mayot, P. Vesseur, M.I. Gracia, P. Medel, 2016. Interaction of a combination of organic acids and mono-glycerides and a probiotic on Campylobacter colonization in broilers. XXV World’s Poultry Congress. September 5-9, 2016. Beijing, China.
• Gracia, M.I. M. Guyard-Nicodème, A. Csorbai, M. Den Hartog, I. Tenk, P. Medel, 2016. Interaction of a combination of organic acids and mono-glycerides and a probiotic with a functional diet on Campylobacter colonization in broilers. XXV World’s Poultry Congress. September 5-9, 2016. Beijing, China.
• Casabuena, O., A. Martín, V. Elgosi, M. G. Molnar, M.I. Gracia, P. Medel, 2016. Effect of feeding a combination of organic acids and mono-glycerides at different dosages and a probiotic on Campylobacter colonization in broilers. XXV World’s Poultry Congress. September 5-9, 2016. Beijing, China.
• Sánchez, J., O. Casabuena, A. Parra, G. Le Pottier, F. Sánchez, P. Medel, 2016. Effect of supplementing a combination of organic acids and mono-glycerides and a probiotic during different times of administration on Campylobacter colonization in broilers. XXV World’s Poultry Congress. September 5-9, 2016. Beijing, China.
In addition, another 5 articles has been published as a part of the invited conference:
• Medel, P., Sánchez F., Rodríguez, E., Martín, Á., 2014. Campylobacter en avicultura de carne: situación actual y estrategias de reducción. Jornadas Profesionales de Avicultura. Sevilla, Spain. Pp: 74.
• Medel, P, F. Sánchez, A. Parra, Á. Martín, Y. Carré, J. Mayot, M. den Hartog, G. Molnar, M. Tenk, M. Guyard, 2015. Problemática actual del Campylobacter en la salud pública. Proccedings of Jornadas Profesionales de Avicultura 2016, Soria, Spain. Pp: 62-68
• Medel, P, F. Sánchez, A. Parra, Á. Martín, Y. Carré, J. Mayot, M. den Hartog, G. Endrodi, M. Tenk, M. Chemaly, 2015. Métodos de control de Campylobacter en la industria avícola. III Congreso Internacional de Seguridad alimentaria. Proceedings of the conference (only in e-format).
• Guyard-Nicòdeme, M., 2016. Why is Campylobacter the number one priority for the poultry production chain? International Association for Food Protection European Annual European meeting, Athens, Greece. Pp: 51
• Medel, P., 2016. Un update about the different state of the art methods to control campylobacter in broilers: the European project CAMPYBRO. International Association for Food Protection European Annual European meeting, Athens, Greece. Pp: 51
5. Articles published in the popular press
There were 6 publications, listed below:
• Martín, Á., Redondo, J.L. Fernández, A., Medel, P., 2014. Campylobacyter es el nuevo reto de Europa en avicultura de carne; 01/03/2014; Albéitar, 17: 10-11. Zaragoza, Spain
• Martín, Á., Redondo, J.L. Fernández, A., Medel, P., 2014. Campylobacter é o novo desafio da europa em avicultura de carne; 01/06/2014; Albéitar (Portuguese edition) 3 (Mai/Jun 2014):14-16
• Foldi, P., Csorbai, A., 2015.Campylobacter a baromfihúsban; 01/06/2014; BAROMFI ÁGAZAT 2014/2 JÚNIUS: 75-78
• Csorbai, A., György, E., György, M., Foldi, P., Medel, P., 2015. Campylobacter: egy baktérium, amit gyéríteni kell!; 04/12/2015; BAROMFI ÁGAZAT 2015/4 15: 84-87
• Le Pottier, G., Elgosi, V., Mayot, J., Carre, Y., 2015. Campylobacter: stratégies de réduction envisagées.; 01/09/2014; Filères avicoles, September 2014, 10-13
• Gracia, M.I. Redondo, J.L. Fernández, A., Martín, a., Medel, P., 2014. Campylobacter en avicultura de carne. Situación actual y estrategias actuales de reducción; 01/10/2014; Selecciones avícolas. Octubre 2014: 6-9
• Csorbai, 2016. Campylobacter-ma ennyit tudunk róla. Baromfiágazat 2016/3: 78-83
• Dossier Campylobacter. Réussir Avicole 220:10-19
6. Press releases
The project has had a big impact in the international media. There were 5 press releases, three of them in the most popular poultry websites, which have clearly contributed to the knowledge of sector about the project (title-date-location):
• PRESS RELEASE OF THE PROJECT - 20/12/2013-www.lafranceagricole.fr;
• PRESS RELEASE OF THE PROJECT - 23/12/2013 - www.agrapresse.fr;
• PRESS RELEASE OF THE PROJECT - 30/12/2013 - www.lequotidienlesmarches.fr;
• PRESS RELEASE OF THE PROJECT - 10/01/2014- Asociación Española de Ciencia Avícola newsletter;
• Aprobación en el 7º Programa Marco del proyecto CAMPYBRO. - 31/01/2014 - Arenys de Mar, Barcelona, Spain. Selecciones Avícolas, 56, 1, pp: 36;
• Una estrategia común contra el Campylobacter en avicultura de carne - 01/03/2014 - Madrid, Spain. Ganadería, 89: 15;
• Topsectoronderzoek naar Campylobacterreductie in pluimveevleessector - 18/03/2014 - http://www.agriholland.nl;
• Onderzoek Campylobacter verlegd naar primaire sector - 19/03/2014 - Boerderij Vandaag, 19/04/2014, page 9, The Netherlands;
• Rem op Campylobacter in pluimvee - 19/03/2014 - http://www.nieuweoogst.nl;
• Onderzoek naar Campylobacter reductie op plumeveebe drijven - 19/03/2014 - http://www.pluimveeweb.nl;
• Nepluvi participeert in grootschalig Europees CAMPYBRO Project - 01/06/2014 - www.nepluvi.nl
• PRESS RELEASE OF THE PROJECT - 16/10/2013- Newsletter of CIDEF, MORDELLES (France),
• Press Release at: http://www.pluimveeweb.nl/artikelen/2014/04/reguliere-vleeskuikens-minst-vaak-Campylobacter-positief/ and
• Campylobacterreductie - 01/04/2014 - Meat & Co. April 2014: 56.
• Voeradditieven effectief tegen campylobacterinfectie. Boerderij Vandaag, 27/01/2016, pp:9
• Strijd tegen Campylobacter. Pluimveehouderij 46e. 12/02/2016;
• Koeleman, E., 2016. Feed additives show promise in Campylobacter reduction. AllaboutFeed, 25/01/2016. http://www.allaboutfeed.net/Feed-Additives/Articles/2016/1/Feed-additives-show-promise-in-Campylobacter-reduction-2750600W/
• Koeleman, E., 2016. Campylobacter reduction: Feed additives role. World Poultry, 26/01/2016. http://www.worldpoultry.net/Nutrition/Articles/2016/1/Campylobacter-reduction-Feed-additives-show-promise-2751797W/
• Progress Versus Campylobacter. The Polutry site. 10 February 2016 http://www.thepoultrysite.com/poultrynews/36543/progress-versus-campylobacter/
• A press note on the Spanish workshop: http://avicultura.info/resultados-del-proyecto-europeo-campybro/ Avinews, Agosto 2016: 128
• 4 press notes on the Dutch workshop
o https://www.pluimveeweb.nl/artikelen/2016/08/campylobacter-congres-2016/
o https://www.nieuweoogst.nu/nieuws/2016/09/01/nederlandse-aanpak-campylobacter-succesvol
o http://www.wur.nl/nl/Expertises-Dienstverlening/Onderzoeksinstituten/livestock-research/Expertisegeb
o https://www.pluimveeweb.nl/artikelen/2016/09/optimisme-terugdringen-campylobacter/
7. Oral presentation to a scientific event/Technical meetings
IMASDE was invited to impart the conference “Campylobacter en avicultura de carne. Situación y estrategias actuales de reducción”, on 26/03/2014, in Jornadas Profesionales de Avicultura, Sevilla (Spain), 25-28 March 2014. Proceedings: 74. (http://www.jornadasavicultura.com/2015/produccion-pollos). IMASDE was also invited to impart the conference “Problemática actual del Campylobacter en la salud pública”, on 10/06/2015, in Jornadas Profesionales de Avicultura, organized by REAL ESCUELA DE AVICULTURA at Soria (Spain), 9-12 June 2015. IMASDE was also invited to impart the conference “Métodos de control de Campylobacter en la industria avícola”, on 26/11/2015, in III Congreso Internacional de Seguridad alimentaria, organized by Ilustre Colegio Oficial de Veterinarios Región de Murcia and Academia de Veterinaria de la Región de Murcia, at Murcia (Spain), 25-27 November 2015. Finally, IMASDE was invited to impart the conference “Impact of Campylobacter contamination in broiler production- Which opportunities can be provided on the part of feeding for a possible reduction of Campylobacter to improve food safety. Current information about CAMPYBRO project”, on 20/09/2016, in 19 Orffa symposium, organized by ORFFA, at Dinklage (Germany), 20 September 2016.
Two other invited conferences were imparted in the international workshop on Campylobacter within The International Association for Food Protection European Annual European meeting, hold in 2016 in Athens (Greece), as explained bellow. The conference titles were:
• Guyard-Nicòdeme, M., 2016. Why is Campylobacter the number one priority for the poultry production chain? International Association for Food Protection European Annual European meeting, Athens, Greece. Pp: 51
• Medel, P., 2016. An update about the different state of the art methods to control campylobacter in broilers: the European project CAMPYBRO. International Association for Food Protection European Annual European meeting, Athens, Greece. Pp: 51
Organization of workshops in International Scientific Meetings
An international workshop on Campylobacter was presented by ANSES within the The International Association for Food Protection (https://www.foodprotection.org/) European Annual European meeting, hold in 2016 in Athens (Greece) between the 11th and 13th May (https://www.foodprotection.org/europeansymposium/).
During the meeting, a workshop focused on Campylobacter was presented by ANSES, in which the results of the project within the context of general measures to control Campylobacter were the main issue.
The workshop was chair by Muriel Guyard and Habila Haddad (ANSES) and the main speakers were Muriel Guyard-Nicòdeme and Pedro Medel (coordinator, IMASDE).
8. Vertical dissemination activities between producers within Poultry Producers Associations
In total, 7 presentations were made. In the first reporting period, there were three presentations: “Campylobacter challenge for EU poultry producers”; on 30/10/2013; in Budapest, Hungary (BTT facilities at Budapest), destined to main poultry producers in Hungary; “Campylobacter challenge for EU poultry producers. First results of CAMPYBRO project” on 11/02/2014 in Madrid (Propollo facilities, Madrid), destined to main poultry producers in Spain and “Campylobacter challenge for EU poultry producers” on 19/11/2014 in Madrid (Propollo facilities, Madrid), destined to main poultry producers in Spain.
In the Second reporting period the project results were presented to the Association members seven times. In Spain, all the presentations took place in the PROPOLLO facilities in Madrid, and the main poultry producers participate into the presentation. Since the poultry production is very integrated in Spain (the main 10 operators have approximately 60% of the production), most of the Spanish production was informed about the project results. All the presentations were given by the coordinator (Pedro Medel) and the PROPOLLO CEO (Ángel Martín). The dates of the meetings were: 03/03/2015, 24/11/2015 and 19/04/2016.
An update of the Campylobacter in the EU poultry sector was made in Houten on 05/11/2015. The public were the quality managers of the 16 bigger poultry producer in the Netherlands and NEPLUVI (Association of the Dutch Poultry Processing Industries). The update included the prevalence in the Netherlands and in the rest of Europe, the intentions for creating a European Process Hygiene Criterion for Campylobacter and the preliminary results of the project (WP1, WP2, WP3 and WP5) presented in the second GA at Stockholm. Also, the members were updated with the management activities of the project and the dissemination activities. The presentation was given by Peter Vesseur.
The project results were presented to the Hungarian companies in several opportunities (a year-bases) in the Second reporting period. All the presentations took place in the Geréby Kúria Hotel at Lajosmizse (Hungary), and most of the poultry producers of the country participate into the presentation (more than 100 both times). All the presentations were given by the coordinator (Pedro Medel) and the BTT CEO (Attila Csorbai). The dates of the meetings were: 03/03/2015, 24/11/2015 and 19/04/2016.
An update of the Campylobacter in the EU poultry sector was done for French producers. First, there was a presentation of the Ecole Superior d’Agriculture of Angers, and about the importance of poultry in this Region of France and in the ESA in particular. After, Julie Mayot presented the epidemiology of Campylobacter and campilobacteriosis. Pedro Medel presented some aspects of the infection, the most practical measures to fight again. Finally, the project CAMPYBRO was presented, and the results until March 2015 were explained. There were approximately 30 attendees, belonging to several poultry companies inside FIA and CIDEF.
9. Participation in international meetings presenting information to decision makers
The International Conference on PREVENTION AND CONTROL OF CAMPYLOBACTER IN THE POULTRY PRODUCTION SYSTEM, was organized by IZSAM G. CAPORALE TERAMO, Ministero della Salute and the Instituto Zooprofilattico Sperimentale delle Venezie, and hold on Milano, the 31st August 2015. In the meeting participated a lot of researchers, policy makers including Italian Ministry of Heatth or the the EC (Klaus Kostenzer), policy maker supporters (ECDC, EFSA), or the National Reference Laboratories of several countries for Campylobacter. The Director of the station of Ploufragan, ANSES, Dr. Gilles Salvat, was invited to give a talk called “Reducing the risk of Campylobacteriosis from poultry: Feed interventions - blocking the colonization of birds”, which summarizes the results of the project Campybro until this moment.
The Food Standards Agency of the UK, was informed about CAMPYBRO project through Francisco Javier Domínguez Orive, Veterinary Director and Head of Foodborne Diseases Control Unit, who was invited as speaker in the workshops of France and Spain. After that, a colleague of him (Adam Hardgrave) was asked to write a chapter for the ACMSF Campylobacter report on ‘Controls (regulatory framework for products of concern, EU targets etc.) and evidence of success in other countries’, and they though that a section on Campybro would probably fit well/be really useful for inclusion. The Terms of Reference for the report is: ‘To assess the actions that have taken place since the publication of the Second Campylobacter Report and make proposals to advise the FSA in evolving its strategy for reducing the incidence and risk of foodborne Campylobacter infection in humans.’ The chapter currently details the sampling regimes and interventions in Norway, Sweden, Denmark, Iceland and the Netherlands. Each section also includes more technical details on the Characteristics of the broiler industry in each country, such as the number of farms, type and number of slaughterhouses/processors, average stocking densities etc. Its content will enter the public domain probably toward the end of the year. A summary of the project was sent the 3rd of August for their revision and edition to be included in the Report.
10. Organization of Conferences
A Workshop was organized by IMASDE “Campylobacter: un reto para la avicultura de carne europea” on 13/12/2013 in Murcia (Spain), destined to Scientific community (resarchers), students and policy makers (Consejería de Agricultura y Consejería de Sanidad de la Región de Murcia).
11. Dissemination of the data in the countries that participate in the project
The project results are interesting for poultry producers and processors. Fortunately, the project partners SME-AGs are the Poultry Producers Associations (Spain, Hungary, The Netherlands and France [2]). Therefore, it has been very easy to disseminate the results of the project within their Associates. In 2016 four technical workshops were done in the four countries that participate in the project (Spain, France, The Netherlands and Hungary). These workshops were technical, and were open to the all sector, including the media, the academia, the industry and the Administration.
List of Websites:
www.campybro.eu
The objective of CAMPYBRO project, under the 7th FRAME PROGRAM of UE (Capacities- Research for SME Associations FP7-SME GA 605385) is to develop practical strategies to decrease the population of Campylobacter jejuni in broilers through two strategies, nutrition and vaccination. The project started in September 2013 and will finish in August 2016, is coordinated by IMASDE AGROALIMENTARIA S.L. (IMASDE), and is formed by 10 partners: 5 poultry producers National Associations (FIA and CIDEF from France, PROPOLLO from Spain, NEPLUVI from The Netherlands, BTT from Hungary), a vaccine laboratory (CZV, Spain), a poultry producer (EXPLOTACIONES AVÍCOLAS JOSÉ LUIS REDONDO S.A. Spain) and a lab (MIKROLAB, Hungary). The Research centers involved are ANSES (France) and IMASDE (Spain).
Into the nutrition strategy the steps were: i) identify single additives that decreased in vivo cecal Campylobacter infection, ii) identify synergisms between the most efficient additives, iii) look for changes in the feed form that also decreased in vivo cecal Campylobacter infection, iv) look for synergisms between feed form and combination of additives, v) optimize the nutrition program (time of administration, dose), vi) test the additives strategy in field (experimental and commercial) conditions with natural contamination, vi) demonstration field trials, (Spain, France, The Netherlands, Hungary). Single additives: A total of 24 single products belonging to the next categories of products: ORGANIC ACIDS (OA, 42%), PLANT EXTRACTS (PE, 33%), PREBIOTICS (PRE, 4%) or PROBIOTICS (PRO, 21%) were tested in vivo through challenge trials. In total, 46% of the products showed a significant reduction in Campylobacter counts anytime. The best results were obtained with a yeast extract, with a probiotic based on B. subtilis, and with monoglycerides of medium chain fatty acids (MCFA). Group of additives: A total of 10 combinations between families of groups of additives (PE, PRO, PRE and OA) were designed and tested. One of the combinations tested (mixture of a combination of organic acids and monoglycerides of MCFA [MGOA] and a B. subtilis-based probiotic [CALS]) dramatically decreased the Campylobacter population. Feed form studies: A total of 4 experiments were done, testing: the diet main cereal (corn, wheat or barley), the inclusion of oat hulls (OH), grinding screen size (Ø2 vs 5mm), feed presentation (mash vs pellets) or whole wheat (ww) inlusion. Only the inclusion of ww and 5% of OH decreased the Campylobacter contamination at 42d. Synergisms between additives and feed form: No synergisms were found, and the additives strategy was selected to continue. Additive program optimization: The combination of MGOA and CALS had a synergistic effect, although this effect was variable: in one trial there was an effect at 35d but not at 42d, in two trials the effect was maintained at 35 and 42d, and in one trial there was not any effect. The yeast extract did not show any effect on Campylobacter cecal population. Field trials: In field experimental conditions, the natural infection did not occur in 3 of 5 experiments, and in the other 2 experiments the combination of MGOA+CALS had not any effect on Campylobacter infection. In Spain, the pattern of natural contamination was very different and there were not any effect of the additives. In Hungary, for unknown reasons, the additive addition not only did not decrease the Campylobacter population, but it increased it. Demonstration activities: In France.... In Spain, the treatment delay the infection in two trials, and the MGOA by water decreased the infection in other trial. In The Netherlands, no natural infection was achieved. In Hungary, there were no effect of treatment on final contamination level.
Vaccine development: a list of 12 potential highly immunogenic vaccine antigens against C. jejuni was obtained through reverse vaccinology. A vaccine protocol was studied using flagellin as a model. Once the model was developed, the new potential antigens were tested, with promising results on Campylobacter cecal reduction of at least five antigens.
Final conclusions: i) a combination of additives (MGOA+CALS) decreased the Campylobacter cecal population in challenge trials, ii) the effect of this combination was not homogenous, iii) the interaction between in vitro trials and in vivo trials was avoided, but there is an interaction between challenge trials and natural contamination infection trials, iv) much more knowledge is needed in Campylobacter epidemiology, since the natural infection did not follow classical patrons of infection, v) the demonstration trials will define if the strategies developed are effective in field conditions in some cases, vi) a potential vaccine could be developed, although its commercial development could be compromised by the price and the vaccination program.
Project Context and Objectives:
Campylobacteriosis is the most frequently reported zoonotic illness at the EU, with 238,851 declared cases in 2014, and a total annual cost of 2.4 billion € per year in the EU27.
However, In order to appraise more realistically the impact of Campylobacteriosis in EU/EEA MS we compiled data from three different sources: an ECDC-funded sero-epidemiological study, reported cases in The European Surveillance System (TESSy) database, and data stemming from literature reviews. ECDC commissioned sero-epidemiological study aimed at estimating the exposure to Campylobacter spp. in selected EU/EEA MS by measuring antibody responses in human serum samples and back-calculating the estimated time of infection. Results from this serological study were anchored to published community studies in order to derive the actual incidence of Campylobacteriosis disease. In the EU/EEA MS, the annual rate of exposure to Campylobacter spp. is estimated to be around 0.83 per person-year, translating in more than 420 million yearly infections. The vast majority of exposed cases does not develop the clinical disease and remain asymptomatic. Based on community studies, the related incidence of Campylobacteriosis disease is 475 per 100 000 (CI 95%: 423-524 per 100 000) or 2.4 million cases per year amongst European citizens. Underestimation of the disease, therefore, is considered to be 11 times the notification rate. Moreover, in a recent burden of disease study (BCoDE 2015), ECDC estimated that about 600 deaths are related to Campylobacteriosis every year, largely among elderly people. Results from BCoDE 2015 also found that Campylobacteriosis is the food and water-borne disease producing the highest number of DALYs (Cassini, 2015 ).
We are therefore faced with a major problem of public health, and the EC has been discussing for a long time the possible measures to be taken to decrease the risk of campilobacteriosis infection. After a stakeholder meeting in Brussels , and the consultation of all the EU countries, very recently has been launched a Regulation proposal amending Regulation (EC) No 2073/2005 as regards Campylobacter in broiler carcasses. This Regulation will be published in short time, and in theory it will be enter into force the 01/09/2016. Based on the EFSA opinions of 2010 and 2011, the Commission commissioned an analysis of setting certain control measures for reduction of Campylobacter in broiler meat at different stages of the food chain. The main conclusion of this analysis is that setting a process hygiene criterion to Campylobacter in broiler carcasses would provide one of the best balances between reducing human Campylobacteriosis attributed to the consumption of poultry meat and economic consequences from the application of the criterion. In view to reduce administrative burden for food business operators, the sampling plan for the criterion on Campylobacter should follow the same testing approach as for the process hygiene criterion set out in Row 2.1.5 of Chapter 2 of the Regulation (EC) No 2073/2005 for Salmonella in poultry carcasses. The same samples used for testing the compliance with criterion 2.1.5 may therefore be used for the Campylobacter analyses. The international standard ISO/TS 10272-2 will be the horizontal method for the enumeration of Campylobacter spp in food and feed stuffs.
When testing against the process hygiene criterion in poultry carcasses in slaughterhouses, neck skins from a minimum of 15 poultry carcasses shall be sampled at random after chilling during each sampling session. A piece of approximately 10 g from neck skin shall be obtained from each poultry carcass. On each occasion the neck skin samples from three poultry carcasses from the same flock of origin shall be pooled before examination in order to form 5 x 25 g final samples. The food business operators of slaughterhouses or establishments producing minced meat, meat preparations, mechanically separated meat or fresh poultry meat shall take samples for microbiological analysis at least once a week. The day of sampling shall be changed each week to ensure that each day of the week is covered.
Campylobacteriosis is produced by Campylobacter. These rods are negative, curved or spiral shaped, gram movable by flagellum are not sporulate and microaerophilic. There are at least 23 species, but the most important are thermophilic, growing at 32-47 º C , and are those that cause human gastroenteritis. The most important are C. jejuni and C. coli, with other minor (C. lari, C. upsaliensis, C. helveticus). Of the reported cases of Campylobacteriosis in the EU, 81.1 % were C. jejuni, C. coli 6.2%, 0.2% C. lari, C. upsaliensis 0.06%, and 0,01% C. fetus . The rest were doubts among C. jejuni and C. coli. Another feature of Campylobacter is their sensitivity to desiccation and extreme temperatures, both heat and cold. The natural reservoir of Campylobacter is the gastrointestinal tract of domestic and wild animals, most notably birds ( chickens, ducks , turkeys, etc. ), pigs, and cattle, and rodents , dogs , cats and insects (mainly flies). Poultry meat is the major source of human infection, and there is a direct correlation between the contaminations of the herds with the carcasses. However, also other foods such as raw milk, are reporting a growing number of cases.
Campylobacter is commensal bacteria that do not produce illness or morbidity in broilers (apparently, it does not produce any immune response), and its capacity of (horizontal) infection is very effective, with the whole farm infected within one week form a few animals infected. Basically Campylobacter lives in the caeca of broilers, and for that reason it is complicated to design nutritional strategies to decrease the colonization, since most of the products have been absorbed or changed during its pass through the gastrointestinal tract. Moreover, this is the main reason of the lack of coincidence between in vitro and in vivo trials. Therefore, there is still no effective, reliable and practical strategy available to prevent or to reduce Campylobacter colonization in broilers.
One of the most important problems to deal with is the lack of knowledge on Campylobacter epidemiology. It is not known the source of Campylobacter in the continuous contamination process within a farm. There are a lot of possible sources, such us the rest or organic matter in drinkers or feeders from the previous flock, the drinking water, the farmers tools, several pests, wild animals, etc. However, no conclusive studies have been done, and in fact, multiple strains has been found in several studies in consecutive contaminated batches, which suggest that there are several sources of contamination, and that the origin is not necessarily the same source between consecutive flocks. Also, there is a lack of knowledge on the transmission of Campylobacter along the food chain. In theory, the strains that appear in the live broilers should be the same than in the slaughterhouse and in the carcasses. But recent research has demonstrated that this is not always true, and the strains appears and disappears along the food chain with a logical explanation.
Starting from the premise that there is no vertical transmission reduction strategies fall into two groups: pre-slaughter and post sacrifice. Within the pre-slaughter strategies, there are different alternatives: Biosecurity, Vaccine or Nutritional strategies. The two last are being developed by the project CAMPYBRO "Control of Campylobacter infection in broiler flocks through two-steps strategy: nutrition and vaccination”.
The biosecurity measures to control Campylobacter have been extensively analyzed by the CAMCON European project (www.camcon-eu.net). This project has generated a good manufacturing practices manual, in which a lot of biosecurity measures are recommended. However, many of them are difficultly applicable in most of the real farms within the EU, mainly because of the farm design and age. In addition, the thinning practice (in which part of the animals are slaughtered at 32-36d and the rest are maintained until the final slaughtering), is a clear break of the biosecurity, and most of the measures lack their efficacy after this process. To avoid the thinning is not possible in practice, because of the huge economic impact for the producers in a very complex context, with very low margins and the threat of imported chickens for third countries.
Also, the most efficient biosecurity measures such as the fly control through nets, have demonstrated their efficacy only after other very strict measures have been implemented, which is not always possible. On the other hand, the measures proposed by CAMPYBRO project (nutrition and vaccination) can be implemented immediately and horizontally to the entire poultry sector in the EU and worldwide. However, in practice, the most logical procedure should be to combine all the available techniques in order to decrease the possibility of contamination.
Regarding the vaccine development, there are different initiatives. The University of Arizona has recently presented Anivax, and they are searching investors to initiate the registration in the USDA. Nothaft et al. (2015 ) engineered the C. jejuni N-glycan heptasaccharide to be expressed on the surface of E. coli cells as a heptasaccharide-LPS fusion. Vaccination of chickens with live E. coli expressing the heptasaccharide fusions resulted in >8 log(10) reduction of C. jejuni colonization compared to the control. However for the development and approval of this potential vaccine there is a long period, and since it is a GMO vaccine, its approval in Europe should be complicated.
The objective of the present project is to develop a two-steps strategy in order to fight against Campylobacter in primary poultry production. The first step, through a nutritional approach, and in a second step, a more medium-term strategy through the development of a vaccine. The nutritional approach consisted in a combination of two strategies: i) nutritional additives, such as plant extracts, organic acids, prebiotics, probiotics, trying to find synergisms between them, and ii) feed presentation (type of cereal, mash vs pellets, particle size, whole wheat or oat hulls inclusion). This nutritional strategy was tested in trials with (challenged) infected broilers first, and then with natural contamination in real conditions (either experimental or commercial). In a second step, the vaccination approach was based on a novel development of a Campylobacter vaccine through the use of reverse vaccinology. First, 12 novel antigens were detected based on their theoretical antigenicity. After, a vaccination model was developed by using falgellin protein. Finally, the new candidates were tested for their capacity of generating immunity and their capacity to reduce the Campylobacter numbers in vivo.
The objectives of the project are to develop i) a short term strategy to decrease the Campylobacter infection in vivo through new nutritional strategies based on a synergic combination of feed additives and feed presentation, ii) a medium term strategy through a novel vaccine to reduce the prevalence of Campylobacter in broiler flocks.
Project Results:
WP1: Efficacy of several compounds against Campylobacter in broilers orally infected looking for synergies
The WP1 “Efficacy of several compounds against Campylobacter in broilers orally infected looking for synergies” has developed the additives strategy through three tasks: T1.1: “In vivo effectiveness of products based on plant extracts, organic acids, prebiotics, and probiotics against Campylobacter”; T1.2: “In vitro effectiveness of mixtures of products: Synergistic effect”; and T1.3 “In vivo effectiveness of product mixtures based on plant extracts, organic acids, prebiotics, and probiotics against Campylobacter”.
The objectives of this WP were to design combinations of additives that effectively decrease the Campylobacter population of infected broilers in vivo. To fulfill this objective, in the T1.1. a total of 24 additives were tested. The 24 products involved belong to the next categories of products: ORGANIC ACIDS ([OA], 42%), PLANT EXTRACTS ([PE], 33%), PREBIOTICS ([PRE], 4%) or PROBIOTICS ([PRO], 21%). The 24 additives were tested in vivo through challenge trials (50% each RTD: ANSES and IMASDE AGROALIMENTARIA, S.L.) in which all the animals were infected with C. jejuny ST-45. After inoculation, Campylobacter jejuny was analyzed at 14/21, 35 or 42d of age, reflecting the commercial practice in different countries across the EU. Campylobacter counts were analyzed by ISO/TS 10272-2:2006. At 14d, the products that decrease the Campylobacter counts were BRC, Biotronic, PoultryStar, Alliin, Adimix, Antaphy and Campylostat. At 21d, only the caprilic+capric as acids or monoglycerides (MG), and the combination of organic acids (OA) plus MG of Medium Chain Fatty Acids (MCFA) had a significant effect. At 35 d, BRC, PoultryStar, Adimix, caprilic+capric as acids or MG, and the combination of OA plus MG of MCFA also reduced the contamination. At 42d, the probiotic Calsporin, the prebiotic XPC, the acid Adimix, MG of caprilic+capric acids, and the combination of OA plus MG of SMCFA decreases the caecal Campylobacter counts. The usual level of contamination in control animals were between 7-9 Log10UFC/g, and the reductions were between 1 and 3 Log10UFC/g. Therefore, in total, 46% of the products (11 of 24) showed a significant reduction in Campylobacter counts in any of the periods tested.
although all of them had a significant effect in vivo, only three products had a significant bacteriostatic effect in vitro: monoglycerides (MG) of Medium Chain Fatty Acids (MCFA), MG-MCFA in combination with organic acids (OA), and the PE Alliin. Interestingly, results did not reveal important synergistic effects between the products tested. However, potential antagonistic effects were also observed.
These results were used to define the 10 treatments of Task 1.3 based on the mixture of five products (MG-MCFA, PE, coated OA, OA+MG-MCFA, and the yeast-wall based PRE) and the two PRO (based on multi-species and B. subtilis). A total of 10 combinations between families of groups of additives (PE, PRO, PRE and OA) were designed and tested in ANSES (6 combinations) and IMASDE (4 combinations). This mixtures combine between 1 and 2 products from three families of products (PE, PRE and OA), plus one PRO.
In the ANSES trial, five combinations decreased the Campylobacter population at 21d (mixture of Lactobutyrin BRC and Calsporin; mixture of Excential Alliin Plus and Calsporin; mixture of Monoglycerides and Calsporin; mixture of Adimix 30 coated, Excential Alliin Plus and Calsporin; and the mixture of Lactobutyrin BRC, XPC and PoultryStar), but only one mixture, (Lactobutyrin BRC, XPC and PoultryStar) significantly decreased Campylobacter counts at 35 days of age and none of them at 42d.
In the IMASDE trial, one of the combinations tested (mixture of Campylostat and Calsporin) clearly decreased the Campylobacter population, mainly avoiding the contamination of birds, with reductions of 3.52 4.09 and 3.98 log10 CFU/g; or 75, 58 and 75% of inoculated birds negative at 21, 35 and 42d, respectively. The other treatments showed numerical but not significant reductions, although the other mixture which contained Campylostat (plus Alliin and PoultryStar) also tended to show a significant reduction at 21d, with 66.7% of birds not infected.
It can be concluded that 7, 2 and 1 combinations of the products tested in T1.1 gave significant reductions at 21, 35 and 42d. Therefore, the combination that showed the best results at 42d (mixture of Campylostat and Calsporin), and some other individual products that showed the biggest reductions in T1.1 alone, will be used in WP3.
Therefore, the objectives of the WP1 were achieved, since several individual compounds and combinations have demonstrated its ability to decrease the Campylobacter population. However, it was expected a higher level of synergisms between individual additives. This effect is not well understood, and it is related with the ecology in the caeca of birds, and the mode of action of each group of additives: acting directly against Campylobacter (such as OA, MCFA, MG-MCFA or PE), o modifying the microflora of caeca for displace the Campylobacter populations (PRO, PRE). Also, although for the moment is only a theory, it was discovered that the best way to decrease the Campylobacter infection is to avoid the contamination, rather than decrease the cecal populations (once the bird is infected, it is very difficult to decrease the populations). This is the case of the combination of Campylostat and Calsporin, which showed that 75, 58 and 75% of inoculated birds were negative at 21, 35 and 42d, respectively.
There were not deviations in connection with the expected works. However, there was a delay, due to two reasons: i) There were some works in the Animal Biosafety Level 2 (ABL2) facilities in the RTD ANSES, which lasted from September to December of 2013. Therefore, there was a delay of three months, since the trials started in January 2014, ii) In IMASDE, the experiment EXP.EU02 has to be repeated. The reason was that the chickens suffer an E. coli infection, which origin is unknown, which was not controlled by the selective medium mCCDA. This causes that most of the plates presented a huge growth of E. coli, which was invasive, that make impossible to count the C. jejuny counts. This make impossible to reach any conclusion after the trial, and the products tested were re-tested in trials EXP.EU04 and EXP.EU05. This caused a parallel delay of three months approximately, from which two has been recovered (last experimental work finished in December). Therefore, all the works have the same delay, but at this time, all the work expected for WP1 has been achieved. The consortium will try to recover the rest of this delay in WP3.
WP2. Feed presentation strategies against Campylobacter
The WP2 “Feed presentation strategies against Campylobacter” has developed the feed form strategy through two tasks: T2.1: “Effect of feed composition, particle size and feed presentation on the prevalence of Campylobacter in broilers orally infected”; and T2.2: “Effect of whole grain feeding on the prevalence of Campylobacter in broilers orally infected”.
The objectives of this WP were to design feed forms that effectively decrease the Campylobacter population of infected broilers in vivo. A total of 4 experiments (2 experiments in each Task) were done. In the first experiment (EXP.03) there were five treatments, based on the main cereal of the diet (corn, wheat or barley), and the inclusion of 5% of oat hulls (OH) in the corn and wheat-based diets. Oat hulls were added by dilution. The second experiment (EXP.06) was done with wheat-based diets, and there were four treatments factorially arranged with two screen sizes of wheat (Ø2 vs 5mm) and two feed presentations (mash vs pelleted). In the third experiment (EXP.07) there were three treatments, a mash wheat-based diet (Control, T1), T1+ whole wheat (ww) at 7.5% from 0-21d and 15% from 21 to 42d (T2), and T2+5% of OH. The fourth experiment (EXP.08) compromised six wheat-based treatments factorially arranged, with two feed forms (mash vs pellet), and three levels of ww: zero, 7.5% from 0-21d and 15% from 21 to 42d, and 15% from 0-21d and 30% from 21 to 42d. All the experiments were performed by IMASDE AGROALIMENTARIA, S.L. and were done in vivo through challenge trials, in which all the animals were infected at 14d with C. jejuny ST-45. After inoculation, Campylobacter jejuny was analyzed in the ceca of animals at 21, 35 or 42d of age, reflecting the commercial practice in different countries across the EU. Experimental feeds were given to animals along the whole experimental period. Campylobacter counts were analyzed by ISO/TS 10272-2:2006 from direct sampling from the ceca of animals. In EXP.03 EXP.06 and EXP.08 several intestinal physiology measures were recorded: the weight of gastrointestinal tract, empty body, proventriculus (whole or empty), gizzard (whole or empty), and caeca; and the pH at proventriculus, gizzard and caeca.
In the first experiment (EXP.03) corn-based diets numerically showed less contamination, but neither cereal type nor oat hulls inclusion significantly decreased the level of contamination.In the second experiment (EXP.06) there were no significant differences in C. jejuny contamination between experimental group. Birds fed pelleted diets showed higher feed consumption and growth. In the third experiment (EXP.07) the inclusion of ww (7.5% between 0-21d, and 15% from 21 to 42d) and 5% of oat hulls significantly decreased the C. jejuny contamination at 42d with respect to a mash diet or a mash diet plus ww (8.10 vs 9.48 and 9.00 log10UFC/g, respectively, P<0.05). In the last experiment (EXP.08) neither the feed form (mash vs pellet) nor ww inclusion affected the Campylobacter level at the ceca of animals.
The intestinal physiology was clearly affected by treatments, with bigger gizzards in animals fed with OH, ww or higher particle size. However, there was no clear relationship with this physiological changes and the level of C. jejuny counts at ceca level. It is concluded that despite of the clear physiological changes derived of the feed form (mash or pellets), particle size, type of cereal, and OH or ww inclusion, no clear reductions in C. jejuny populations in the ceca were observed, although the inclusion of ww and OH to a mash diet decreased significantly the contamination at 42d in EXP.07.
Therefore, the objectives of the WP2 were achieved, since one effective combination of feed form was obtained: mash diets in overall tended to show lower Campylobacter counts, and the addition of ww and OH to mash diets also decreased the Campylobacter cecal population.
WP3: Interactions between products and feed presentation against Campylobacter. Synergies.
In the WP3 “Interactions between products and feed presentation against Campylobacter. Synergies”. the experiments were done with the best combination of products as a result of WP1, combined with the best Functional diet as a result of WP2. The products tested in this WP were: a combination of monoglycerides of medium chain fatty acids and organic acids (Campylostat, MGOA); three probiotics, two based on Bacillus subtilis: C-3102 DSM 15544 [Calsporin®, CALS] and DSM 17299 [GalliPro®, GAL], and a multispecies probiotic [PoultryStar®, PS]; and a yeast extract (XPC). The Functional diet consisted in the addition of 5% oat hulls [OH] and 7.5/12/15% Whole Wheat [WW].
A total of 7 experiments (6 in IMASDE AGROALIMENTARIA S.L. –IMASDE- and 1 in ANSES) were done. Two of six experiments in IMASDE came from repetitions for several causes, and this causes a delay of 4 months in the deliverable. In the first experiment (EXP. 10) there were four treatments in a factorial design with the addition of MGOA (1.5% form 0-10d and 2.5% thereafter), CALS (0.01%), or a combination of both. All diets were Functional. Due to unknown reasons, animals were not infected neither at 21 nor at 35d after inoculation at 14d, and they were re-inoculated at 39d. At 42d, animals fed MGOA either alone or in combination with CALS, decreased the number of infected animals to middle (P<0.05). However, this trial was repeated because the first inoculation process was unsuccessful. In the second experiment (EXP.11) there were four treatments factorially arranged based on two types of diet: a Control diet (C) and a functional (F) diet (with 7.5 12 and 15% of whole wheat form 1-10, 10-21 and 21-42d, respectively); with or without the addition of MGOA (1.5% from 1-10d and 2.5% thereafter) + CALS (0.01%). At 35d, feed supplemented with MGOA+CALS was able to reduce the colonization of Campylobacter both in control and functional diets (6.573 3.705 6.464 4.915 log10 CFU/g for Control, Control+MGOA+CALS, Functional and Functional+MGOA+CALS, respectively, P=0.036). This reduction was mainly because of the number of not infected birds in animals fed the diets supplemented with MGOA and CALS (14.30 71.40 7.10 and 35.70% of not infected birds for Control, Control+MGOA+CALS, Functional and Functional+MGOA+CALS, respectively, P=0.001). However, this effect of MGOA was lost at 42d of age (7.444 7.188 6.882 7.082 log10 CFU/g for Control, Control+MGOA+CALS, Functional and Functional+MGOA+CALS, respectively, P=0.351). In this experiment an additional treatment wqas included to evaluate the product GAL. The combination of MGOA +GAL showed the same effect (or even better) that MGOA+CALS at 21 and 35d, but its effect was also lost at 42d. In the third experiment (EXP.12) there were five treatments depending on the time of administration of the combination of MGOA (1.5% from 0-10d and 2.5% thereafter) + CALS (0.01%): a Control diet (C), and C plus MGOA+CALS in four periods: <21d, >21d, >10d, 0-42d for treatments 2 to 5, respectively. In this experiment the environmental conditions were very poor and the trial was repeated (EXP.15). In the fourth experiment (EXP.13) there were four treatments factorially arranged: T1, a Control diet (C); T2, C plus MGOA at 0.5% from 0-10d and 2.5% thereafter; T3, C plus CALS at 0.01%, and T4, C plus the addition of MGOA (1.5% from 0-10d and 2.5% thereafter)+CALS (0.01%). At 21d of age, broilers fed MGOA (alone or with CALS) showed less infection than broilers fed control diet or the control diet supplemented with CALS (4.463 2.374 4.656 2.266 log10 CFU/g for T1 to T4, respectively, P<0.001). Those differences came from the proportion of negative animals (43.8 93.8 37.5 and 93.8% for T1 to T4, respectively, P<0.001). At 35d of age (21d after challenge), broilers fed MGOA+CALS showed less infection than broilers fed the rest of treatments (6.816 6.557 6.394 4.451 log10 CFU/g for T1 to T4, respectively, P=0.024). Those differences came from the proportion of negative animals (6.30 6.30 12.50 43.80% for T1 to T4, respectively, P=0.012). Finally, at 42d, broilers fed MGOA+CALS showed less infection than control animals, showing the rest of treatments an intermediate Campylobacter population (8.514 7.299 8.276 and 6.887 log10 CFU/g for T1 to T4, respectively, P=0.001).
In the fifth experiment (EXP.14) there were four treatments depending on the MGOA dose: T1, a Control diet (C); T2, C plus MGOA at 0.5% from 0-10d and 1% thereafter and CALS at 0.01%; T3, C plus MGOA at 0.5% from 0-10d and 2% thereafter and CALS at 0.01%; T4, C plus MGOA at 0.5% from 0-10d and 3% thereafter and CALS at 0.01%. At 21d of age, the level of infection was very poor and there were not effect of treatment on Campylobacter. At 35d of age, birds fed the diets with the higher levels of MGOA from 10 to 42 d (2 or 3%) showed a clear reduction of Campylobacter infection (6.450 5.678 3.780 3.670 log10 CFU/g for T1 to T4, respectively, P=0.009). Those differences came from the proportion of negative animals (12.5 25.0 62.5 and 68.8% for T1 to T4, respectively, P=0.002). At 42d of age, again birds fed the diets with the higher levels of MGOA from 10 to 42 d (2 or 3%) showed a clear reduction of Campylobacter infection (6.441 5.741 3.979 and 4.156 log10 CFU/g for T1 to T4, respectively, P=0.009) and again, those differences came from the proportion of negative animals (7.1 25.0 62.5 56.3% for T1 to T4, respectively, P=0.005). In the sixth experiment (EXP.15) there were four treatments depending on the time of administration of the combination of MGOA (0.5% from 0-10d and 2.5% thereafter)+CALS (0.01%): a Control diet (C), and C plus MGOA + CALS in four periods: 0-42d, <21d, >21d, for treatments 2 to 4, respectively. At 21d of age, birds fed diets containing MGOA and CALS (T2 and T3) showed less contamination than the animals fed control diet (T1 and T4) (7.852 6.596 6.799 and 7.214 log10 CFU/g for T1 to T4, respectively, P=0.026). However, surprisingly this effect was lost at 35 and 42d.
It can be concluded that the combination of at least 2% of MGOA plus a probiotic based on B. subtilis (either CALS or GAL) administrated during the whole fattening period decrease the Campylobacter populations at 35 and 42d, but for unknown reasons, this effect is inconsistent. The next step will be testing this combination of products in field conditions with natural infected animals.
WP4. Application of different nutritional strategies against Campylobacter in experimental farm and field trials.
In the WP4 “Application of different nutritional strategies against Campylobacter in experimental farm and field trials”, the experiments were done with the best combination of products as a result of WP1 and WP3, since the feed form tested in WP2 or the combination of the most effective feed form and additives did not shown any synergistic effect. The products or combinations tested were: i) a combination of monoglycerides of medium chain fatty acids and organic acids (Campylostat, MGOA); ii) two probiotics based on Bacillus subtilis: C-3102 DSM 15544 [Calsporin®, CALS] and DSM 17299 [GalliPro®, GAL]; and iii) a yeast extract (XPCTM). There were three tasks: T4.1. Effect of different strategies against Campylobacter on performance parameters and level of infection of broilers chickens in experimental farm; T4.2. Effect of different strategies against Campylobacter on performance parameters and level of infection of broilers chickens in commercial farms; and T4.3. Effect of different strategies against Campylobacter on performance parameters and level of infection of turkeys in commercial farms.
In Task 4.1 a total of five experiments were done (EXP.16 to EXP.20) in the experimental facilities (two different farms) of IMASDE AGROALIMENTARIA S.L. –IMASDE- and one in ANSES. Those experiments differ with the field conditions in the experimental unit: in the experimental farm the experimental unit is a box within a single barn (all treatments together), and in the field trials the experimental unit is the barn (treatments separated in different barns). In EXP.16 the experimental design was a completely randomized design with 6 different experimental treatments: T1, Control (C); T2, T3 and T4, C + MGOA at different dosages + CALS; T5, C + MGOA+ GAL and T6, C + XPC (yeast extract). Each treatment was applied to 12 pens of 20 birds. The natural Campylobacter infection only appeared in some boxes at 35d, and was forced to the rest through the dissemination of litter, but the additives supplementation had not effect at 41d (6.68 6.92 6.84 6.80 6.23 and 6.64 log10 cfu/g for T1 to T6, respectively, P=0.306). In EXP.17 the experimental design was a completely randomized design with 6 different experimental treatments: T1, Control (C); T2, T3 and T4, C + MGOA at different dosages + CALS; T5, T6 and T7, C + MGOA at different dosages + GAL and T8, C + XPC. Each treatment was applied to 12 pens of 20 birds. Unexpectedly, all analyzed broiler caeca at 35 and 42 days of age were Campylobacter spp. free. In EXP.18 the experimental design was a completely randomized design with 3 different experimental treatments: T1, Control (C); T2, C + MGOA + CALS, and T3, C + [(MGOA + CALS) only 0-10d] + XPC (1-42d). Each treatment was applied to 8 pens of 22 birds. Although the hygienic barriers were very low and the trial was done in summer time, the natural Campylobacter infection was not implemented (additionally to the caeca samples, a fecal swab of all the animals was taken and were all negative). In EXP.19 the experimental design was a completely randomized design with 4 different experimental treatments (supplied by drinking water at 46-47d/48-49d): T1, Control (C); T2, C + 0.1/0.2% of MGOA; T3, C + 0.3/0.6% MGOA and T4, C + 0.5/1.0% MGOA. Each treatment was applied to 2 pens of 22 birds. The MGOA supplementation decreased the water consumption. Campylobacter infection was implemented, but water treatment did not show any effect on Campylobacter counts (9.04 9.08 9.15 and 9.27 log10 cfu/g for T1 to T4, respectively, P=0.756). In EXP.20 the experimental design was a completely randomized design with 3 different experimental treatments: T1, Control (C); T2, C + MGOA (1.5%) + CALS (0.01%), and T3, C + CALS (0.1% all periods) + MGOA (3%, only in the finisher diet). Each treatment was applied to 8 pens of 22 birds. Although the infection was detected in only nine replicates at 35d control (T1: 2.63 3.61 4.17 and 6.95; T2: 2.75 and 3.16; T3: 2.43 3.30 and 4.79 log10 cfu/g), when the analysis was repeated at 42 days of age (78 samples in total), all the samples except 3, were Campylobacter spp. free (T2: 0.47 and 3.31 T3: 3.29 log10 cfu/g).
Unfortunately, although we detected repeatedly Campylobacter in the two experimental farms previously to the project, the natural infection was not achieved in most of the cases. Some factor related with the structural disposition of the boxes could be the cause of the lack of infection.
In the first trial of ANSES, the additives increased the Campylobacter population both at 35 and 42d. On the contrary in the second trial, the additives avoid the contamination in most of the birds, but unfortunately the effect was lost either at 35 or 42d.
In Task 4.2 the trials were carried out in Spain (three different farms with twin buildings, one Control and the other Treated with MGOA+CALS), and Hungary (one single farm with three pairs of twin buildings with the same design). In Spain, previously to the feeding trial, a survey was carried out between 23 EXPLOTACIONES AVÍCOLAS JOSÉ LUIS REDONDO S.A. (REDONDO) farms at 42/29d. Most farms (21; 91.3%) were positive with more than 107 cfu/g. In the first farm, broilers were Campylobacter free until the last week, in which they reached 7log10 cfu/g, but no differences among treatments were found. However, carcasses form birds fed the supplemented diets showed more contamination than the controls birds (3.37 vs 3.92 log10 cfu/g for control and treated animals, respectively, P=0.002). In the second farm, the infection was very high at 26d, and it naturally decreased until 47d. Unexpectedly, the decrease in the control barn was higher than in the treated one, so the final infection was higher for treated animals than for control animals (5.31 vs 6.46 log10 cfu/g for control and treated animals, respectively, P=0.009). However, the treated animals presented lower contamination in the neck skin (1.67 vs 0.58 log10 cfu/g for control and treated animals, respectively, P=0.020). In the third farm, both barns were positive at 32d, and the infection was maintained high until the end of the fattening period without any difference between treatments, neither in live animals nor in the neck skin. So, from the Spanish trials we can conclude that the infection patron is completely different among farms, and that the additive addition had not a positive effect on Campylobacter infection neither in live animals nor in the carcasses.
In the Hungarian trials, the infection was only achieved at the end of the trial, and unexpectedly, diet supplementation with additives significantly increased the Campylobacter infection, since Control animals clearly showed lower contamination (4.433 vs 8.912 log10 cfu/g for Control and Treated animals, respectively, P<0.001).
In Task 4.3 two strategies were followed: MGOA+CALS in the feed and the MGOA in the water during the withdrawal period. In females (12-13 weeks of age), the addition of MGOA+CALS in feed surprisingly increased the Campylobacter population in two of the three farms tested, but in males (18/19 weeks) the addition of the additives did not modify the Campylobacter cecal population. On the other hand, the addition of MGOA through the water in females decreased the Campylobacter population in one farm but no in other, and in males it increased the Campylobacter population in one farm but no in other.
It can be concluded that i) for unknown reasons, the natural infection was only implanted in part of the trials, which could not be explained with the actual state of art on the Campylobacter epidemiology, ii) the combination of at least 1.5% of MGOA plus a probiotic based on B. subtilis (either CALS or GAL) or the yeast extract XPC administrated during the whole fattening period, did not decrease the Campylobacter populations at 35 and 42d under natural conditions.
WP5 Development of a novel vaccine against Campylobacter based on reserve vaccinology
In order to evaluate the protective potential of the selected antigens, we had to develop an experimental vaccine model. Since we would like to test several antigens, the way of producing the vaccine for each potential vaccine antigen should be as easy as possible. We have a huge experience in DNA vaccination at ANSES. Therefore we decided to start with DNA vaccination, which is an easy way to produce vaccine prototypes. Flagellin A was used as a vaccine antigen model since it was described in the literature to induce some protective immune response in chickens.
In order to analyze the immune responses induced by the vaccination and infection, we developed and optimized ELISAs to quantify the levels of systemic antibodies in the blood (IgY type antibodies) and mucosal antibodies in the bile (IgA-type antibodies).
In a first assay we evaluated a vaccine protocol based only on DNA vaccination. In total 155 Ross broilers divided into 5 groups were used. 2 adjuvants were compared: CpG-ODN and interleukin 2. These adjuvants were selected because they were massively used in DNA vaccination in chickens. The DNA vaccine was injected through the sub-cutaneous route, a route also largely used in chicken vaccination. A prime injection was performed in one-day old chicks. A boost injection was performed in 10-days old chicks. The challenge with Campylobacter jejuni 81-176 strain was performed on day 21. Cecal loads of Campylobacter were measured on days 28 and 42. This DNA vaccination protocol did not induce detectable levels of antibodies at any time-point of the assay. No reduction of Campylobacter loads were observed at any of the time-points tested.
Nevertheless this assay was useful to design the next assay. First we observed a negative effect of interleukin 2 on Campylobacter load; only the CpG-ODN will be used as DNA vaccine adjuvant afterward. Second, the route of injection will be modified (intramuscular). Third, the vaccine regimen will be strengthened by including a boost-injection with the protein form of the vaccine. Fourth, we observed massive presence of maternal derived antibodies in the chickens; this should be taken into account for the next assays.
In the second assay, the vaccine was injected via the intramuscular route. We compared the DNA-DNA regimen to the DNA-protein regimen. CpG-ODN was used as adjuvant for the DNA vaccine and montanide as adjuvant for the protein vaccine. We included also groups of specific pathogen free (SPF) Leghorn hens which are from a Campylobacter-free breeding in order to study the impact of the maternal derived antibodies on the vaccine efficacy. In total 104 chickens divided in 6 groups were used. The prime and the boost injections were performed on 1-day and 7-day old chicks, respectively. The challenge with the same strain of Campylobacter as assay 1 was performed on day 15. For the Ross broilers, the DNA / protein regimen induced the production of specific antibodies against Campylobacter, but no reduction of the Campylobacter load on day 42. The DNA/DNA regimen was inefficient for all the parameters tested. For the SPF Leghorn hens, the DNA / protein vaccine regimen did not only induce the production of specific antibodies, but also permitted to eliminate entirely Campylobacter in the caecum. These results are showing that the DNA/protein vaccine regimen is highly powerful to induce a protective immune response, but the SPF Leghorn hens are not the targeted chickens. The vaccine has to be efficient in Ross broilers.
Therefore we performed a third assay with only Ross broilers where the prime-injection of the vaccine was done on day 5 (less maternal antibodies are present at this time point) and the boost on day 12. We also compared the DNA/protein regimen to the protein/protein one. In total, 128 chickens divided into 6 groups were used. None of these regimens induced the reduction of Campylobacter loads. The levels of antibodies were higher in the DNA/protein boost regimen performed on days 5 and 12.
In an additional assay we performed the DNA prime injection of day 14 and the boost injection on day 21. Unfortunately the challenge was inefficient; therefore we could not measure the protective effect of this vaccine regimen. Nevertheless, the level of antibodies against Campylobacter was equivalent on day 42 to the ones of the chickens injected on days 5 and 12.
Therefore the vaccine regimen that will be used in the assays to evaluate the protective potentials of the potential vaccine antigens will be the prime-boost regimen injected on days 5 and 12. The challenge with Campylobacter is performed on day 19.
The in vitro test to evaluate if the proteins are recognized or not by antibodies against Campylobacter has been developed by using the flagellin A as a protein model and a commercial polyclonal vaccine against Campylobacter jejuni.
We applied this method to the 12 potential vaccine antigens identified in task 5.1 (D5.1). Unfortunately, no staining was observed when the cells were transfected with all these antigens. Afterward, we produced a rabbit polyclonal antibody specifically directed against Campylobacter jejuni 81-176, the strain used in the project. Also no staining was obtained with any of the newly identified potential vaccine antigens. We do not know at this stage why this test was not successful for the newly identified potential vaccine antigens.
In conclusion, we were able under certain conditions to stain cells transfected with plasmids encoding Flagellin A. But this was not the case for other potential vaccine antigens. The in vitro test to select the antigens for the vaccine efficacy tests was therefore not useful. But this was not a barrier for the rest of the project since we used the alternative way to select the proteins to be tested for their protective potentials: a part of the proteins that have the strongest probability to be an antigenic protein were selected for the in vivo assessment.
We added some additional criteria for the selection. In some cases (YP_001001371.1 YP_001000204.1 YP_001000248.1) the proteins were not selected even if they were highly immunogenic because we found recently that these proteins were not common to many Campylobacter strains. These results were obtained now because there are now much more sequences of genomes of Campylobacter available. It is in fact important for the vaccine development that the vaccine proteins are shared among different Campylobacter strains in order to achieve a protection against several strains. In other cases (YP_001000153.1 YP_001000945.1) we were not able to produce all the components of the vaccine (protein form of the vaccine). In total 6 proteins were selected to test their protective potentials.
Two assays were performed for this part. We based the design of the assays on the results of task 5.3: DNA-prime / protein boost regimen injected on days 5 and 12, challenge with Campylobacter on day 19. Furthermore, we focused the determination of the Campylobacter loads on day 42, the slaughter date. In fact, the main goal of the vaccine is to get a reduction of Campylobacter load at this time point. The assays were designed to analyze at least 15 individual caeca from each batch. The chickens used are Ross broilers coming from a conventional hatchery.
The protocols of the in vivo experiments were designed in collaboration with Eugenia Puentos Colorado and Alberto Parra of CZV.
The first trial was performed with 3 potential vaccine antigens: YP_001000562.1 YP_001001115.1 and YP_999769.1. The vaccine potentials of these candidates were compared to a group injected with placebos (empty plasmid / PBS). 4 groups of chickens were used here. The vaccination protocol is initially the one of deliverable 5.2. The vaccine was injected through the intra-muscular route. On day 5, the antigens were injected in their DNA forms (300 µg); 50 µg CpG-ODN were added as adjuvants. On day 12, the boost was performed with the protein forms of the antigens (100 µg); montanide was used as adjuvant. On day 19, the chickens were challenged with the C. jejuni 81-176 strain (which was able to colonize the chickens in the previous assays – see deliverable 5.2). Unfortunately we observed no Campylobacter colonization of caecas 4 days later. Therefore we decided to modify the protocol in performing a second challenge with the same strain on day 28. Again, no colonization was observed. We finally decided to perform a challenge with the C. jejuni C97ANSES640 strain on day 42 and Campylobacter colonization was obtained. We stopped the assay on day 49 and analyzed the levels of Campylobacter colonization in each chicken. The induction of specific antibodies in the blood was analyzed weekly throughout the assay; mucosal specific antibodies were researched in the bile on day 49.
Our data showed that 2 of the 3 antigens of Campylobacter jejuni tested were able to induce the production of specific antibodies against Campylobacter (YP_001000562.1 - flagellin family protein and YP_001001115.1 - FlgK). No modifications of bile IgA were observed on day 49. Only YP_001001115.1 – FlgK induced under these particular experimental conditions used here a moderate but significant reduction of Campylobacter load in the caecum 1.4 log10). But the results should be interpreted with caution because the experimental conditions were particular following the failure of colonization observed after the first challenge. In total 3 challenges with 2 different Campylobacter strains were performed.
A second assay was performed with 6 vaccine antigens tested individually or mixed. Furthermore, we used as a challenge strain C. jejuni C97ANSES640 since it was able to induce high colonization levels in chickens in assay 1 (third challenge). Furthermore, this strain is representative of the strains isolated in chickens. So the modification of the strain does not modify the goal of the project.
Five out of the 6 antigens induced the production of antibodies against Campylobacter. For the last antigen (YP_999769.1 (FlgE-1)), we do not know now if significant levels of antibodies were produced because the ELISA used to measure the production of antibodies was not optimized for antibodies against this particular protein. More importantly, 4 out of 6 antigens induced significant reductions of Campylobacter loads in the caecum. The pool of the 6 antigens did not induce any reduction of Campylobacter loads. This may be due to the fact that low quantities of each antigen were used.
Two of these antigens were of particular efficacy and interest: YP_001000437.1 induced a reduction of Campylobacter loads of about 3.6 log10 and YP_999817.1 induced the highest reduction of Campylobacter loads of about 4.2 log10. In fact, a reduction of at least 3 log10 of Campylobacter in chickens may decrease by more than 90 % the risks of campylobacteriosis in humans (Romero-Barrios P. et al, Food Control 2013), indicating that the results we obtained are of main importance for future vaccine development to control Campylobacter in chickens.
Due to the delays of the beginning of the project, we were not able to perform a third assay to confirm the results we obtained. This assay has been postponed after the end of Campybro. It is expected that the assay starts the 16th of September 2016. Consequently the PhD student will finish her PhD training after the end of the project (probably during the first quarter of 2017).
WP6. Evaluation of the developed nutritional strategies in different geographical situations.
In the WP6 “Evaluation of the developed nutritional strategies in different geographical situations”, the experiments were done in practical farm conditions in the four countries that participate into the project: France, Hungary, Spain and The Netherlands. The products or combinations tested were: i) a combination of monoglycerides of medium chain fatty acids and organic acids (Campylostat, MGOA); and ii) a probiotic based on Bacillus subtilis: C-3102 DSM 15544 (Calsporin®, CALS). There was only a task: T6.1. Evaluation of developed nutritional strategies in South, Central, and East European conditions. In France, the trial was done in two farms with 2 twin buildings each (26,400 broilers per building, 96,900 in total) located at Sillé-le-Guillaume. The trial started the 23/06/2016 and lasted the 29th of July. The experimental feeds were done in the company LDC at Sillé-le-Guillaume, and there were two treatments: Control (C), and C + MGOA (0.5% 0-10d and 1.5% thereafter) + CALS (0.01% all the period). In The Netherlands, the trial was done in one farm with 3 twin buildings (63,400, 63,400 and 48,060 broilers per building, 174,860 in total) located at Grootschermer. The experimental feeds were done in the company Agrifirm at Mepple. The trial started the 01/07/2016 and lasted the 05/08/2016 and there were two treatments: Control (C), and C + MGOA (0.5% 0-7d and 1.5% thereafter) + CALS (0.01% all the period). In Spain there were three trials. The first trial was done in Cosuenda (Zaragoza) in one farm with twin buildings (4,500 birds each, 9,000 broilers in total). The experimental feed was done in Nanta (Casetas, Zaragoza). The trial started the 20/06/2016 and lasted 01/08/2016 and there were two treatments: Control, and C + MGOA (0.5% 0-10d and 1.5% thereafter) + CALS (0.01% all the period). The second trial was done in Fuente Álamo (Murcia) in one farm with three twin buildings (19,000 birds each, 57,000 broilers in total). The experimental feed was done in Avilesa (Torre Pacheco, Murcia). The trial started the 16/05/2016 and lasted the 04/07/2016 and there were three treatments: Control, C + MGOA (2.5% from 33 to 50d) + CALS (0.01% from 33 to 50d), C+ MGOA in drinking water at 0.06% from 43 to 50d. The third trial was done in Lominchar (Toledo) in one farm with two twin buildings (18,500 birds each, 37,000 broilers in total). The experimental feed was done in Explotaciones Avícolas José Luis Redondo S.A. (Pantoja, Toledo). The trial started the 17/05/2016 and lasted the 01/07/2016 and there were two treatments: Control, and C + MGOA (0.5% 0-10d and 1.5% thereafter) + CALS (0.01% all the period). In Hungary, the trial was done in one farm (Varjasi Farm Kft.) located at Újkígyós with 6 twin buildings (18,552 broilers per building, 110,760 in total) and there were two treatments: Control, and C + MGOA (0.5% 0-7d and 1.5% thereafter) + CALS (0.01% all the period). The experimental feeds were done in the company Agrifirm at Győr. The trial started the 23/06/2016 and lasted the 02/08/2016 and there were two treatments: Control, and C + MGOA (0.5% 0-10d and 1.5% thereafter) + CALS (0.01% all the period).
In France, in one of the farms the additives addition decreased the cecal Campylobacter counts (8.20 vs 7.18 Log10 cfu/g, P<0.001). In the other farm, the samples from control animals were incorrectly stored and showed difficulties in the analysis.
In The Netherlands, although the trial was done in the summer, unfortunately the natural infection started in one of the barns just in the final part of the trial, and the efficacy of the additives could not be tested.
In Spain, in the first trial the addition of MGOA (0.5% from 0 to 7d and 1.5% thereafter) + CALS (0.01%) in the feed decreased the Campylobacter infection at 24d (6.859 vs 6.006 Log10cfu/g for CONTROL and TREATED, respectively; P=0.019). However, the opposite effect was found at 35d, when birds fed the TREATED diet showed more Campylobacter counts than CONTROL birds (6.935 vs 7.387 Log10 cfu/g for CONTROL and TREATED, respectively; P=0.046). At 42d, there were no differences between treatments, although TREATED animals tended to show more Campylobacter counts than the CONTROL birds (6.502 vs 6.827 Log10cfu/g for CONTROL and TREATED, respectively; P=0.093). In the second trial, the MGOA application in water significantly decreased the Campylobacter contamination (8.25a 8.28a 8.14a 7.18b Log10 cfu/g for CONTROL, TREATED feed before thinning (37d), TREATED feed (50d), TREATED water, respectively, P=0.04). In the third trial, treatment decreased significantly the Campylobacter infection at 32d (7.25 vs 6.04 Log10 cfu/g for CONTROL and TREATED, respectively, P=0.046) and showed numerically lower values at 39 (6.21 vs 5.45 Log10 cfu/g for CONTROL and TREATED, respectively, P=0.131) and 42d (6.69 vs 5.71 Log10 cfu/g for CONTROL and TREATED, respectively, P=0.152).
In Hungary, There were not differences between treatments neither at 35d nor at the end of the experiment (7.708 vs 7.500 and 8.326 vs 8.805 Log10 cfu/g for CONTROL and TREATED, P=0.383 and 0.133 at 35 and 40d, respectively).
The addition of MGOA and CALS had a limited effect on Campylobacter infection and performance in all the countries studied. In France, in one of the farms the additives addition decreased the cecal Campylobacter counts (8.20 vs 7.18 Log10 cfu/g, P<0.001). In the other farm, the samples from control animals were incorrectly stored and showed difficulties in the analysis. In the Netherlands, natural infection did not occur during the experiment. In Spain, treatment delays the infection in two of the experiments, and decreases the final figures when MGOA was administrated in the water the last week before slaughtering in the other experiment. In Hungary, any effect was detected. Therefore, the solution developed by the project CAMPYBRO and tested in filed conditions in four countries in Europe, has a limited effect on campylobacter infection. Further investigations are needed to understand much better the epidemiology of Campylobacter for develop new strategies to decrease the contamination in broiler chickens.
Potential Impact:
The objective of CAMPYBRO project, under the 7th FRAME PROGRAM of UE (Capacities- Research for SME Associations FP7-SME GA 605835) is to develop practical strategies for EU poultry producers to decrease the population of Campylobacter jejuni in poultry, as a methodology to decrease the contamination of poultry carcasses. The expected results are practical strategies based on nutrition and vaccination which decrease the level of infection of Campylobacter jejuny in poultry batches.
The final conclusions are: i) the mixture of a combination of organic acids and monoglycerides of medium chain fatty acids [MGOA] and a B. subtilis-based probiotic [CALS]) decreased the Campylobacter cecal population in challenge trials, ii) the effect of this combination on Campylobacter reduction was not homogenous, iii) the interaction between in vitro trials and in vivo trials was avoided, but it look like there is an interaction between challenge trials and natural contamination infection trials, iv) much more knowledge is needed in Campylobacter epidemiology, since the natural infection did not follow classical patrons of infection, v) in the demonstration trials variable results were found, with a significant effect in some cases, a delay of infection in others and no effect in other cases, vi) a potential vaccine could be developed based on the antigens discovered.
The potential impact of the projects results are: i) Improved food safety and public health: the reduction of Campylobacter population in broilers may decrease the percentage of carcasses infected and the level of infection, which will cause an improvement of food safety, since campylobacteriosis is the most important zoonosis in the UE. The expected impact for a 1-2 Log10UFC/g is a reduction of 50-90%, and 90% for a 3 Log10UFC/g reduction. The expected costs are related to direct illness effects (laboratory diagnosis, consultations, medical cares, hospitalization, etc.) and indirect costs (work inefficacy, no-working days, etc.); ii) Meet future regulatory requirements: the European Commission launched a Proposal Regulation amending Regulation (EC) No 2073/2005, as regards Campylobacter in broiler carcasses. This proposal will limit the percentage of samples (sampling: once per week, 15 birds at random form one batch, pooled in 5 samples 3 birds) with more than 103 cfu Campylobacter/g of neck skin to 20% in 2017, 14% in 2018 and 2019, and 10% after 2020. These limits will be unachievable for many producers in most of the countries, especially in the summer. For that reason, the results of the project are also key for the EU Commission in order to know if it is feasible to decrease the Campylobacter infection in vivo through nutrition, vaccination or both; iii) To improve the competitiveness of EU poultry industry. Producing poultry meat with lower levels of Campylobacter will improve the sector competitiveness, with higher possibilities of exporting products, and avoiding possible future food safety barriers, iv) Improvement of health, welfare and productivity of chickens, and health and welfare of farmers, this project will have a significant effect on the quality of life of these workers due to a considerable lower risk of infection. v) Decrease in antimicrobial resistance: A decrease in Campylobacter population could decrease the risk of transmission of antimicrobial resistance both in broilers and humans (because of less cases of Campylobacteriosis treated with antibiotics).
However, the solution developed by the project CAMPYBRO and tested in filed conditions in four countries in Europe, has a limited and very variable effect on Campylobacter infection. Some relevant advances have been achieved on the vaccination area for the future development of a vaccine. Further investigations are needed to understand much better the epidemiology of Campylobacter for develop new strategies to decrease the contamination in broiler chickens.
Dissemination activities
The main communication activities within the first and second reporting period are: a webpage (www.campybro.eu) 8 newsletters, 5 scientific peer reviewed articles, 32 scientific communications, 6 invited conferences, 8 articles in the popular press, 23 press releases, a workshop per participant country (Spain, The Netherlands, Hungary and France), 10 presentation of the results to the industry of the participant countries and a scientific workshop were published regarding the Campylobacter issue and the project.
1. Website
The website www.campybro.eu has been created and updated with the last news about the project and the Campylobacter issue in poultry in general.
2. Newsletters of the project
There have been published 8 newsletters (3 per year) during the project.
3. Peer reviewed scientific publications (published)
It has been published 5 papers. Full articles appear in the Annex 2. The impact factor (2015) of the publications, according to research gate (www.researchgate.net) was: i) Poultry Science: 2.14 ii) Journal of Applied Microbiology: 2.38 iii) Clinical and Developmental Immunology: 3.87 iv) plos-one: 3.54. In the publications it is worth to be mentioned that the main authors were the researchers of the RTDs: ANSES (M. Guyard-Nicodème, D. Dory, M. Chemaly, M. Meunier, M., A. Keita, S. Quesne, M. Amelot, T. Poezevara, B. Le Berre, E. Hirchaud) and IMASDE (M. I. Gracia, C. Millán, J. Sánchez, Ó. Casabuena, P. Medel), but also other researchers from most of the partners: CIDEF (Y. Carre), BTT (A. Csorbai), FIA (J. Mayot), NEPLUVI (P. Vesseur), PROPOLLO (Á. Martín), CVZ (A. Parra). It is expected to send at least two more publications once the project will finish.
• Gracia, M. I., C. Millán, J. Sánchez, M. Guyard-Nicodème, J. Mayot, Y. Carre, A. Csorbai, M. Chemaly, P. Medel, 2016. Efficacy of feed additives against Campylobacter in live broilers during the entire rearing period: Part B. Poult. Sci. 95: 886-892. DOI: 10.3382/ps/pev346.
• Guyard-Nicodème, M., A. Keita, S. Quesne, M. Amelot, T. Poezevara, B. Le Berre, J. Sánchez, P. Vesseur, Á. Martín, P. Medel, M. Chemaly, 2016. Efficacy of feed additives against Campylobacter in live broilers during the entire rearing period. Poult. Sci. 95: 298-305. DOI: 10.3382/ps/pev303.
• Meunier, M., M. Guyard-Nicodème, D. Dory, M. Chemaly, 2016. Control Strategies against Campylobacter at the Poultry Production Level: Biosecurity Measures, Feed Additives and Vaccination. Journal of Applied Microbiology 120: 1139-1173. DOI: 10.1111/jam.12986.
• Meunier, M., M. Guyard-Nicodème , E. Hirchaud, A. Parra, M. Chemaly, D. Dory, 2016. Ientification of Novel Vaccine Candidates against Campylobacter through Reverse Vaccinology. Clinical and Developmental Immunology 2016 Article ID 5715790: 1-9. DOI: http://dx.doi.org/10.1155/2016/5715790
• Gracia, M. I., J. Sánchez, C. Millán, Ó. Casabuena, P. Vesseur, Á. Martín, F. J. García-Peña, P. Medel, 2016. Effect of Feed Form and Whole Grain Feeding on Gastrointestinal Weight and the Prevalence of Campylobacter jejuni in Broilers Orally Infected. PLoS ONE 11(8): e0160858. doi:10.1371/journal.pone.0160858.
4. Scientific publications (abstracts in Scientific Meetings or congress)
The results of the projects have been presented in the next scientific meetings covering the FIVE CONTINENTS:
• XIVth European POULTRY CONFERENCE. World's Poultry Science Association. 23-27 June, 2014, Stavanguer, Norway.
• 3rd International conference on Responsible use of Antibiotics in Animals. Amsterdam. 29th September-1st October, 2014. Amsterdam. The Netherlands.
• LI symposio científico de avicultura. 2-3 October, 2014. Alfara del Patriarca, Valencia, Spain.
• 11th Journées de la Recherche Avicole et palmipèdes a Foie Gras. 25-26 March 2015. Tours, France.
• European Symposium on Food Safety. 20-22 April 2015. Cardiff, Wales, UK.
• XXII European Symposium on the Quality of Poultry Meat » and the « XVI European Symposium on the Quality of Eggs and Egg Products. 10-13 May 2015. Nantes. France.
• Poultry Science Association 104th annual Meeting. July 27-30, 2015. Louisville, Kentucky, USA.
• 20th European Symposium on Poultry Nutrition. 24-27 August 2015. Prague, Chez Republic.
• 19th world Veterinary Poultry Association Congress. 7-11 September 2015. Cape Town, South Africa.
• 18th International Conference on Campylobacter, Helicobacter and related organisms. 1-5 November 2015. Rotoura, New Zealand.
• European Symposium on Food Safety. 11-13 may 2016. Athens, Greece.
• World Poultry Congress. 5-9 September 2016. Beijing, China.
All these publications are available on request to the Project Officer or Scientific evaluator.
A total of 32 communications were published in the Meetings listed above: The bibliographic references are:
• Millán, C., Casabuena, Ó., Porras, S., Sánchez, J., Gracia, M.I. 2014. Effect of feeding caproic and caprylic as acids or monoglycerides on Campylobacter counts in broilers. XIVth European POULTRY CONFERENCE. World's Poultry Science Associaction. Pp: 247
• Martín, Á., Mayot, J., den Hartog, M., Carre, Y., Molnar, G., Sánchez, F., Fernández, A., Tenk, M., Chemaly, M., Dory, D., Medel, P., 2014. CAMPYBRO PROJET: Strategies to fight the biggest zoonoses in the EU without antibiotics therapy. 3rd International conference on Responsible use of Antibiotics in Animals. Pp: 23
• Millán, C., Sánchez, J., Casabuena, O., Gracia, M.I. 2014. LI symposio científico de avicultura Efecto de la adición de un derivado de Allium sativum y de un probiótico sobre la población fecal de Campylobacter jenuni en broilers infectados experimentalmente. Asociación Española de Ciencia Avícola. Proceedings CD.
• Chemaly, M., Mayot, J., 2015. Les moyens de lute vis-à-vis de Campylobacter chez le poulet de chair: mythe ou realite?. 11th Journées de la Recherche Avicole et palmipèdes a Foie Gras. Tours, 25-26 March 2015.
• Guyard-Nicodème, M., Quesne, S., Poezevara, T., Le Berre, B., Amelot, M., Chemaly, M., 2015. A large scale study to test a wide variety of additives in broilers’ feed to decrease Campylobacter shedding. European Symposium on Food Safety. 20-22 April 2015. Cardiff, Wales, UK.
• Guyard-Nicodème, M., Houard, E., Poezevara, T., Chemaly, M., 2015. Combinations of broilers’ feed additives against Campylobacter. XXII European Symposium on the Quality of Poultry Meat » and the « XVI European Symposium on the Quality of Eggs and Egg Products. 10-13 May 2015. Nantes. France.
• Meunier, M., Guyard- Nicodème, M., Béven, V., Vigouroux, E., Chemaly, M., Dory, D., 2015. Optimization of a vaccination protocol against Campylobacter for poultry. XXII European Symposium on the Quality of Poultry Meat » and the « XVI European Symposium on the Quality of Eggs and Egg Products. 10-13 May 2015. Nantes. France.
• Chemaly M., 2015. Is this a fiction to tackle Campylobacter in the broiler food chain? XXII European Symposium on the Quality of Poultry Meat » and the « XVI European Symposium on the Quality of Eggs and Egg Products. 10-13 May 2015. Nantes. France.
• Gracia, M. I., O. Casabuena, F. Sánchez, J. Mayot, P. Medel, 2015. Effect of feeding a live yeast and a combination of organic acids and essential oils on Campylobacter colonization in broilers. Poult. Sci. 94 (E-Suppl. 1): 117.
• Sánchez, J., C. Millán, F. Sánchez, Y. Carre, P. Medel, 2015. Effect of type of cereal and oat hulls addition on cecal morphology and Campylobacter jejuni colonization of broilers orally infected. Poult. Sci. 94 (E-Suppl. 1): 119-120.
• Millán, C., A. Martín, M. G. Molnar, J. Sánchez, P. Medel, 2015. Effect of feed presentation and whole wheat addition on gastrointestinal morphology in orally infected broilers with C. jejuni. Poult. Sci. 94 (E-Suppl. 1): 82.
• Casabuena, O., V. Elgosi, M. Den Hartog, M.I. Gracia, P. Medel, 2015. Effect of particle size and feed presentation on gastrointestinal morphology in infected broilers with C. jejuni. Poult. Sci. 94 (E-Suppl. 1): 82.
• Casabuena, O., M. Chemaly, M. Den Hartog, P. Vesseur, M. I. Gracia. 2015. Effect of feeding different combinations of organic acids, mono-glycerides and probiotics on Campylobacter colonization in broilers. Pages 173-175 in Full Papers of the 20th European Symposium on Poultry Nutrition, Prague, Czech Republic.
• Gracia, M. I., F. J. García-Peña, M. Guyard, G. Le Pottier, M. Tenk. 2015. Effect of particle size and feed presentation (mash vs pellets) on cecal morphology and Campylobacter jejuni colonization of broilers orally infected. Pages 561-563 in Full Papers of the 20th European Symposium on Poultry Nutrition, Prague, Czech Republic.
• Millán, C., A. Martín, J. Mayot, J. Sánchez, M. I. Gracia. 2015. Effect of type of cereal and oat hulls addition on gastrointestinal morphology in broilers orally infected with Campylobacter jejuni. Pages 434-436 in Full Papers of the 20th European Symposium on Poultry Nutrition, Prague, Czech Republic.
• Sánchez, J., A. Parra, Y. Carre, A. Csorbai, M. I. Gracia. 2015. Effect of supplementing via drinking water a blend of mono-glycerides, alone or in combination with organic acids, on Campylobacter colonization in broilers. Pages 267-269 in Full Papers of the 20th European Symposium on Poultry Nutrition, Prague, Czech Republic.
• Millán, C., O. Casabuena, O. Benito, S. Porras, J. Sánchez, M. I. Gracia, 2015. Effect of feeding two different combinations of flavouring compounds on Campylobacter colonization in broilers. Proceedings of the 19th World Veterinary Poultry Association Congress, Cape Town, South Africa.
• Millán, C., O. Casabuena, I. Muñoz, S. Porras, J. Sánchez, M. I. Gracia, 2015. Effect of feeding two different combinations of essential oils on Campylobacter colonization in broilers. Proceedings of the 19th World Veterinary Poultry Association Congress, Cape Town, South Africa.
• Millán, C., O. Casabuena, M. Guyard, M. Tenk, P. Medel, 2015. Effect of feeding a combination of a yeast product and a probiotic, alone or in combination with a blend of mono-glycerides, on Campylobacter colonization in broilers. 18th International Workshop Campylobacter, Helicobacter & Related Organisms (CHRO) 1-5 November, 2015 Rotorua, New Zealand. O142. Pp: 113.
• Millán, C., P. Vesseur, E. Puentes, O. Casabuena, P. Medel, 2015. Effect of feed presentation (mash vs pellets) and whole wheat addition on cecal morphology and Campylobacter jejuni colonization of broilers orally infected. 18th International Workshop Campylobacter, Helicobacter & Related Organisms (CHRO) 1-5 November, 2015 Rotorua, New Zealand. O125. Pp: 102.
• Gracia, M.I. M.Chemally Á. Martín, A. Csorbai, P. Medel, 2015. Effect of oat hulls addition and whole wheat addition on cecal morphology and Campylobacter jejuni colonization of broilers orally infected. 18th International Workshop Campylobacter, Helicobacter & Related Organisms (CHRO) 1-5 November, 2015 Rotorua, New Zealand. P120. Pp: 178.
• Meunier, M., M. Guyard-Nicodème, E. Vigouroux, T. Poézëvara, V. Beven, S. Quesne, D. Dory, A. Parra, M. Chemaly, 2015. Sequential optimization of an avian vaccine protocol against Campylobacter. 18th International Workshop Campylobacter, Helicobacter & Related Organisms (CHRO) 1-5 November, 2015 Rotorua, New Zealand. O139. Pp: 111.
• Medel, 2016. Effect of feeding a combination of organic acids, mono-glycerides and a probiotic on Campylobacter colonization in broilers. European Symposium on Food Safety. 11-13 may 2016. Athens, Greece. P2-54. Pp 128.
• Millán, C., M. Chemaly, J. Mayot, P. Vesseur, M.I. Gracia, P. Medel, 2016. Interaction of a combination of organic acids and mono-glycerides and a probiotic on Campylobacter colonization in broilers. XXV World’s Poultry Congress. September 5-9, 2016. Beijing, China.
• Gracia, M.I. M. Guyard-Nicodème, A. Csorbai, M. Den Hartog, I. Tenk, P. Medel, 2016. Interaction of a combination of organic acids and mono-glycerides and a probiotic with a functional diet on Campylobacter colonization in broilers. XXV World’s Poultry Congress. September 5-9, 2016. Beijing, China.
• Casabuena, O., A. Martín, V. Elgosi, M. G. Molnar, M.I. Gracia, P. Medel, 2016. Effect of feeding a combination of organic acids and mono-glycerides at different dosages and a probiotic on Campylobacter colonization in broilers. XXV World’s Poultry Congress. September 5-9, 2016. Beijing, China.
• Sánchez, J., O. Casabuena, A. Parra, G. Le Pottier, F. Sánchez, P. Medel, 2016. Effect of supplementing a combination of organic acids and mono-glycerides and a probiotic during different times of administration on Campylobacter colonization in broilers. XXV World’s Poultry Congress. September 5-9, 2016. Beijing, China.
In addition, another 5 articles has been published as a part of the invited conference:
• Medel, P., Sánchez F., Rodríguez, E., Martín, Á., 2014. Campylobacter en avicultura de carne: situación actual y estrategias de reducción. Jornadas Profesionales de Avicultura. Sevilla, Spain. Pp: 74.
• Medel, P, F. Sánchez, A. Parra, Á. Martín, Y. Carré, J. Mayot, M. den Hartog, G. Molnar, M. Tenk, M. Guyard, 2015. Problemática actual del Campylobacter en la salud pública. Proccedings of Jornadas Profesionales de Avicultura 2016, Soria, Spain. Pp: 62-68
• Medel, P, F. Sánchez, A. Parra, Á. Martín, Y. Carré, J. Mayot, M. den Hartog, G. Endrodi, M. Tenk, M. Chemaly, 2015. Métodos de control de Campylobacter en la industria avícola. III Congreso Internacional de Seguridad alimentaria. Proceedings of the conference (only in e-format).
• Guyard-Nicòdeme, M., 2016. Why is Campylobacter the number one priority for the poultry production chain? International Association for Food Protection European Annual European meeting, Athens, Greece. Pp: 51
• Medel, P., 2016. Un update about the different state of the art methods to control campylobacter in broilers: the European project CAMPYBRO. International Association for Food Protection European Annual European meeting, Athens, Greece. Pp: 51
5. Articles published in the popular press
There were 6 publications, listed below:
• Martín, Á., Redondo, J.L. Fernández, A., Medel, P., 2014. Campylobacyter es el nuevo reto de Europa en avicultura de carne; 01/03/2014; Albéitar, 17: 10-11. Zaragoza, Spain
• Martín, Á., Redondo, J.L. Fernández, A., Medel, P., 2014. Campylobacter é o novo desafio da europa em avicultura de carne; 01/06/2014; Albéitar (Portuguese edition) 3 (Mai/Jun 2014):14-16
• Foldi, P., Csorbai, A., 2015.Campylobacter a baromfihúsban; 01/06/2014; BAROMFI ÁGAZAT 2014/2 JÚNIUS: 75-78
• Csorbai, A., György, E., György, M., Foldi, P., Medel, P., 2015. Campylobacter: egy baktérium, amit gyéríteni kell!; 04/12/2015; BAROMFI ÁGAZAT 2015/4 15: 84-87
• Le Pottier, G., Elgosi, V., Mayot, J., Carre, Y., 2015. Campylobacter: stratégies de réduction envisagées.; 01/09/2014; Filères avicoles, September 2014, 10-13
• Gracia, M.I. Redondo, J.L. Fernández, A., Martín, a., Medel, P., 2014. Campylobacter en avicultura de carne. Situación actual y estrategias actuales de reducción; 01/10/2014; Selecciones avícolas. Octubre 2014: 6-9
• Csorbai, 2016. Campylobacter-ma ennyit tudunk róla. Baromfiágazat 2016/3: 78-83
• Dossier Campylobacter. Réussir Avicole 220:10-19
6. Press releases
The project has had a big impact in the international media. There were 5 press releases, three of them in the most popular poultry websites, which have clearly contributed to the knowledge of sector about the project (title-date-location):
• PRESS RELEASE OF THE PROJECT - 20/12/2013-www.lafranceagricole.fr;
• PRESS RELEASE OF THE PROJECT - 23/12/2013 - www.agrapresse.fr;
• PRESS RELEASE OF THE PROJECT - 30/12/2013 - www.lequotidienlesmarches.fr;
• PRESS RELEASE OF THE PROJECT - 10/01/2014- Asociación Española de Ciencia Avícola newsletter;
• Aprobación en el 7º Programa Marco del proyecto CAMPYBRO. - 31/01/2014 - Arenys de Mar, Barcelona, Spain. Selecciones Avícolas, 56, 1, pp: 36;
• Una estrategia común contra el Campylobacter en avicultura de carne - 01/03/2014 - Madrid, Spain. Ganadería, 89: 15;
• Topsectoronderzoek naar Campylobacterreductie in pluimveevleessector - 18/03/2014 - http://www.agriholland.nl;
• Onderzoek Campylobacter verlegd naar primaire sector - 19/03/2014 - Boerderij Vandaag, 19/04/2014, page 9, The Netherlands;
• Rem op Campylobacter in pluimvee - 19/03/2014 - http://www.nieuweoogst.nl;
• Onderzoek naar Campylobacter reductie op plumeveebe drijven - 19/03/2014 - http://www.pluimveeweb.nl;
• Nepluvi participeert in grootschalig Europees CAMPYBRO Project - 01/06/2014 - www.nepluvi.nl
• PRESS RELEASE OF THE PROJECT - 16/10/2013- Newsletter of CIDEF, MORDELLES (France),
• Press Release at: http://www.pluimveeweb.nl/artikelen/2014/04/reguliere-vleeskuikens-minst-vaak-Campylobacter-positief/ and
• Campylobacterreductie - 01/04/2014 - Meat & Co. April 2014: 56.
• Voeradditieven effectief tegen campylobacterinfectie. Boerderij Vandaag, 27/01/2016, pp:9
• Strijd tegen Campylobacter. Pluimveehouderij 46e. 12/02/2016;
• Koeleman, E., 2016. Feed additives show promise in Campylobacter reduction. AllaboutFeed, 25/01/2016. http://www.allaboutfeed.net/Feed-Additives/Articles/2016/1/Feed-additives-show-promise-in-Campylobacter-reduction-2750600W/
• Koeleman, E., 2016. Campylobacter reduction: Feed additives role. World Poultry, 26/01/2016. http://www.worldpoultry.net/Nutrition/Articles/2016/1/Campylobacter-reduction-Feed-additives-show-promise-2751797W/
• Progress Versus Campylobacter. The Polutry site. 10 February 2016 http://www.thepoultrysite.com/poultrynews/36543/progress-versus-campylobacter/
• A press note on the Spanish workshop: http://avicultura.info/resultados-del-proyecto-europeo-campybro/ Avinews, Agosto 2016: 128
• 4 press notes on the Dutch workshop
o https://www.pluimveeweb.nl/artikelen/2016/08/campylobacter-congres-2016/
o https://www.nieuweoogst.nu/nieuws/2016/09/01/nederlandse-aanpak-campylobacter-succesvol
o http://www.wur.nl/nl/Expertises-Dienstverlening/Onderzoeksinstituten/livestock-research/Expertisegeb
o https://www.pluimveeweb.nl/artikelen/2016/09/optimisme-terugdringen-campylobacter/
7. Oral presentation to a scientific event/Technical meetings
IMASDE was invited to impart the conference “Campylobacter en avicultura de carne. Situación y estrategias actuales de reducción”, on 26/03/2014, in Jornadas Profesionales de Avicultura, Sevilla (Spain), 25-28 March 2014. Proceedings: 74. (http://www.jornadasavicultura.com/2015/produccion-pollos). IMASDE was also invited to impart the conference “Problemática actual del Campylobacter en la salud pública”, on 10/06/2015, in Jornadas Profesionales de Avicultura, organized by REAL ESCUELA DE AVICULTURA at Soria (Spain), 9-12 June 2015. IMASDE was also invited to impart the conference “Métodos de control de Campylobacter en la industria avícola”, on 26/11/2015, in III Congreso Internacional de Seguridad alimentaria, organized by Ilustre Colegio Oficial de Veterinarios Región de Murcia and Academia de Veterinaria de la Región de Murcia, at Murcia (Spain), 25-27 November 2015. Finally, IMASDE was invited to impart the conference “Impact of Campylobacter contamination in broiler production- Which opportunities can be provided on the part of feeding for a possible reduction of Campylobacter to improve food safety. Current information about CAMPYBRO project”, on 20/09/2016, in 19 Orffa symposium, organized by ORFFA, at Dinklage (Germany), 20 September 2016.
Two other invited conferences were imparted in the international workshop on Campylobacter within The International Association for Food Protection European Annual European meeting, hold in 2016 in Athens (Greece), as explained bellow. The conference titles were:
• Guyard-Nicòdeme, M., 2016. Why is Campylobacter the number one priority for the poultry production chain? International Association for Food Protection European Annual European meeting, Athens, Greece. Pp: 51
• Medel, P., 2016. An update about the different state of the art methods to control campylobacter in broilers: the European project CAMPYBRO. International Association for Food Protection European Annual European meeting, Athens, Greece. Pp: 51
Organization of workshops in International Scientific Meetings
An international workshop on Campylobacter was presented by ANSES within the The International Association for Food Protection (https://www.foodprotection.org/) European Annual European meeting, hold in 2016 in Athens (Greece) between the 11th and 13th May (https://www.foodprotection.org/europeansymposium/).
During the meeting, a workshop focused on Campylobacter was presented by ANSES, in which the results of the project within the context of general measures to control Campylobacter were the main issue.
The workshop was chair by Muriel Guyard and Habila Haddad (ANSES) and the main speakers were Muriel Guyard-Nicòdeme and Pedro Medel (coordinator, IMASDE).
8. Vertical dissemination activities between producers within Poultry Producers Associations
In total, 7 presentations were made. In the first reporting period, there were three presentations: “Campylobacter challenge for EU poultry producers”; on 30/10/2013; in Budapest, Hungary (BTT facilities at Budapest), destined to main poultry producers in Hungary; “Campylobacter challenge for EU poultry producers. First results of CAMPYBRO project” on 11/02/2014 in Madrid (Propollo facilities, Madrid), destined to main poultry producers in Spain and “Campylobacter challenge for EU poultry producers” on 19/11/2014 in Madrid (Propollo facilities, Madrid), destined to main poultry producers in Spain.
In the Second reporting period the project results were presented to the Association members seven times. In Spain, all the presentations took place in the PROPOLLO facilities in Madrid, and the main poultry producers participate into the presentation. Since the poultry production is very integrated in Spain (the main 10 operators have approximately 60% of the production), most of the Spanish production was informed about the project results. All the presentations were given by the coordinator (Pedro Medel) and the PROPOLLO CEO (Ángel Martín). The dates of the meetings were: 03/03/2015, 24/11/2015 and 19/04/2016.
An update of the Campylobacter in the EU poultry sector was made in Houten on 05/11/2015. The public were the quality managers of the 16 bigger poultry producer in the Netherlands and NEPLUVI (Association of the Dutch Poultry Processing Industries). The update included the prevalence in the Netherlands and in the rest of Europe, the intentions for creating a European Process Hygiene Criterion for Campylobacter and the preliminary results of the project (WP1, WP2, WP3 and WP5) presented in the second GA at Stockholm. Also, the members were updated with the management activities of the project and the dissemination activities. The presentation was given by Peter Vesseur.
The project results were presented to the Hungarian companies in several opportunities (a year-bases) in the Second reporting period. All the presentations took place in the Geréby Kúria Hotel at Lajosmizse (Hungary), and most of the poultry producers of the country participate into the presentation (more than 100 both times). All the presentations were given by the coordinator (Pedro Medel) and the BTT CEO (Attila Csorbai). The dates of the meetings were: 03/03/2015, 24/11/2015 and 19/04/2016.
An update of the Campylobacter in the EU poultry sector was done for French producers. First, there was a presentation of the Ecole Superior d’Agriculture of Angers, and about the importance of poultry in this Region of France and in the ESA in particular. After, Julie Mayot presented the epidemiology of Campylobacter and campilobacteriosis. Pedro Medel presented some aspects of the infection, the most practical measures to fight again. Finally, the project CAMPYBRO was presented, and the results until March 2015 were explained. There were approximately 30 attendees, belonging to several poultry companies inside FIA and CIDEF.
9. Participation in international meetings presenting information to decision makers
The International Conference on PREVENTION AND CONTROL OF CAMPYLOBACTER IN THE POULTRY PRODUCTION SYSTEM, was organized by IZSAM G. CAPORALE TERAMO, Ministero della Salute and the Instituto Zooprofilattico Sperimentale delle Venezie, and hold on Milano, the 31st August 2015. In the meeting participated a lot of researchers, policy makers including Italian Ministry of Heatth or the the EC (Klaus Kostenzer), policy maker supporters (ECDC, EFSA), or the National Reference Laboratories of several countries for Campylobacter. The Director of the station of Ploufragan, ANSES, Dr. Gilles Salvat, was invited to give a talk called “Reducing the risk of Campylobacteriosis from poultry: Feed interventions - blocking the colonization of birds”, which summarizes the results of the project Campybro until this moment.
The Food Standards Agency of the UK, was informed about CAMPYBRO project through Francisco Javier Domínguez Orive, Veterinary Director and Head of Foodborne Diseases Control Unit, who was invited as speaker in the workshops of France and Spain. After that, a colleague of him (Adam Hardgrave) was asked to write a chapter for the ACMSF Campylobacter report on ‘Controls (regulatory framework for products of concern, EU targets etc.) and evidence of success in other countries’, and they though that a section on Campybro would probably fit well/be really useful for inclusion. The Terms of Reference for the report is: ‘To assess the actions that have taken place since the publication of the Second Campylobacter Report and make proposals to advise the FSA in evolving its strategy for reducing the incidence and risk of foodborne Campylobacter infection in humans.’ The chapter currently details the sampling regimes and interventions in Norway, Sweden, Denmark, Iceland and the Netherlands. Each section also includes more technical details on the Characteristics of the broiler industry in each country, such as the number of farms, type and number of slaughterhouses/processors, average stocking densities etc. Its content will enter the public domain probably toward the end of the year. A summary of the project was sent the 3rd of August for their revision and edition to be included in the Report.
10. Organization of Conferences
A Workshop was organized by IMASDE “Campylobacter: un reto para la avicultura de carne europea” on 13/12/2013 in Murcia (Spain), destined to Scientific community (resarchers), students and policy makers (Consejería de Agricultura y Consejería de Sanidad de la Región de Murcia).
11. Dissemination of the data in the countries that participate in the project
The project results are interesting for poultry producers and processors. Fortunately, the project partners SME-AGs are the Poultry Producers Associations (Spain, Hungary, The Netherlands and France [2]). Therefore, it has been very easy to disseminate the results of the project within their Associates. In 2016 four technical workshops were done in the four countries that participate in the project (Spain, France, The Netherlands and Hungary). These workshops were technical, and were open to the all sector, including the media, the academia, the industry and the Administration.
List of Websites:
www.campybro.eu