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The influence of probiotic supplementation in broiler chickens on population and carcass contamination with Campylobacter spp. - Field study
Research in Veterinary Science 118 (2018) 312–316
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The influence of probiotic supplementation in broiler chickens on population and carcass contamination with Campylobacter spp. - Field study
T
⁎
Marcin Smialeka, , Szymon Burchardtb, Andrzej Koncickia a b
Department of Poultry Diseases, University of Warmia and Mazury, ul. Oczapowskiego 13/13, 10-719 Olsztyn, Poland JHJ Sp. Z.O.O., Nowa Wieś 11, 63-308 Gizałki, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Broiler chickens Probiotic supplementation Campylobacter spp. Humoral immunity
Campylobacter spp. is a food-borne pathogen occurring all over the world. According to European Food Safety Authority, in Europe, in 2015 the number of recorded and confirmed cases of Campylobacter spp. infections in humans has reached approximately 230,000. Poultry and poultry meat are considered to be the main sources of human infection, which triggers the discussion about the possibility of imposing obligatory control of Campylobacter spp. population at the level of primary poultry production. Recently, the use of probiotics in poultry is considered as a very promising alternative that could reduce infection rate in broiler chickens with Campylobacter spp. Although, there were some approaches made in vivo, up to date, there were no studies that would evaluate those issues under field conditions. A study was carried out in order to determine the feasibility of reducing infection rate in broiler chickens with Campylobacter spp. raised at a commercial farm, by the addition of multispecies probiotic (Lavipan, JHJ, Poland) that composed of Lactococcus lactis, Carnobacterium divergens, Lactobacillus casei, Lactobacillus plantarum and Saccharomyces cerevisae to the feed. Results of our study indicate that probiotic (Lavipan) added to a feed for broiler chickens was capable to reduce the extent of Campylobacter spp. invasion in the gastrointestinal tract of birds and, resultantly, to diminish contamination level in bird environment, which eventually contributed to the improved hygienic parameters of analyzed poultry carcasses. Additionally, this probiotic displayed promising immunomodulatory properties that may improve the effectiveness of the specific prophylaxis program applied in a flock of broiler chickens.
1. Introduction In recent years, campylobacteriosis has emerged as the most frequently reported food-borne disease in humans in Europe (EFSA, 2016; Ghareeb et al., 2012; Hue et al., 2011; Miller et al., 2005; Mohan, 2015; Nachamkin et al., 2002; Nielsen et al., 2000; Zhang, 2008). According to the European Food Safety Authority, in 2015 the number of recorded and confirmed cases of Campylobacter spp. infections in humans has reached approximately 230,000 (EFSA, 2016). What is even more disturbing is the fact that the number of recorded cases of campylobacteriosis has been successively increasing - for instance the number of cases from 2015 was equal to the half of the total number of cases recorded between 2008 and 2014 (EFSA, 2016). The high number of campylobacteriosis cases is associated with the fact that these bacteria exist widely in nature. Campylobacter spp. are considered to be a part of saprophytic microflora in a digestive tract of
many wild and domestic animal species, including poultry, especially grown commercially (EFSA, 2016; Ghareeb et al., 2012; Mohan, 2015). Poultry and poultry meat are considered to be the main sources of infection to humans, but the role of beef and pork meat is also emphasized in campylobacteriosis epidemiology (EFSA, 2016). For instance in 2015, 46.7% of the 6707 samples of fresh broiler meat were found to be positive for Campylobacter spp., which was higher than in 2014 (EFSA, 2016). The very important aspect is that infections of poultry with these bacteria are generally subclinical (Lee and Newell, 2006; Mohan, 2015; Zhang, 2008). The above situation gives rise to an ongoing discussion about the possibility of imposing obligatory control and reducing Campylobacter spp. population at the level of primary poultry production. In the light of the above, one of the alternatives that can easily be implemented at the level of primary production is the use of probiotics. Probiotics, which are one of the oldest feed additives, are live
Abbreviations: CH, Chicken House; EFSA, European Food Safety Association; ELISA, Enzyme-linked Immunosorbent Assay; IB, Infectious Bronchitis; IBD, Infectious Bursal Disease; MD, Mareks Disease; ND, Newcastle Disease; OD, Optical Density; REO, Reoviruses; S., Salmonella; SD, Standard Deviation; S/P, Sample to Positive Ratio ⁎ Corresponding author. E-mail addresses: [email protected] (M. Smialek), [email protected] (S. Burchardt), [email protected] (A. Koncicki). https://doi.org/10.1016/j.rvsc.2018.03.009 Received 7 February 2017; Received in revised form 30 April 2017; Accepted 13 March 2018 0034-5288/ © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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droppings). Similarly, after the main experiment (production cycle 2) had been completed, the control study was performed twice (production cycles 3 and 4). Environmental samples (n = 4 / CH) comprised of 10 pooled dropping samples collected from different parts of a chicken house. Environmental samples were collected at 37 days of birds life in each production cycle.
microorganisms. If supplemented in the right amount, they can beneficially affect the host's health by: (1) ensuring a favorable balance between commensal and pathogenic microbiota in the gastrointestinal tract, (2) increasing digestibility and assimilability of nutrients, as well as (3) exerting immunomodulatory and immunostimulatory effects (Alkhalf et al., 2010; Balevi et al., 2001; Chichlowski et al., 2007; Dhama et al., 2011; Farnell et al., 2006; Fellah et al., 2014; Galdeano and Perdigón, 2006; Koenen et al., 2004; Samanya and Yamauchi, 2002). It has been demonstrated that the use of probiotics and eventually their domination in birds digestive tract may reduce the growth of pathogenic bacteria such as Staphylococcus aureus, E. coli, Salmonella (S.) enteritidis and S. typhimurium, Clostridium perfrigens, Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolica, Candida albicans, as well as coccidia (Bengmark, 1998; Dalloul et al., 2005; Dhama et al., 2011; Fuller, 2001; Ghareeb et al., 2012; Lee et al., 2007; Mohan, 2015; Nurmi and Rantala, 1973; Patterson and Burkholder, 2003; Willis and Reid, 2008). In the light of the above, regular supplementation of poultry with probiotics may both minimize the risk of infection and disease in birds as well as decrease the risk of poultry meat contamination with such pathogens as Salmonella spp., Campylobacter spp., Listeria monocytogenes and/or E.coli. Up to date, only a few studies have evidenced a possible role of probiotics in preventing the shedding of Campylobacter spp. at the level of primary production. Although, there were some approaches made in order to evaluate the potential influence of probiotic supplementation on the rate of broiler chickens infection with Campylobacter spp. and shedding of the bacteria in vivo (Ghareeb et al., 2012; Willis and Reid, 2008), there were no studies that would evaluate those issues under field conditions. Considering the above, a study was carried out in order to determine the feasibility of reducing Campylobacter spp. infection rate in broiler chickens raised at a commercial farm, by feed supplementation of Lavipan – a multispecies probiotic product (JHJ, Poland), in the final concentration of 0,5 kg of probiotic per every 999.5 kg of the feed.
2.2. Experimental layout During production cycle 2 (Experiment), chickens from CH2 (group L) received a Lavipan probiotic (JHJ, Poland) in the feed in the final dose of 0.5 kg of probiotic per every 999.5 kg of the feed during the entire production cycle. At the same time, birds from CH1 (group K) received the same feed but without the probiotic. Throughout the experiment, veterinary prophylaxis and therapy schedule and program were the same for CH1 and CH2 (as well as for CH1 and CH2 during three other production cycles). Before birds were placed in CH1 or CH2, 23 birds were selected at random from a transport truck in order to obtain blood samples for the serological evaluation of the time of vaccination against Infectious Bursal Disease (IBD), based on the Deventer formula, as well as for the evaluation of the level of maternally-derived antibodies against Infectious Bronchitis (IB) virus and reoviruses (REO). Vaccination program against Marek Disease (MD), IB and IBD that was executed at the farm in each production cycle is summarized in Table 2. At day 37 of birds life, 20% of the chickens from CH1 and CH2 were transferred for slaughter. At the slaughterhouse, 23 blood samples were collected from birds from K and L group independently for the serological evaluation of the level of antibodies against IB, IBD and REO. At the same time, 5 samples of freshlysqueezed feces from ileum and ceca of the chickens from group K (n = 5) and L (n = 5) were collected for microbiological examination and Campylobacter spp. enumeration. Prior to feces samples collection, abdominal skin was disinfected (40% ethanol) and intestines were dissected with the use of sterile surgical equipment (individual equipment was prepared for each bird). Additionally, samples of pectoral muscles (10 g), cut superficially (max. cut depth < 10 mm), with overlaying skin were collected from birds of group K (n = 4) and L (n = 4) for microbiological examination. At the same time, environmental samples were collected from CH1 and CH2 (n = 4 / CH). In each production cycle, the samples collected for microbiological examination were transferred on ice to a laboratory in < 3 h after they had been collected.
2. Materials and methods 2.1. Farm The study was carried out under field conditions on a chicken farm with four poultry houses (CH - chicken house) in four consecutive production cycles. It was conducted in CH1 and CH2 and the number of birds in those houses in each production cycle was (approx.): CH1 – 22,000 and CH2 – 18,000 chickens. Study layout is summarized in Table 1. In each production cycle (1 - retrospective, 2 - experiment, 3 and 4 - control), both CH1 and CH2 were settled with Ross 308 broiler chicks purchased from one hatchery and from one hatch. Feed and water were given to the birds ad libitum. Feed was provided by its producer - Wipasz sp. z.o.o. (Poland). The retrospective microbiological study was conducted on a farm (production cycle 1) in order to establish the presence of Campylobacter spp. and the diversity of its population between CH1 and CH2. This study was conducted with environmental samples (freshly excreted
2.3. Probiotic During production cycle 2 (Experiment) chickens from CH2 received in feed Lavipan probiotic product (JHJ, Poland) which comprises of selected stains of lactic acid bacteria: Lactococcus lactis IBB 500 (origin - chicken feces), Carnobacterium divergens S-1 (origin - carp gut), Lactobacillus casei ŁOCK 0915 (origin - chicken feces) and Lactobacillus plantarum ŁOCK 0862 (origin - turkey feces) in the amount of 1 × 109 colony forming units (CFU/g) each and Saccharomyces cerevisae ŁOCK 0141 (origin - plant silage) in the amount of 1 × 107 CFU/g.
Table 1 Study layout summarized. Only in production cycle 2 birds from chicken house 2 (CH2) received probiotic (Lavipan, JHJ, Poland) in feed. Sample type
1/retrospective Environmental sample Feces from intestines Pectoral muscles Blood a
Table 2 Immunoprophylaxis programme executed at the experimental farm during 4 production cycles.
Production cycle no.
+ − − −
a
2/experiment
3/control
4/control
+ + + +
+ − − −
+ − − −
Disease
Day of life
Vaccine strain
MD IB
In. ovo 1 14 16 (Based on Deventer formula)
Rispens + HVT H-120 1/96 Winterfield 2512
IBD
“+” indicates samples collected during each production cycle.
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– –
– –
2.5. Serology Infectious Bronchitis, IBD and REO serological evaluations were performed with commercial ELISA kits (IDEXX, USA). Successive steps of the procedure were performed according to the manufacturer's recommendations. ELISA was carried out with the use of the Eppendorf epMotion 5075 LH automated pipetting station (Eppendorf, Germany), BioTek ELx405 automatic plate washer (BioTek, USA) and BioTek ELx800 plate reader. The sample to positive (S/P)-ratio was calculated based on the ODs and used to express the mean (S/P)-ratio ± SD per group.
–
– 7,1 × 103 ± 13,4 × 103
–
30,9 × 103 ± 9,1 × 103⁎ 0,47 × 103 ± 0,25 × 103 7,1 × 104 ± 1,1 × 104⁎ 1 × 104 ± 0,32 × 104 2,8 × 103 ± 0,37 × 103⁎
Microbiological examination was performed in accordance with PNEN ISO 6887-1 protocol. Ten-fold serial dilutions were prepared from each sample. Next dilutions were plated on Brilliance CampyCount Agar (Oxoid, Basingstoke, UK) followed by incubation at 41.5 °C for 48 h under microaerobic conditions (CapmyGen, Oxoid, Basingstoke, UK). Every sample were prepared in triplicate. After incubation grown colonies were counted and the final results were expressed as mean CFU/ml ± SD. For each positive plate randomly chosen typical Campylobacter colonies were subcultured onto plates of Columbia blood agar (Oxoid, Basingstoke, UK) for further characterization according to the PN-EN ISO 10272-1 protocol.
2.6. Statistical analysis
– Significant differences (Manna–Whitney U test, * as p < 0,05) in comparison to the control group (K).
– –
Campylobacter spp. is a food-borne pathogen occurring all over the world (EFSA, 2016; Nielsen et al., 2000; Nachamkin et al., 2002; Miller et al., 2005; Unicomb et al., 2006). The main symptoms of campylobacteriosis in human include diarrhea, cramping, abdominal pain, and fever. Campylobacter jejuni is one of the most common causes of human gastroenteritis in many countries (Fallon et al., 2003; Hänninen et al., 2003). It remains inexplicit whether the Campylobacter spp. genus bacteria are transferred vertically from a laying hen to its progeny, however the infection of a laying hen flock with these bacteria may result in eggshell contamination, which in turn may constitute the source of infection to the newly-hatched chicks (Doyle, 1984; Shanker et al., 1986). The likelihood of bacteria isolation from broiler chickens has been demonstrated to increase with bird age (Agunos et al., 2014; Mohan, 2015; Zhang, 2008), which results from the number of factors being potential sources of infection with these bacteria. The route of birds infection is usually through the gastrointestinal tract and sources of infection include contaminated litter, rodents, flies, farm staff, and other farm animals kept at or near the production farm (Agunos et al., 2014). This points to the contribution of biosecurity aspects of a farm as an element enabling the reduction of both the incidence and the rate of Campylobacter spp. infections in poultry. However, considering the multiplicity of factors being sources and vectors of infection (and the likely vertical transmission), it seems that today the biosecurity regime alone may prove ineffective in controlling infections induced by these microorganisms. Up to date, only a few studies have evidenced the possible role of probiotics in preventing the Campylobacter spp. colonization and carcass contamination at the level of primary production. Recent studies have shown that an avian - specific multiprobiotic was able to reduce the Campylobacter jejuni and cecal colonization in broiler chickens (Ghareeb et al., 2012; Willis and Reid, 2008).
⁎
0 4,15 × 10 ± 4,98 × 10
3
– –
1
37,2 × 105 ± 0,45 × 105⁎ 3 × 105 ± 0,45 × 105
Environmental sample Feces from intestines Pectoral muscles
CH1 (K) CH2 CH1
74 × 103 ± 161 × 103
Statistical analysis was performed with GraphPad Prism 6.05 with the use of Mann Whitney U test. Differences were considered statistically significant at p < 0,05. 3. Results and discussion
2
25,3 × 103 ± 1,55 × 103
CH2 CH1 CH2 CH1 CH2 (L)
2.4. Microbiological studies
1
Production cycle no. Sample type
Table 3 Results of microbiological studies. Results are presented as the mean Campylobacter spp. CFU/ml ± SD.
3
4
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Campylobacter spp. in the environment of chicken houses CH1 and CH2. Results obtained reflected correlations observed in the retrospective study, i.e. a statistically significantly higher count of the analyzed bacteria was found in the environmental samples from CH2 than in these from CH1, in both production cycles. The reason behind differences noted in Campylobacter spp. populations between CH1 and CH2 in production cycles 1, 3 and 4 is somehow inexplicable. It could be speculated that they were due to differences in humidity levels in both chicken houses, with a greater problem with moist litter being observed in CH2. On the other hand, these differences could result from the internal system of farm management, the mode and order of handling procedures in each chickens house, or even from its location in respect of other chicken houses (the CH2 is an internal house, whereas the CH1 is an external one). Those aspects are currently under further investigation. The above-mentioned issues of Campylobacter spp. on-farm transmission have been widely reviewed previously (Agunos et al., 2014). During our study on-farm workers were not informed about the project, so the internal management and biosecurity measures did not change during the entire study.
Table 4 Results of serological analysis for day old and 37-day old broiler chickens. Levels of specific antibodies against IB, IBD and REO in chickens serum were evaluated. Results are presented as mean S/P ± SD with the number of samples reacting positive (pos) and negative (neg) in each test. Assay
Group (age)
S/P
SD
pos
neg
IB
K + L (hatch day) K (37 days) L (37 days) K + L (hatch day) K (37 days) L (37 days) K + L (hatch day) K (37 days) L (37 days)
0,51 0,32 0,34 2,35 1,23 0,81 1,94 0,27 0,47⁎
0,30 0,29 0,33 1,08 1,03 0,67 0,416 0,2 0,32
21 13 13 22 19 18 23 14 20
2 10 10 1 4 5 0 9 3
REO
IBD
⁎ Significant differences (Manna–Whitney U test, * as p < 0,05) in comparison to the control group (K).
The inhibitory effects of Lactococcus lactis IBB 500, Carnobacterium divergens S-1, Lactobacillus casei ŁOCK 0915 and Lactobacillus plantarum ŁOCK 0862 as well as yeasts Saccharomyces cerevisae ŁOCK 0141, on S. enteritidis and S. typhimurium growth in vitro has been evaluated previously and those probiotic strains revealed very promising potential to reduce the growth of pathogenic bacteria (Burchardt, personal communication, 2015).
3.2. The influence of Lavipan probiotic product on humoral immunity Probiotics significantly improve functions of the immune system of animals, including birds (Alkhalf et al., 2010; Balevi et al., 2001; Chichlowski et al., 2007; Dhama et al., 2011; Farnell et al., 2006; Fellah et al., 2014; Galdeano and Perdigón, 2006; Koenen et al., 2004; Samanya and Yamauchi, 2002). In the case of poultry, probiotics have been demonstrated to increase total amount of antibodies, including the vaccine-induced specific antibodies (Stringfellow et al., 2011; Talebi et al., 2008). Results of serological analyses were presented in Table 4. Statistically significantly higher vaccine-induced titers of antibodies against Gumboro disease virus were demonstrated in the L group of birds at 37 days of life, in comparison to the control group. These results are consistent with findings reported by Talebi et al. (2008), who demonstrated higher vaccine induced titers of anti-IBD and anti-ND (Newcastle disease) antibodies in broiler chickens administered a Lactobacillus-based multiprobiotic throughout the entire production cycle. It may, therefore, be concluded that the Lavipan probiotic product administered to broiler chickens has an immunomodulatory effect. In addition, our study indicated a tendency for the reduction of seroconversion degree towards REO in the group receiving the Lavipan preparation. Though these differences were statistically insignificant (p = 0.21) (Table 4), they suggest that this probiotic may be capable of minimizing infections with these viruses per os, by reducing the degree of its replication at the gate of infection. No differences were found in the vaccine-induced level of anti-IBV antibodies between groups K and L (Table 4). The differences in levels of antibodies against IB and IBD viruses between groups L and K may be explained by: (Agunos et al., 2014) difference in the method of vaccine application with water (IBD) or in aerosol (IB), (Alkhalf et al., 2010) various immunogenicity of vaccinal viruses, and (Balevi et al., 2001) the degree and target site of vaccine virus replication.
3.1. Influence of Lavipan probiotic product on population of Campylobacter spp. Results of microbiological analyses were summarized in Table 3. In the production cycle 1, the population number of Campylobacter spp. in the environment of CH2 was statistically significantly higher (p < 0.05) than in CH1. The differences noted between CH1 and CH2 during the retrospective study allowed choosing birds from CH2 as the study group – L in the exact experiment. The scope of microbiological tests in the Experiment was extended to three sampling sites, i.e. feces from ileum and cecum, environmental samples, and pectoral muscles. Muscle samples were collected with overlaying skin, as muscle contamination with Campylobacter spp. proceeds from the environment through the skin. The whole sampling scheme was, therefore, aimed at mimicking the route of pectoral muscles contamination with Campylobacter spp. excreted with droppings to the environment. In the Experiment, a statistically significantly lower (p < 0.05) count of Campylobacter spp. was determined in environmental samples from CH2 than from CH1 (Table 3). A lower mean population number was assayed in the intestinal samples of birds from group L compared to birds from group K, however the difference was statistically insignificant (Table 3). In contrast to birds from group K (growth of Campylobacter spp. recorded in 100% of the samples), 1 out of 4 intestinal samples of birds from group L was negative for Campylobacter spp. No growth of Campylobacter spp. was confirmed in all samples of pectoral muscles of birds from group L, whereas in group K positive results were found in 50% of the samples (Table 3). Hue et al. (2011) demonstrated that the level of carcasses contamination with Campylobacter spp. genus bacteria was directly correlated with the degree of intestine invasion with these bacteria. Considering the above and findings from our study, it may be concluded that – by reducing the possibility of Campylobacter spp. invasion in intestine lumen and, resultantly, by minimizing bird environment contamination with these bacteria – the Lavipan probiotic product contributed to the improvement of hygienic parameters of the samples of pectoral muscles. The Experiment was followed by two control studies (production cycles 3 and 4), where Lavipan preparation was no longer administered, which were aimed at determining changes in the population of
4. Conclusions It has been demonstrated earlier that probiotics inhibit the growth of pathogenic microflora in the gastrointestinal tract through competence exclusion. Simultaneously, they are increasingly indicated as an alternative method for the risk minimization of food product contamination with Campylobacter spp. bacteria through the reduction of infection degree at the primary production level (Mohan, 2015). The analysis of study results allows concluding that the Lavipan probiotic product (JHJ, Poland), that comprised of selected stains of lactic acid bacteria: Lactococcus lactis IBB 500, Carnobacterium divergens S-1, Lactobacillus casei ŁOCK 0915 and Lactobacillus plantarum ŁOCK 0862 in 315
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the amount of 1 × 109 CFU/g each and Saccharomyces cerevisae ŁOCK 0141 in the amount of 1 × 107 CFU/g, added to feed mixture for broiler chickens was capable to reduce the extent of Campylobacter spp. invasion in the gastrointestinal tract of birds and, resultantly, to diminish contamination level in bird environment, which eventually contributed to the improved hygienic parameters of the analyzed poultry carcasses. Considering the fact that the alarming epidemiological situation of campylobacteriosis in humans is, to a large extent, associated with these bacteria prevalence in slaughter poultry population, apart from the biosecurity and hygienic-technological aspects of food product manufacture, the application of the analyzed probiotic may contribute to improved consumer safety through its effect on Campylobacter spp. population reduction. In addition, Lavipan probiotic product display promising immunomodulatory properties that may successfully improve the effectiveness of the specific prophylaxis program applied in a flock of broiler chickens. Further studies are, however, needed to establish the exact mechanism of the effect of this probiotic on the immunological system of birds.
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Conflict of interest statement The authors declared no conflicts of interest. Funding Publication supported by KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal - Safe Food”, decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015. Acknowledgement The authors would like to thank MSc Szczucińska Ewa and DVW Kaczorek Edyta from Department of Microbiology and Clinical Immunology, University of Warmia and Mazury, Olsztyn, Poland, for the help with microbiological study. References Agunos, A., Waddell, L., Léger, D., Taboada, E., 2014. A systematic review characterizing on-farm sources of Campylobacter spp. for broiler chickens. PLoS One 9, e104905. http://dx.doi.org/10.1371/journal.pone.0104905. Alkhalf, A., Alhaj, M., Al-Homidan, I., 2010. Influence of probiotic supplementation on immune response of broiler chicks. Egypt Poult. Sci. 30, 271–280. Balevi, T., Uçan, U.S., Cokun, B., Kurtoglu, V., Cetingul, I.S., 2001. Effect of dietary probiotic on performance and humoral immune response in layer hens. Br. Poult. Sci. 42, 456–461. Bengmark, S., 1998. Ecological control of the gastrointestinal tract - the role of probiotic flora. Gut 42, 2–7. Chichlowski, M., Croom, W.J., Edens, F.W., McBride, B.W., Qiu, R., Chiang, C.C., Daniel, L.R., Havenstein, G.B., Koci, M.D., 2007. Microarchitecture and spatial relationship between bacteria and ileal, cecal, and colonic epithelium in chicks fed a direct-fed microbial, primalac, and salinomycin. Poult. Sci. 86, 1121–1132. Dalloul, R.A., Lillehoj, H.S., Tamim, N.M., Shellem, T.A., Doerr, J.A., 2005. Induction of local protective immunity to Eimeria acervulina by a Lactobacillus based probiotic. Comp. Immunol. Microbiol. Infect. Dis. 28, 351–361. Dhama, K., Verma, V., Sawant, P.M., Tiwari, R., Vaid, R.K., Chauhan, R.S., 2011. Applications of probiotics in poultry: enhancing immunity and beneficial effects on production performances and health - a review. J. Immunol. Immunopathol. 13, 1–19. Doyle, M.P., 1984. Association of Campylobacter jejuni with laying hens and eggs. Appl. Environ. Microbiol. 47, 533–536. EFSA, 2016. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015. EFSA J. 14, 4634.
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The influence of probiotic supplementation in broiler chickens on population and carcass contamination with Campylobacter spp. - Field study
T
⁎
Marcin Smialeka, , Szymon Burchardtb, Andrzej Koncickia a b
Department of Poultry Diseases, University of Warmia and Mazury, ul. Oczapowskiego 13/13, 10-719 Olsztyn, Poland JHJ Sp. Z.O.O., Nowa Wieś 11, 63-308 Gizałki, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Broiler chickens Probiotic supplementation Campylobacter spp. Humoral immunity
Campylobacter spp. is a food-borne pathogen occurring all over the world. According to European Food Safety Authority, in Europe, in 2015 the number of recorded and confirmed cases of Campylobacter spp. infections in humans has reached approximately 230,000. Poultry and poultry meat are considered to be the main sources of human infection, which triggers the discussion about the possibility of imposing obligatory control of Campylobacter spp. population at the level of primary poultry production. Recently, the use of probiotics in poultry is considered as a very promising alternative that could reduce infection rate in broiler chickens with Campylobacter spp. Although, there were some approaches made in vivo, up to date, there were no studies that would evaluate those issues under field conditions. A study was carried out in order to determine the feasibility of reducing infection rate in broiler chickens with Campylobacter spp. raised at a commercial farm, by the addition of multispecies probiotic (Lavipan, JHJ, Poland) that composed of Lactococcus lactis, Carnobacterium divergens, Lactobacillus casei, Lactobacillus plantarum and Saccharomyces cerevisae to the feed. Results of our study indicate that probiotic (Lavipan) added to a feed for broiler chickens was capable to reduce the extent of Campylobacter spp. invasion in the gastrointestinal tract of birds and, resultantly, to diminish contamination level in bird environment, which eventually contributed to the improved hygienic parameters of analyzed poultry carcasses. Additionally, this probiotic displayed promising immunomodulatory properties that may improve the effectiveness of the specific prophylaxis program applied in a flock of broiler chickens.
1. Introduction In recent years, campylobacteriosis has emerged as the most frequently reported food-borne disease in humans in Europe (EFSA, 2016; Ghareeb et al., 2012; Hue et al., 2011; Miller et al., 2005; Mohan, 2015; Nachamkin et al., 2002; Nielsen et al., 2000; Zhang, 2008). According to the European Food Safety Authority, in 2015 the number of recorded and confirmed cases of Campylobacter spp. infections in humans has reached approximately 230,000 (EFSA, 2016). What is even more disturbing is the fact that the number of recorded cases of campylobacteriosis has been successively increasing - for instance the number of cases from 2015 was equal to the half of the total number of cases recorded between 2008 and 2014 (EFSA, 2016). The high number of campylobacteriosis cases is associated with the fact that these bacteria exist widely in nature. Campylobacter spp. are considered to be a part of saprophytic microflora in a digestive tract of
many wild and domestic animal species, including poultry, especially grown commercially (EFSA, 2016; Ghareeb et al., 2012; Mohan, 2015). Poultry and poultry meat are considered to be the main sources of infection to humans, but the role of beef and pork meat is also emphasized in campylobacteriosis epidemiology (EFSA, 2016). For instance in 2015, 46.7% of the 6707 samples of fresh broiler meat were found to be positive for Campylobacter spp., which was higher than in 2014 (EFSA, 2016). The very important aspect is that infections of poultry with these bacteria are generally subclinical (Lee and Newell, 2006; Mohan, 2015; Zhang, 2008). The above situation gives rise to an ongoing discussion about the possibility of imposing obligatory control and reducing Campylobacter spp. population at the level of primary poultry production. In the light of the above, one of the alternatives that can easily be implemented at the level of primary production is the use of probiotics. Probiotics, which are one of the oldest feed additives, are live
Abbreviations: CH, Chicken House; EFSA, European Food Safety Association; ELISA, Enzyme-linked Immunosorbent Assay; IB, Infectious Bronchitis; IBD, Infectious Bursal Disease; MD, Mareks Disease; ND, Newcastle Disease; OD, Optical Density; REO, Reoviruses; S., Salmonella; SD, Standard Deviation; S/P, Sample to Positive Ratio ⁎ Corresponding author. E-mail addresses: [email protected] (M. Smialek), [email protected] (S. Burchardt), [email protected] (A. Koncicki). https://doi.org/10.1016/j.rvsc.2018.03.009 Received 7 February 2017; Received in revised form 30 April 2017; Accepted 13 March 2018 0034-5288/ © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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droppings). Similarly, after the main experiment (production cycle 2) had been completed, the control study was performed twice (production cycles 3 and 4). Environmental samples (n = 4 / CH) comprised of 10 pooled dropping samples collected from different parts of a chicken house. Environmental samples were collected at 37 days of birds life in each production cycle.
microorganisms. If supplemented in the right amount, they can beneficially affect the host's health by: (1) ensuring a favorable balance between commensal and pathogenic microbiota in the gastrointestinal tract, (2) increasing digestibility and assimilability of nutrients, as well as (3) exerting immunomodulatory and immunostimulatory effects (Alkhalf et al., 2010; Balevi et al., 2001; Chichlowski et al., 2007; Dhama et al., 2011; Farnell et al., 2006; Fellah et al., 2014; Galdeano and Perdigón, 2006; Koenen et al., 2004; Samanya and Yamauchi, 2002). It has been demonstrated that the use of probiotics and eventually their domination in birds digestive tract may reduce the growth of pathogenic bacteria such as Staphylococcus aureus, E. coli, Salmonella (S.) enteritidis and S. typhimurium, Clostridium perfrigens, Listeria monocytogenes, Campylobacter jejuni, Yersinia enterocolica, Candida albicans, as well as coccidia (Bengmark, 1998; Dalloul et al., 2005; Dhama et al., 2011; Fuller, 2001; Ghareeb et al., 2012; Lee et al., 2007; Mohan, 2015; Nurmi and Rantala, 1973; Patterson and Burkholder, 2003; Willis and Reid, 2008). In the light of the above, regular supplementation of poultry with probiotics may both minimize the risk of infection and disease in birds as well as decrease the risk of poultry meat contamination with such pathogens as Salmonella spp., Campylobacter spp., Listeria monocytogenes and/or E.coli. Up to date, only a few studies have evidenced a possible role of probiotics in preventing the shedding of Campylobacter spp. at the level of primary production. Although, there were some approaches made in order to evaluate the potential influence of probiotic supplementation on the rate of broiler chickens infection with Campylobacter spp. and shedding of the bacteria in vivo (Ghareeb et al., 2012; Willis and Reid, 2008), there were no studies that would evaluate those issues under field conditions. Considering the above, a study was carried out in order to determine the feasibility of reducing Campylobacter spp. infection rate in broiler chickens raised at a commercial farm, by feed supplementation of Lavipan – a multispecies probiotic product (JHJ, Poland), in the final concentration of 0,5 kg of probiotic per every 999.5 kg of the feed.
2.2. Experimental layout During production cycle 2 (Experiment), chickens from CH2 (group L) received a Lavipan probiotic (JHJ, Poland) in the feed in the final dose of 0.5 kg of probiotic per every 999.5 kg of the feed during the entire production cycle. At the same time, birds from CH1 (group K) received the same feed but without the probiotic. Throughout the experiment, veterinary prophylaxis and therapy schedule and program were the same for CH1 and CH2 (as well as for CH1 and CH2 during three other production cycles). Before birds were placed in CH1 or CH2, 23 birds were selected at random from a transport truck in order to obtain blood samples for the serological evaluation of the time of vaccination against Infectious Bursal Disease (IBD), based on the Deventer formula, as well as for the evaluation of the level of maternally-derived antibodies against Infectious Bronchitis (IB) virus and reoviruses (REO). Vaccination program against Marek Disease (MD), IB and IBD that was executed at the farm in each production cycle is summarized in Table 2. At day 37 of birds life, 20% of the chickens from CH1 and CH2 were transferred for slaughter. At the slaughterhouse, 23 blood samples were collected from birds from K and L group independently for the serological evaluation of the level of antibodies against IB, IBD and REO. At the same time, 5 samples of freshlysqueezed feces from ileum and ceca of the chickens from group K (n = 5) and L (n = 5) were collected for microbiological examination and Campylobacter spp. enumeration. Prior to feces samples collection, abdominal skin was disinfected (40% ethanol) and intestines were dissected with the use of sterile surgical equipment (individual equipment was prepared for each bird). Additionally, samples of pectoral muscles (10 g), cut superficially (max. cut depth < 10 mm), with overlaying skin were collected from birds of group K (n = 4) and L (n = 4) for microbiological examination. At the same time, environmental samples were collected from CH1 and CH2 (n = 4 / CH). In each production cycle, the samples collected for microbiological examination were transferred on ice to a laboratory in < 3 h after they had been collected.
2. Materials and methods 2.1. Farm The study was carried out under field conditions on a chicken farm with four poultry houses (CH - chicken house) in four consecutive production cycles. It was conducted in CH1 and CH2 and the number of birds in those houses in each production cycle was (approx.): CH1 – 22,000 and CH2 – 18,000 chickens. Study layout is summarized in Table 1. In each production cycle (1 - retrospective, 2 - experiment, 3 and 4 - control), both CH1 and CH2 were settled with Ross 308 broiler chicks purchased from one hatchery and from one hatch. Feed and water were given to the birds ad libitum. Feed was provided by its producer - Wipasz sp. z.o.o. (Poland). The retrospective microbiological study was conducted on a farm (production cycle 1) in order to establish the presence of Campylobacter spp. and the diversity of its population between CH1 and CH2. This study was conducted with environmental samples (freshly excreted
2.3. Probiotic During production cycle 2 (Experiment) chickens from CH2 received in feed Lavipan probiotic product (JHJ, Poland) which comprises of selected stains of lactic acid bacteria: Lactococcus lactis IBB 500 (origin - chicken feces), Carnobacterium divergens S-1 (origin - carp gut), Lactobacillus casei ŁOCK 0915 (origin - chicken feces) and Lactobacillus plantarum ŁOCK 0862 (origin - turkey feces) in the amount of 1 × 109 colony forming units (CFU/g) each and Saccharomyces cerevisae ŁOCK 0141 (origin - plant silage) in the amount of 1 × 107 CFU/g.
Table 1 Study layout summarized. Only in production cycle 2 birds from chicken house 2 (CH2) received probiotic (Lavipan, JHJ, Poland) in feed. Sample type
1/retrospective Environmental sample Feces from intestines Pectoral muscles Blood a
Table 2 Immunoprophylaxis programme executed at the experimental farm during 4 production cycles.
Production cycle no.
+ − − −
a
2/experiment
3/control
4/control
+ + + +
+ − − −
+ − − −
Disease
Day of life
Vaccine strain
MD IB
In. ovo 1 14 16 (Based on Deventer formula)
Rispens + HVT H-120 1/96 Winterfield 2512
IBD
“+” indicates samples collected during each production cycle.
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– –
– –
2.5. Serology Infectious Bronchitis, IBD and REO serological evaluations were performed with commercial ELISA kits (IDEXX, USA). Successive steps of the procedure were performed according to the manufacturer's recommendations. ELISA was carried out with the use of the Eppendorf epMotion 5075 LH automated pipetting station (Eppendorf, Germany), BioTek ELx405 automatic plate washer (BioTek, USA) and BioTek ELx800 plate reader. The sample to positive (S/P)-ratio was calculated based on the ODs and used to express the mean (S/P)-ratio ± SD per group.
–
– 7,1 × 103 ± 13,4 × 103
–
30,9 × 103 ± 9,1 × 103⁎ 0,47 × 103 ± 0,25 × 103 7,1 × 104 ± 1,1 × 104⁎ 1 × 104 ± 0,32 × 104 2,8 × 103 ± 0,37 × 103⁎
Microbiological examination was performed in accordance with PNEN ISO 6887-1 protocol. Ten-fold serial dilutions were prepared from each sample. Next dilutions were plated on Brilliance CampyCount Agar (Oxoid, Basingstoke, UK) followed by incubation at 41.5 °C for 48 h under microaerobic conditions (CapmyGen, Oxoid, Basingstoke, UK). Every sample were prepared in triplicate. After incubation grown colonies were counted and the final results were expressed as mean CFU/ml ± SD. For each positive plate randomly chosen typical Campylobacter colonies were subcultured onto plates of Columbia blood agar (Oxoid, Basingstoke, UK) for further characterization according to the PN-EN ISO 10272-1 protocol.
2.6. Statistical analysis
– Significant differences (Manna–Whitney U test, * as p < 0,05) in comparison to the control group (K).
– –
Campylobacter spp. is a food-borne pathogen occurring all over the world (EFSA, 2016; Nielsen et al., 2000; Nachamkin et al., 2002; Miller et al., 2005; Unicomb et al., 2006). The main symptoms of campylobacteriosis in human include diarrhea, cramping, abdominal pain, and fever. Campylobacter jejuni is one of the most common causes of human gastroenteritis in many countries (Fallon et al., 2003; Hänninen et al., 2003). It remains inexplicit whether the Campylobacter spp. genus bacteria are transferred vertically from a laying hen to its progeny, however the infection of a laying hen flock with these bacteria may result in eggshell contamination, which in turn may constitute the source of infection to the newly-hatched chicks (Doyle, 1984; Shanker et al., 1986). The likelihood of bacteria isolation from broiler chickens has been demonstrated to increase with bird age (Agunos et al., 2014; Mohan, 2015; Zhang, 2008), which results from the number of factors being potential sources of infection with these bacteria. The route of birds infection is usually through the gastrointestinal tract and sources of infection include contaminated litter, rodents, flies, farm staff, and other farm animals kept at or near the production farm (Agunos et al., 2014). This points to the contribution of biosecurity aspects of a farm as an element enabling the reduction of both the incidence and the rate of Campylobacter spp. infections in poultry. However, considering the multiplicity of factors being sources and vectors of infection (and the likely vertical transmission), it seems that today the biosecurity regime alone may prove ineffective in controlling infections induced by these microorganisms. Up to date, only a few studies have evidenced the possible role of probiotics in preventing the Campylobacter spp. colonization and carcass contamination at the level of primary production. Recent studies have shown that an avian - specific multiprobiotic was able to reduce the Campylobacter jejuni and cecal colonization in broiler chickens (Ghareeb et al., 2012; Willis and Reid, 2008).
⁎
0 4,15 × 10 ± 4,98 × 10
3
– –
1
37,2 × 105 ± 0,45 × 105⁎ 3 × 105 ± 0,45 × 105
Environmental sample Feces from intestines Pectoral muscles
CH1 (K) CH2 CH1
74 × 103 ± 161 × 103
Statistical analysis was performed with GraphPad Prism 6.05 with the use of Mann Whitney U test. Differences were considered statistically significant at p < 0,05. 3. Results and discussion
2
25,3 × 103 ± 1,55 × 103
CH2 CH1 CH2 CH1 CH2 (L)
2.4. Microbiological studies
1
Production cycle no. Sample type
Table 3 Results of microbiological studies. Results are presented as the mean Campylobacter spp. CFU/ml ± SD.
3
4
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Campylobacter spp. in the environment of chicken houses CH1 and CH2. Results obtained reflected correlations observed in the retrospective study, i.e. a statistically significantly higher count of the analyzed bacteria was found in the environmental samples from CH2 than in these from CH1, in both production cycles. The reason behind differences noted in Campylobacter spp. populations between CH1 and CH2 in production cycles 1, 3 and 4 is somehow inexplicable. It could be speculated that they were due to differences in humidity levels in both chicken houses, with a greater problem with moist litter being observed in CH2. On the other hand, these differences could result from the internal system of farm management, the mode and order of handling procedures in each chickens house, or even from its location in respect of other chicken houses (the CH2 is an internal house, whereas the CH1 is an external one). Those aspects are currently under further investigation. The above-mentioned issues of Campylobacter spp. on-farm transmission have been widely reviewed previously (Agunos et al., 2014). During our study on-farm workers were not informed about the project, so the internal management and biosecurity measures did not change during the entire study.
Table 4 Results of serological analysis for day old and 37-day old broiler chickens. Levels of specific antibodies against IB, IBD and REO in chickens serum were evaluated. Results are presented as mean S/P ± SD with the number of samples reacting positive (pos) and negative (neg) in each test. Assay
Group (age)
S/P
SD
pos
neg
IB
K + L (hatch day) K (37 days) L (37 days) K + L (hatch day) K (37 days) L (37 days) K + L (hatch day) K (37 days) L (37 days)
0,51 0,32 0,34 2,35 1,23 0,81 1,94 0,27 0,47⁎
0,30 0,29 0,33 1,08 1,03 0,67 0,416 0,2 0,32
21 13 13 22 19 18 23 14 20
2 10 10 1 4 5 0 9 3
REO
IBD
⁎ Significant differences (Manna–Whitney U test, * as p < 0,05) in comparison to the control group (K).
The inhibitory effects of Lactococcus lactis IBB 500, Carnobacterium divergens S-1, Lactobacillus casei ŁOCK 0915 and Lactobacillus plantarum ŁOCK 0862 as well as yeasts Saccharomyces cerevisae ŁOCK 0141, on S. enteritidis and S. typhimurium growth in vitro has been evaluated previously and those probiotic strains revealed very promising potential to reduce the growth of pathogenic bacteria (Burchardt, personal communication, 2015).
3.2. The influence of Lavipan probiotic product on humoral immunity Probiotics significantly improve functions of the immune system of animals, including birds (Alkhalf et al., 2010; Balevi et al., 2001; Chichlowski et al., 2007; Dhama et al., 2011; Farnell et al., 2006; Fellah et al., 2014; Galdeano and Perdigón, 2006; Koenen et al., 2004; Samanya and Yamauchi, 2002). In the case of poultry, probiotics have been demonstrated to increase total amount of antibodies, including the vaccine-induced specific antibodies (Stringfellow et al., 2011; Talebi et al., 2008). Results of serological analyses were presented in Table 4. Statistically significantly higher vaccine-induced titers of antibodies against Gumboro disease virus were demonstrated in the L group of birds at 37 days of life, in comparison to the control group. These results are consistent with findings reported by Talebi et al. (2008), who demonstrated higher vaccine induced titers of anti-IBD and anti-ND (Newcastle disease) antibodies in broiler chickens administered a Lactobacillus-based multiprobiotic throughout the entire production cycle. It may, therefore, be concluded that the Lavipan probiotic product administered to broiler chickens has an immunomodulatory effect. In addition, our study indicated a tendency for the reduction of seroconversion degree towards REO in the group receiving the Lavipan preparation. Though these differences were statistically insignificant (p = 0.21) (Table 4), they suggest that this probiotic may be capable of minimizing infections with these viruses per os, by reducing the degree of its replication at the gate of infection. No differences were found in the vaccine-induced level of anti-IBV antibodies between groups K and L (Table 4). The differences in levels of antibodies against IB and IBD viruses between groups L and K may be explained by: (Agunos et al., 2014) difference in the method of vaccine application with water (IBD) or in aerosol (IB), (Alkhalf et al., 2010) various immunogenicity of vaccinal viruses, and (Balevi et al., 2001) the degree and target site of vaccine virus replication.
3.1. Influence of Lavipan probiotic product on population of Campylobacter spp. Results of microbiological analyses were summarized in Table 3. In the production cycle 1, the population number of Campylobacter spp. in the environment of CH2 was statistically significantly higher (p < 0.05) than in CH1. The differences noted between CH1 and CH2 during the retrospective study allowed choosing birds from CH2 as the study group – L in the exact experiment. The scope of microbiological tests in the Experiment was extended to three sampling sites, i.e. feces from ileum and cecum, environmental samples, and pectoral muscles. Muscle samples were collected with overlaying skin, as muscle contamination with Campylobacter spp. proceeds from the environment through the skin. The whole sampling scheme was, therefore, aimed at mimicking the route of pectoral muscles contamination with Campylobacter spp. excreted with droppings to the environment. In the Experiment, a statistically significantly lower (p < 0.05) count of Campylobacter spp. was determined in environmental samples from CH2 than from CH1 (Table 3). A lower mean population number was assayed in the intestinal samples of birds from group L compared to birds from group K, however the difference was statistically insignificant (Table 3). In contrast to birds from group K (growth of Campylobacter spp. recorded in 100% of the samples), 1 out of 4 intestinal samples of birds from group L was negative for Campylobacter spp. No growth of Campylobacter spp. was confirmed in all samples of pectoral muscles of birds from group L, whereas in group K positive results were found in 50% of the samples (Table 3). Hue et al. (2011) demonstrated that the level of carcasses contamination with Campylobacter spp. genus bacteria was directly correlated with the degree of intestine invasion with these bacteria. Considering the above and findings from our study, it may be concluded that – by reducing the possibility of Campylobacter spp. invasion in intestine lumen and, resultantly, by minimizing bird environment contamination with these bacteria – the Lavipan probiotic product contributed to the improvement of hygienic parameters of the samples of pectoral muscles. The Experiment was followed by two control studies (production cycles 3 and 4), where Lavipan preparation was no longer administered, which were aimed at determining changes in the population of
4. Conclusions It has been demonstrated earlier that probiotics inhibit the growth of pathogenic microflora in the gastrointestinal tract through competence exclusion. Simultaneously, they are increasingly indicated as an alternative method for the risk minimization of food product contamination with Campylobacter spp. bacteria through the reduction of infection degree at the primary production level (Mohan, 2015). The analysis of study results allows concluding that the Lavipan probiotic product (JHJ, Poland), that comprised of selected stains of lactic acid bacteria: Lactococcus lactis IBB 500, Carnobacterium divergens S-1, Lactobacillus casei ŁOCK 0915 and Lactobacillus plantarum ŁOCK 0862 in 315
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the amount of 1 × 109 CFU/g each and Saccharomyces cerevisae ŁOCK 0141 in the amount of 1 × 107 CFU/g, added to feed mixture for broiler chickens was capable to reduce the extent of Campylobacter spp. invasion in the gastrointestinal tract of birds and, resultantly, to diminish contamination level in bird environment, which eventually contributed to the improved hygienic parameters of the analyzed poultry carcasses. Considering the fact that the alarming epidemiological situation of campylobacteriosis in humans is, to a large extent, associated with these bacteria prevalence in slaughter poultry population, apart from the biosecurity and hygienic-technological aspects of food product manufacture, the application of the analyzed probiotic may contribute to improved consumer safety through its effect on Campylobacter spp. population reduction. In addition, Lavipan probiotic product display promising immunomodulatory properties that may successfully improve the effectiveness of the specific prophylaxis program applied in a flock of broiler chickens. Further studies are, however, needed to establish the exact mechanism of the effect of this probiotic on the immunological system of birds.
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Conflict of interest statement The authors declared no conflicts of interest. Funding Publication supported by KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal - Safe Food”, decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015. Acknowledgement The authors would like to thank MSc Szczucińska Ewa and DVW Kaczorek Edyta from Department of Microbiology and Clinical Immunology, University of Warmia and Mazury, Olsztyn, Poland, for the help with microbiological study. References Agunos, A., Waddell, L., Léger, D., Taboada, E., 2014. A systematic review characterizing on-farm sources of Campylobacter spp. for broiler chickens. PLoS One 9, e104905. http://dx.doi.org/10.1371/journal.pone.0104905. Alkhalf, A., Alhaj, M., Al-Homidan, I., 2010. Influence of probiotic supplementation on immune response of broiler chicks. Egypt Poult. Sci. 30, 271–280. Balevi, T., Uçan, U.S., Cokun, B., Kurtoglu, V., Cetingul, I.S., 2001. Effect of dietary probiotic on performance and humoral immune response in layer hens. Br. Poult. Sci. 42, 456–461. Bengmark, S., 1998. Ecological control of the gastrointestinal tract - the role of probiotic flora. Gut 42, 2–7. Chichlowski, M., Croom, W.J., Edens, F.W., McBride, B.W., Qiu, R., Chiang, C.C., Daniel, L.R., Havenstein, G.B., Koci, M.D., 2007. Microarchitecture and spatial relationship between bacteria and ileal, cecal, and colonic epithelium in chicks fed a direct-fed microbial, primalac, and salinomycin. Poult. Sci. 86, 1121–1132. Dalloul, R.A., Lillehoj, H.S., Tamim, N.M., Shellem, T.A., Doerr, J.A., 2005. Induction of local protective immunity to Eimeria acervulina by a Lactobacillus based probiotic. Comp. Immunol. Microbiol. Infect. Dis. 28, 351–361. Dhama, K., Verma, V., Sawant, P.M., Tiwari, R., Vaid, R.K., Chauhan, R.S., 2011. Applications of probiotics in poultry: enhancing immunity and beneficial effects on production performances and health - a review. J. Immunol. Immunopathol. 13, 1–19. Doyle, M.P., 1984. Association of Campylobacter jejuni with laying hens and eggs. Appl. Environ. Microbiol. 47, 533–536. EFSA, 2016. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2015. EFSA J. 14, 4634.
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