Effect of yeast mannan-rich fractions on reducing Campylobacter colonization in broiler chickens

 C 2017 Poultry Science Association Inc.

Effect of yeast mannan-rich fractions on reducing Campylobacter colonization in broiler chickens A. Corrigan,∗,1 N. Corcionivoschi,† and R. A. Murphy∗ ∗

Alltech Biotechnology, Meath, Ireland, +353 (0)18026258; and † Agri-Food and Biosciences Institute, Newforge Lane, Belfast, UK, +44 (0)2890 255662

Primary audience: Poultry Producers, Nutritionists, Veterinarians SUMMARY Campylobacteriosis is considered to be the most frequently reported cause of zoonotic illness globally, with poultry being the main source of infection. Reducing the colonization level of Campylobacter spp. in broilers entering the processing unit could make an effective contribution at reducing the incidence of zoonotic transmission of this pathogen. It is essential to search for new, natural, and sustainable strategies to reduce the incidence of this bacterium in the broiler cecum. The aim of this study was to examine whether dietary supplementation of broilers with 3 different yeast mannan-rich fraction (MRF) supplements (supplements 1 to 3) reduced the level of natural Campylobacter spp. colonization in the broiler cecum. Birds were allowed to naturally become colonized with Campylobacter spp. from the environment. Weight gains and feed conversion ratios were measured throughout production. All 3 MRF based supplements resulted in higher weight gains over 35 d when compared with the control. Broiler pens were tested at d 21 post hatch using the boot swab method and confirmed Campylobacter spp. presence in the flock. At d 35 post hatch, colonization levels were measured using serial dilution plate counts and quantitative polymerase chain reaction (qPCR) of cecal material. Enumeration of Campylobacter colonization level in cecal content by qPCR showed that both supplement 2 and supplement 3 significantly reduced the levels of Campylobacter spp. colonization in the broiler cecum at 35 days. In conclusion, the results of this study demonstrate that MRF based supplements have the ability to significantly reduce Campylobacter colonization level in the broiler cecum while also offering an improvement in weight gain. Key words: qPCR, natural challenge, prebiotic, MOS, cecum 2017 J. Appl. Poult. Res. 00:1–8 http://dx.doi.org/10.3382/japr/pfx002

DESCRIPTION OF PROBLEM Campylobacter is regarded as the most important causal pathogen of foodborne gastrointestinal disease in humans, with the majority of infections caused by C. jejuni [1]. Campylobacter species are widely distributed in most warm-blooded animals and are prevalent in the intestines of food animals such as poultry, 1

Corresponding author: [email protected]

cattle, and pigs [2]. Source attribution studies have identified contaminated poultry products as the largest reservoir of this pathogen and the most likely source of human infection [3, 4]. Campylobacter spp. can colonize the poultry gastrointestinal tract in large numbers, frequently at levels higher than 106 to 108 colony forming units (CFU)/g of cecal content [5]. Colonization is typically highest in the broiler cecum and primarily present in the mucous layer [6]. Typically, Campylobacter colonization can

2 be detected in a broiler flock at around 14 d of age and all broilers can be colonized by the end of rearing in a positive flock [5]. Experimentally, it was shown that a low dose (less than 40 CFU) of C. jejuni is sufficient for a bird’s full colonization, which can then lead to full flock contamination in 48 hours [7]. In 2008, a survey by the European Food Safety Authority (EFSA) reported that approximately 86% of broiler carcasses across Europe harbored Campylobacter [8]. One way to reduce the incidence of human campylobacteriosis acquired from poultry-associated sources would be by decreasing Campylobacter levels at the broiler farm [9]. European farmers have recognized the issue and have co-operated extensively by including interventions to minimize the carriage of Campylobacter through the food chain. Despite these interventions, Campylobacter contamination levels remain high and poultry producers are still under significant pressure from retailers to produce broiler meat free from Campylobacter contamination. The most effective intervention on farm to date has included improved biosecurity with no single solution being recognized as successful [10]. While Campylobacter-negative broiler flocks can be achieved through these tremendous biosecurity efforts, taking this route is currently not feasible for the vast majority of broiler farms [10]. Consequently, the use of dietary interventions using natural, non-antibiotic, compounds represents one possible approach to reduce the carriage of this pathogen in the host. A natural alternative for the control of Campylobacter colonization that is economically viable for the producer and does not constitute a risk for the health of humans, animals, or the environment is most desirable. In spite of much research, no natural, antibiotic free, nutritional Campylobacter control strategies have been successfully identified and implemented in the poultry industry. Many studies in the literature have shown that some natural compounds have bioactivity against Campylobacter proliferation with few showing success in animal studies [11, 12]. This topic has been reviewed extensively by Meunier et al. [13]. The current study aimed to examine the effects of 3 yeast-based mannan-rich supplements at reducing Campylobacter colonization in broilers. A

JAPR: Research Report natural Campylobacter challenge was used, as opposed to an artificial challenge with a high dose pure culture Campylobacter strain, in an effort to reflect typical Campylobacter colonization in a commercial setting. We used both bacteriological- and molecular-based methods to enumerate Campylobacter colonization levels in the broiler cecum following 35 d of dietary supplementation. Weight gains and feed conversion ratios of the supplemented groups also were measured.

MATERIALS AND METHODS Experimental Design, Sample Collection, and Preservation A total of 492 day-of-hatch male broiler chickens was used in this study. Clean concretefloor pens were used to house the birds in a medium scale trial facility on-site at Agri-Food Biosciences Institute (AFBI) (Belfast, UK). Animals were randomly split into 4 groups of 3 pens, with 12 pens in total (41 birds/pen; 123 birds/group) using a randomized complete block design. The pens were divided into 4 groups: group 1, fed a basal diet; groups 2 to 4, fed a basal diet that included supplements 1, 2, and 3, respectively, at the manufacturers’ recommended R inclusion levels (supplement 1 = Natustat NS R  [14], supplement 2 = Actigen -pak [15], and R supplement 3 = Powertract [16]). These supplements were mannan-rich fractions extracted from the yeast cell wall of Saccharomyces cerevisae. Basal diets were prepared by a commercial feed mill and consisted primarily of wheat and soybean meal, as outlined in Table 1. Starter diets were fed from d zero to d 10, grower diets from d 11 to 25, and finisher diets from d 26 to d 35. Feed and water were provided ad-libitum throughout the study. Each pen was dressed with fresh litter for bedding from d zero. The temperature was initially set at 30◦ C per d up to d 10 and then decreased linearly by 1◦ C every second day. During the experiment the birds received a lighting regimen of 16 h light and 8 h darkness until d 35. All conditions were kept uniform for all 4 groups. All birds were weighed and the values averaged by pen on d 0, 7, 21, and 35. Feed intake was measured in order

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Table 1. Basal diet composition of starter, grower, and finisher rations. Ingredients

Starter

Grower

Finisher

Wheat Full fat whole soya Soybean meal Limestone Di-calcium phosphate Soyabean oil Salt Sodium bi-carbonate DL Methionine L-Lysine Threonine Vitamin-mineral premix

54.62 12.00 25.00 0.72 1.65 4.00 0.20 0.20 0.49 0.37 0.25 0.50

57.55 12.00 21.00 0.70 2.00 5.00 0.20 0.17 0.44 0.32 0.13 0.50

61.30 12.00 17.00 0.50 2.15 5.50 0.20 0.16 0.38 0.28 0.03 0.50

Nutrient analysis % or as indicated Metabolizable Energy (Kcal/kg) Crude Protein Lysine Methionine + Cysteine Calcium Available Phosphorous

2,999.00 23.12 1.45 1.09 0.97 0.49

3,081.00 21.53 1.31 1.00 0.91 0.41

3,133.80 20.04 1.17 0.91 0.85 0.41

Vitamin-Mineral Premix1 Copper (mg) Iodine (mg) Iron (mg) Manganese (mg) Selenium (mg) Zinc (mg) R (g)2 Synergen Vitamin A (IU) Vitamin D3 (IU) Vitamin E (IU) Vitamin K (mg) Thiamin (B1) (mg) Riboflavin (B2) (mg) Niacin (mg) Pantothenic Acid (mg) Pyridoxine (B6) mg Biotin (mg) Folic Acid (mg) Vitamin B12 (ug) Vitamin C (mg) Choline (mg)

16 1.1 30 110 0.3 105 200 13,000 5,000 80 3 3 9 60 20 5 0.25 2 200 200 500

16 1.1 30 110 0.3 105 200 11,000 4,750 60 3 2.5 7 55 15 4 0.225 1.8 175 200 450

16 1.1 30 110 0.3 105 200 10,000 4,500 50 2.5 2 6 50 12 3 0.2 1.6 150 200 400

1 2

Vitamin-mineral premix manufactured for this trial by Target feeds, Shropshire UK. R Synergen is a byproduct of enzyme production with Aspergillus niger produced by Alltech, Inc.

to assess feed conversion ratios and weight gain. The intact cecal pouches of 10 randomly caught birds per pen were removed immediately after euthanization at d 35, and the cecal contents were placed in sterile tubes. The tubes were then flash-frozen in liquid nitrogen, lyophilized, and stored at −80◦ C for further analysis. All procedures were subject to the approval of the local Animal Welfare Ethics Review Board and sub-

sequent approval by a Home Office Inspector. All procedures were carried out under the strict guidelines of the Animal (Scientific Procedures) Act 1986. Bacteriological Analysis Prior to bird placement and at 21 d post hatch, screening of the broiler shed was carried out

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4 to test for Campylobacter spp. presence using the boot swab method and confirmed their presence. Briefly, boot socks were walked around the shed and were then placed in a stomacher bag with 50 mL of maximum recovery diluents (MRD) and stomachered at 260 rpm for one minute [17]. One hundred microliters of the suspension were then transferred to each of 2 plates of Modified Charcoal-CefoperazoneDeoxycholate Agar agar (mCCD) [18] and incubated in a microaerobic atmosphere (85% N2, 10% CO2, and 5% O2) at 41.5 ± 1◦ C and examined after 44 h ± 4 h for typical and/or suspect colonies of Campylobacter spp. [19]. In addition, to enumerate Campylobacter colonization levels of the broiler ceca at 35 d post hatch, cecal contents of 12 birds per group were analyzed using plate count methods. The enumeration method was based on those described in British Standard BS EN ISO 10272:2006 [20]. Briefly, 1 g of cecal content was added to 9 mL of MRD and vortexed vigorously followed by the preparation of serial dilutions down to 10−8 in MRD. First, one mL of the 10−1 dilution was spread across 3 mCCDA plates and for each dilution 100 μl of suspension was spread onto duplicate mCCDA. Plates were incubated for 48 h (±4 h) at 41.5◦ ± 1◦ C in a microaerobic atmosphere. After incubation the plates were examined for typical Campylobacter morphology and plates with less than 150 colonies were used for enumeration. DNA Extraction DNA was extracted from cecal contents using the QIAamp DNA Stool Mini Kit according to the manufacturer’s instructions [21]. Genomic DNA concentration was determined at a wavelength of 260 nm using a NanoDrop [22]. For qPCR analysis, DNA samples were diluted in sterile water to a concentration of 10 ng/μl. Campylobacter Enumeration Quantitative PCR experiments were carried out with a Light Cycler 96 system and the instrument software was used to define the cycle threshold (CT) values using the second derivative method. For determination of Campylobacter spp., the genus primers 5 -CTGCTTAA-

CACAAGTTGAGTAGG-3 , 5 -TTCCTTAGGTACCGTCAGAA-3 , and Campy Probe 5 FAM-TGTCATCCTCCACGCGGCGTTGCTGC-TAMRA-3 were used. PCR reaction conditions are outlined in [23]. The total reaction volume was 20 μl containing: Roche Faststart Essential DNA Probe Master (2X) 10 μl, primers 10 pmol/reaction, primer probe 5 pmol/reaction, template DNA up to 50 ng/reaction, and ultrapure water up to 20 μl. All assays included positive, negative, extraction, and internal amplification controls. Three independent experiments were performed. Statistical Analysis Weight gains and feed conversion ratios were analyzed for statistical differences among groups using the Tukey’s honest significant difference (HSD) test. Results from microbiological analysis and real-time qPCR were subjected to log transformation and assessed for statistical differences using an unpaired student t test. A value of P < 0.05 was considered significant in all cases.

RESULTS AND DISCUSSION Animal Performance Three nutritional supplements were tested for their effect on bird performance by measuring body weight gain and feed conversion (Table 2). Results from this analysis have shown that following 10 d supplementation with the starter diet, body weight gains were significantly increased in supplement 2 (P < 0.05) (Table 2). Significant increases in body weight gains also were observed in both supplement 2 and supplement 3 (P < 0.05) for the grower diets when compared to the control (Table 2). No differences in body weight gains were noted in the supplemented groups for the finisher diets compared with the control (Table 2). Dietary supplementation with all supplements resulted in higher weight gains over 35 d when compared with the control. Supplements 2 and 3 showed the greatest improvement in weight gain with 190 g and 125 g improvements in average bird weights over 35 d, respectively, when compared with the control. Feed conversion ratios were not different

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Table 2. Effect of experimental diets on growth performance of broilers (mean ± SD). Treatment Starter pphase (Day 0 to10) Control Supplement 1 Supplement 2 Supplement 3 P-value Grower Phase (Day 11 to 25) Control Supplement 1 Supplement 2 Supplement 3 P-value Finisher Phase (Day 26 to 35) Control Supplement 1 Supplement 2 Supplement 3 P-value Overall (Day 0 to 35) Control Supplement 1 Supplement 2 Supplement 3 P-value

Body weight (g)

Total gain (g/head)

Average daily gain (g/head)

Feed intake (g/head)

Feed:gain ratio (Kg:Kg)

238 ± 22a 256 ± 6a,c 272 ± 9b,c 262 ± 9a,c 0.044

198 ± 22a 216 ± 6a,c 232 ± 9b,c 222 ± 9a,c 0.044

20 ± 2a 21 ± 1a,c 23 ± 1b,c 22 ± 1a,c 0.044

246 ± 11 246 ± 7 257 ± 3 247 ± 13 NS

1.25 ± 0.10 1.14 ± 0.02 1.11 ± 0.03 1.11 ± 0.03 NS

1243 ± 7a 1311 ± 33a,c 1411 ± 64b,c 1401 ± 78b,c 0.027

1005 ± 26 1054 ± 34 1139 ± 70 1139 ± 69 NS

67 ± 2 70 ± 2 76 ± 5 76 ± 5 NS

1615 ± 37 1537 ± 169 1801 ± 161 1732 ± 46 NS

1.61 ± 0.06 1.45 ± 0.11 1.58 ± 0.05 1.52 ± 0.07 NS

2380 2394 2570 2505

± 18 ± 197 ± 91 ± 54 NS

1136 ± 13 1083 ± 164 1159 ± 36 1104 ± 31 NS

114 ± 1 108 ± 16 116 ± 4 110 ± 3 NS

2172 ± 48 2284 ± 85 2330 ± 152 2275 ± 104 NS

1.91 ± 0.04 2.14 ± 0.26 2.01 ± 0.11 2.06 ± 0.07 NS

2380 2394 2570 2505

± 18 ± 197 ± 91 ± 54 NS

2340 ± 10 2354 ± 114 2530 ± 53 2465 ± 31 NS

67 ± 0 67 ± 3 72 ± 2 70 ± 1 NS

4032 ± 46 4068 ± 143 4388 ± 179 4253 ± 35 NS

1.72 ± 0.02 1.73 ± 0.02 1.74 ± 0.04 1.73 ± 0.03 NS

Means within a column for each treatment phase not sharing a common superscript differ significantly (P < 0.05). Each value represents the mean of n = 3 pens per diet with 36, 30, and 30 birds per pen, per growing phase, resepectively. Comparisons between groups were made using Tukeys HSD test and a probability of P < 0.05 was considered for statistical significance. NS = not significant. a–c

between control and supplemented groups over 35 days. The inclusion of yeast-derived, mannan-rich fractions as dietary supplements in broilers traditionally plays a role in improved production performance parameters [24–26]. The results shown here suggest a positive role in terms of production performance benefits as a result of dietary inclusion, as body weight gains were significantly improved in starter and grower diets. While most studies report performance benefits for mannan-rich fraction inclusion in the diets over 42 d, performance benefits also have been reported during the starter and grower phases [27, 28]. Previous meta-analysis studies have shown that mannan-rich dietary interventions lead to significant improvements in body weight gains and feed conversion ratio over 42 d when included in broiler diets with an average 5.41% increase in body weight reported in broilers [25, 29]. Following 35 d supplementation, supplements 2 and 3 showed an 8 and 5% increase

in body weight, respectively, compared to the control. While the interpretation of these results is limited due to the bird numbers, the performance improvements observed here and in previous studies suggest that these dietary supplements do not have a negative impact on performance and could provide benefits for largescale production units. Bacteriological Analysis and Campylobacter Enumeration Direct plate counts of cecal content from 12 birds per group were carried out at 35 d post hatch to enumerate Campylobacter colonization levels of the broiler ceca (Table 3) and results indicated that all 4 groups had been successfully colonized by Campylobacter. All 3 supplemented groups had lower levels of Campylobacter colonization when compared with the control, with treatments 2 and 3 showing a 0.4 and 0.3 log unit reduction, respectively. In

JAPR: Research Report

6 Table 3. Effect of dietary supplementation on mean Campylobacter concentration in cecal content at 35 d post hatch measured using both plate counts and qPCR methods. Treatment

Plate count (CFU/g)

qPCR counts (gene copy no./g)

Control Supplement 1 Supplement 2 Supplement 3 P-value

9.03 ± 8.62 9.01 ± 8.56 8.59 ± 8.34 8.70 ± 8.50 NS

6.58 ± 1.54a 6.18 ± 1.01a,c 5.05 ± 0.89b,c 5.35 ± 1.05b,c P < 0.001

 Values

represent the mean ± SEM of 12 individual ceca per group for plate counts and n = 30 ceca per treatment group for qPCR. a–c Means within a column for each method not sharing a common superscript differ significantly (P < 0.05). Comparisons between control and each treatment were made using unpaired student t test and a probability of P < 0.05 was considered for statistical significance. NS = not significant.

order to increase the sensitivity of detection of Campylobacter spp., real-time quantitative PCR enumeration was carried out on DNA extracted from 30 cecal samples per group at 35 d post hatch. Our results show that all 3 supplements had reduced Campylobacter colonization levels in the ceca when compared with the control (Table 3). Supplement 2 and supplement 3 performed best showing statistically significant reductions in the levels of Campylobacter colonization compared with the control (P < 0.05). When compared with the control, supplement 2 showed a 1.5 log reduction in Campylobacter colonization level and supplement 3 showed a 1.2 log reduction. Ingestion of C. jejuni numbers as few as 40 CFU can be sufficient for successful colonization of chicks with subsequent fecal shedding allowing for full flock colonization rapidly [5]. A natural Campylobacter challenge method, as opposed to an artificial challenge with a pure culture of Campylobacter, was used in this study to reduce strain variation in colonization dynamics [5, 30] and to be most reflective of colonization conditions in commercial production. Bacteriological examination at d 35 confirmed the presence of Campylobacter in the broiler ceca indicating that this was a suitable method to elicit a natural Campylobacter challenge. This method eliminates extrinsic factors that can affect an artificial Campylobacter challenge and has previously been successfully used by Gormley et al. [31].

The main objective of this study was to determine the effect of dietary supplementation at reducing Campylobacter colonization level in broilers at the end of the production cycle. The results from this study have shown that by using a plate count method, the levels of Campylobacter colonization in the broiler ceca were reduced in all 3 supplements compared with the control at d 35 post hatch, but not significantly. Previous studies have shown that culture-based methods provide a good indication of Campylobacter colonization but can be inaccurate. The inaccuracy may be due to the selective nature of enrichment and plating or due to the fact that Campylobacter cells are able to enter a viable but unculturable state [32]. As such, we additionally chose to use sensitive molecular based qPCR techniques to enumerate Campylobacter colonization level and detect differences between control and supplemented groups. This technique shows improved accuracy at enumerating Campylobacter regardless of the physiological state of the Campylobacter cells compared with plate counts but has the potential to amplify DNA from dead but intact cells [33]. Results from qPCR analysis mirrored those from plate counting, showing that all 3 supplements reduced cecal Campylobacter colonization level. These results however, showed a much greater reduction in cecal Campylobacter colonization level from supplements 2 and 3 compared to plate counts and were shown to be significant. These results indicate that qPCR may be a rapid and reliable alternative to plate counts, which is sensitive enough to detect differences between nutritional treatments in order to assess their ability to reduce Campylobacter colonization level in broilers. The main component of each of the 3 supplements used in this study was a yeast mannanrich fraction. There are limited studies in which mannan-rich fractions have been shown to inhibit Campylobacter colonization in broilers [34]. Mannan oligosaccharides have been shown to significantly decrease the levels of Campylobacter counts in cecal contents at 34 d post hatch when included in the diet of broilers; however, these results indicated only a 0.5 log reduction [35]. In-vitro studies have also shown that mannan oligosaccharides have a negative effect on the ability of Campylobacter to colonize intestinal cells [36]. The exact mode of action by

CORRIGAN ET AL.: MANNANS LOWER CAMPYLOBACTERS which MRF can reduce Camplyobacter is not known. Mannan-rich fractions may act indirectly by altering the bacterial community composition [37, 38] or intestinal structure [35], or by altering the immune system [39], which in turn may be responsible for the reduction in Campylobacter in broiler ceca. The yeast mannan-rich fraction, supplement 2, used in this study showed a 1.5-log reduction in Campylobacter colonization level compared with the control under the trial conditions in this experiment. Quantitative risk assessments have indicated that reducing the Campylobacter counts from poultry carcasses by 2-log units could reduce the incidence of human campylobacteriosis by up to 30 times [40]. Given that the nutritional intervention strategy used in this study has reduced the cecal Campylobacter colonization level in broilers at the end of the production cycle and taken together with mitigation strategies in processing units, the levels of Campylobacter colonization on poultry carcasses may have the ability to be reduced below this critical threshold using this nutritional supplement.

CONCLUSIONS AND APPLICATIONS 1. The results of this study demonstrate that yeast mannan-rich supplements have the ability to significantly reduce Campylobacter colonization levels in the broiler ceca while also offering an improvement in weight gains. 2. The dietary supplements used in this experiment significantly improved weight gains in starter and grower diets and could provide benefits for commercial production units. 3. Further studies will need to be undertaken to evaluate the effectiveness of supplements 2 and 3 at reducing Campylobacter colonization levels in commercial production conditions.

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