Development of a feed additive to reduce caecal Campylobacter jejuni in broilers at slaughter age: from in vitro to in vivo, a proof of concept

Authors


Correspondence

Ester Grilli, Dipartimento di Scienze Mediche Veterinarie, via tolara di Sopra 50, 40064, Ozzano Emilia, BO, Italy. E-mail: ester.grilli@unibo.it

Abstract

Aim

In vitro and in vivo challenge studies were undertaken to develop an in-feed additive of microencapsulated propionic, sorbic acids and pure botanicals to control Campylobacter jejuni in broilers at slaughter age.

Methods and Results

Organic acids (OA) and pure botanicals were tested in vitro against Camp. jejuni, whereas in vivo, chickens were fed either a control diet, or increasing doses of the additive for 42 days (experiment 1); in the second experiment, chickens received the additive at 0·1 or 0·3% from day 0 to 21 or from day 22 to 42. The additive consistently reduced Camp. jejuni caecal counts at any given dose (exp. 1) or inclusion plan (exp. 2). Moreover, it was able to reduce the number of goblet cells and modify mucin glycoconjugates biosynthesis pattern.

Conclusions

We developed an additive that was effective in reducing Camp. jejuni in slaughter-age chickens even at low doses (0·1%). That efficacy was the result of the synergistic action between OA and botanicals.

Significance and Impact of the Study

This study provides a strategy to reduce Camp. jejuni in broilers and, as a consequence, to improve the safety of the food chain. Moreover, data suggest that a treatment limited to the last weeks before slaughter would allow to save on inclusion of the additive throughout the whole production cycle.

Introduction

Campylobacter is one of the leading causes of foodborne illness both in the United States and in Europe (EFSA 2009; CDC 2011), and undercooked poultry meat is considered the primary route of infection (EFSA 2010). Carcass contamination is correlated to intestinal colonization, and it is estimated that a 2 log10 reduction in intestinal Campylobacter would decrease the human risk of infection by 30-fold (Van Deun et al. 2008). Campylobacter was recognized as an emerging foodborne pathogen relatively recently (Bryant 1983), and research conducted on strategies to reduce Campylobacter in food animals has mostly been conducted in the past 10 years (Newell et al. 2011). Efforts in reducing Campylobacter have largely been directed towards biosecurity improvement and in-feed/water additives like phages, bacteriocins, pre- and probiotics, organic acids (OA) and botanicals, but none of them, alone, was proven to be completely effective, reliable and reproducible (Lin 2009; Hermans et al. 2011).

Multi-hurdle technologies, that is, the combination of different feeding strategies like OA and botanicals, can effectively reduce Salmonella in broilers, provided that they can reach the large intestine where infection occurs (Grilli et al. 2011).

The aim of this study was to develop a feed additive that combines OA and pure botanicals in a microcapsule and to test its efficacy against Campylobacter jejuni through in vitro prescreening and in vivo experiments. To our knowledge, this is a unique study because the combination of OA and botanicals in a lipid embedding matrix has never been tested against Campylobacter in chickens. Moreover, as diet components and feed additives can alter mucin composition and carbohydrate expression of goblet cell glycoconjugates, in association with a reduced intestinal number of Campylobacter jejuni (Fernandez et al. 2000), we wanted to investigate whether the feed additive had an effect on mucin biosynthesis of goblet cells.

This stepwise procedure allowed us to narrow down from a long list of possible candidate OA and botanicals to propionic acid, sorbic acid, thymol and eugenol and to demonstrate their synergistic activity in vitro. In vivo doses and modes of employment of the blend were also studied to provide the end-user with sound science in support of a practical application of this novel technology.

Materials and methods

Campylobacter cultures and growth conditions

Campylobacter jejuni ATCC 33291 (Oxoid, Basingstoke, UK) and strain 923/2010 (IZSLER biobank, Forlì, Italy), isolated from a laying hen flock, were used in the in vitro antimicrobial assay and in the in vivo experiments. Campylobacter jejuni 33291 strain was grown in brain heart infusion broth (BHI; Oxoid) through two subsequent 48-h incubations before enumeration on BHI agar at 42°C in microaerophilic conditions. Campylobacter jejuni 923/2010 was titrated on modified charcoal cefoperazone deoxycholate agar (mCCDA-Preston; Oxoid), and dilutions were made in Ringer lactate solution (Oxoid).

Chemicals and test solutions

Pure botanical and organic acid objects of the study were carvacrol, eugenol, salicylaldehyde, vanillin and lactic acid (Fluka, Sigma-Aldrich Corporation, St Louis, MO, USA); camphor, thymol, benzoic acid, caprylic acid, citric acid, formic acid, fumaric acid, d-gluconic acid, heptanoic acid, iso-butyric acid, dl-malic acid, propionic acid, sorbic acid, succinic acid, tartaric acid and n-valeric acid (Sigma-Aldrich), respectively. Acetic acid was purchased by Carlo Erba Reagents (Rodano, Milano, Italy). Twofold dilutions of each substance dissolved in BHI were prepared over a range of 1000–3·9 mmol l−1 and of 125–0·49 mmol l−1 for OA and pure botanicals, respectively. Solutions of heptanoic acid, salicylaldehyde, carvacrol, thymol, camphor, eugenol and vanillin were prepared in BHI with the addition of ethanol 70% at a final concentration of ethanol ≤5% (v/v) to increase their solubility. All the solutions were adjusted to pH 6·5 and filter-sterilized with a membrane pore size of 0·22 μm (Millipore, Billerica, MA, USA).

Antimicrobial assay 1

The minimal inhibitory concentrations (MIC) of the test substances were determined using a broth dilution method. Briefly, all tests were performed in 5-ml disposable tubes containing appropriate dilution of each substance and 48-h cultures of Camp. jejuni 33291 at 5 log10 CFU ml−1. The tubes were incubated in microaerophilic conditions at 42°C for 48 h. Bacterial growth was indicated by the presence of turbidity in the tube and confirmed by spreading 100 μl of broth of each tube onto BHI agar. After 48 h of incubation, the growth of viable cells was observed. The MIC was defined as the lowest concentration effective in killing more than 99·9% of the initial inoculum. Control strains grown with 5% (v/v) of 70% ethanol were also included in each test to exclude an inhibition due to ethanol.

Antimicrobial assay 2

Based on the results of the first antimicrobial assay, propionic and sorbic acid, and thymol and eugenol were selected and tested also on Camp. jejuni 923/2010. Then, appropriate concentrations were mixed (at a mass ratio of 58, 19·5, 18·6 and 3·9%, respectively) and dissolved in BHI broth with 10% of 70% ethanol. This stock solution was buffered to pH 6·5, filter-sterilized and then diluted into fresh sterile BHI to reach final concentrations of 0·25, 0·2, 0·15, 0·1, 0·05, 0·04, 0·03, 0·02% and 0. Campylobacter strains (33291 and 923/2010) were incubated with the solutions at a concentration of 105 CFU ml−1 at 42°C, and the assay was performed as previously described.

Feed additive

The experimental blend of propionic and sorbic acid, and eugenol and thymol tested in vitro was microencapsulated through a spray-chilling with lipid embedding matrix method (EU Patent no. 1391155 B1, Canadian Patent no. 2433484, US Patent no. 7 258 880; Vetagro Spa, Reggio Emilia, Italy). The ingredients were protected to allow their slow release in the gastrointestinal tract of birds (Piva et al. 2007).

Chickens' growth conditions

Chickens used in both experiments were housed in isolation units (0·9 m2; HM1500®, Mountair-Andersen B.V., Sevenum, Netherlands) equipped with bedding straw, drinkers, heating lamps and a filtered air supply. The light/dark hours programme was 23 : 1 during the first days and then gradually decreased to 16 : 8 within 1 week.

Female Ross 308s were fed a mash diet, which was previously tested for Campylobacter spp absence. Moreover, before the start of each experiment, 25 chickens were euthanized to check Campylobacter in liver, spleen, ceca and yolk sac. Both studies were conducted at the Istituto Zooprofilattico di Forlì in accordance with the published guidelines for animal welfare and protection (directive no. 86/609/EEC and Italian Law Act, Decreto Legislativo no. 116, issued on 27 January 1992).

Experimental setup

Experiment 1

Seventy-five-day-old chickens were divided in five isolation units, each one assigned to one of the following dietary treatments: positive control (challenged, not treated with the blend, CTR), and challenged and treated with 0·1, 0·3, 0·5 and 1% of the experimental feed additive. Birds received the additive since the beginning of the study (day 0) via the feed, which was allowed ad libitum as well as water supply. At day 19, cloacal swabs were randomly collected to verify the absence of Camp. jejuni, and at day 21, birds were challenged via endoesophageal inoculation with 1 ml of saline solution containing 107 CFU of Camp.jejuni 923/2010. At day 22, day 23 and day 24 of the study, another set of cloacal swabs was collected to verify Camp. jejuni infection and at 14 and 21 days postchallenge (day 35 and 42 of the study, respectively), five and ten animals per unit were euthanized, and ceca contents were collected to perform Camp. jejuni counts.

Experiment 2

Seventy-five-day-old chickens were sourced from a local hatchery and randomly allocated into five isolation units and assigned to one of the following dietary treatments: a challenged control not treated with the additive (CTR), or the additive added at 0·1 and 0·3% from 0 to 21 day (group 1 and 2, respectively) or from 22 to 42 day (group 3 and 4, respectively). Feed and water were provided ad libitum. The challenge and sampling for Campylobacter were conducted as described in experiment 1.

Moreover, at sacrifice, ceca samples were collected from each animal of CTR, group 3 and 4, fixed in 4% paraformaldehyde in 0·01 mol l−1 phosphate-buffered saline (PBS) pH 7·4 for 24 h at 4°C, dehydrated in a graded series of ethanol, cleared with xylene and embedded in paraffin. Serial microtome sections (4 μm thickness) were obtained from each sample to perform histochemical and immunohistochemical analyses.

Campylobacter analysis

Samples collected from both experiments were assessed for Campylobacter presence following amended ISO procedure (10272:1995). Briefly, cloacal swabs or 5 g of caecum content was pre-enriched in Preston broth (Oxoid) at 42°C for 18 h and streaked onto mCCDA (48 h at 42°C). Direct counts of Campylobacter were performed by serially diluting 1-g samples in saline solution and plating out onto mCCDA. After 48-h incubation at 42°C, the number of colonies was counted and biochemical tests were performed for confirmation.

Histochemistry and histometry

The number and the histochemical characterization of goblet cells, as well as the caecal mucin profile, was determined by staining the sections with the Alcian blue 8GX pH 2·5-periodic acid Schiff (AB-PAS) sequence, which reveals neutral (PAS-reactive, purple stained) and acid (AB-reactive, azure stained) glycoconjugates. The number of goblet cells per 100 enterocytes was determined in 10 full-length intestinal folds of each sample (at 200×) utilizing an Olympus BX51 light microscope, equipped with the DP software for the image analysis (Olympus, Italia, Milano, Italy). In addition, separate counts were performed on AB-reactive, PAS-reactive and AB-PAS-reactive mucous cells to describe the mucin profile in the different experimental conditions. Finally, the high iron diamine–alcian blue 8GX pH 2·5 (HID-AB) sequence was applied, which demonstrates sulphated (diamine-positive, brown-black stained) and sialylated (AB-reactive, azure stained) glycoconjugates, respectively. This latter histochemical stain aimed at further characterizing the acidic glycoconjugates. The observer was unaware of the origin of the sections.

Immunohistochemistry and immunofluorescence

The presence and quantification of spiral-shaped bacteria belonging to Campylobacter genus in the caecal intestinal folds was evaluated as follows.

Deparaffinized rehydrated sections were treated with 3% H2O2 in distilled water to block the endogenous peroxidase activities, and they were then rinsed with PBS and incubated with Normal Goat Serum (Dako, Italia, Milano, Italy) diluted 1 : 20 for 30 min to reduce nonspecific background staining. Subsequently, sections were incubated with rabbit (polyclonal) anti-Helicobacter pylori (code B0471; Dako) antibody (primary antibody) diluted 1 : 40 in PBS pH 7·4 plus 1% bovine serum albumin (BSA) overnight at 4°C. The antibody detects spiral-shaped bacteria belonging to more than one genus (Helicobacter spp, Campylobacter spp; Ceelen et al. 2007). Antigen–antibody complexes were detected with anti-rabbit EnVision+ System Labelled Polymer-HRP (Dako North America Inc, USA) applied for 60 min at room temperature. Appropriate washings with PBS were performed at each step, and all incubations were carried out in a moist chamber. The reaction products were revealed with a predosed diaminobenzidine liquid chromogen (DAB, code K3468; Dako North America, Inc., Carpinteria, CA) as the substrate. When appropriate, the reaction was stopped by immersion in deionized water, and the sections were slightly counterstained with Mayer's haematoxylin, dehydrated and coverslipped.

The intestinal folds (ifs) revealing in their lumen the presence of immunohistochemically identified Campylobacter spp micro-organisms (Campylobacter-IR ifs) were counted with the light microscope and DP image analyzer in at least 40 ifs/section, and the results were expressed as percentages of positive intestinal folds.

Other paraffin-embedded sections were de-waxed and incubated with the above-mentioned primary antibody (same dilution and same conditions as above). The sections were then washed in PBS, and subsequently the secondary antibody, DyLight®488 Anti-rabbit IgG (9 μg ml−1 of 10 mmol l−1 HEPES, 0·15 mol l−1 NaCl, pH 7·5, 0·08% sodium azide; Vector Laboratories Inc., Peterborough, UK), was applied for 1 h at room temperature. Finally, the sections were embedded in Vectashield Mounting Medium (Vector Laboratories Inc.) and examined using a Confocal Laser Scanning Microscope (FluoView FV300; Olympus) equipped with the FluoView software for image analysis (Olympus). The immunoreactive structures were excited using Argon/Helio-Neon-Green lasers with the excitation and barrier filter set for the used fluorochrome. For quantification of Campylobacter spp peaks of immunofluorescence, the laser power and photomultiplier tube voltage were constant so that fluorescence intensities of various samples could be compared. Images were digitized under constant gain and laser offset, with no postcapture modifications. Before quantification, the images were digitally zoomed three times, and ten intestinal folds per section were selected for measurements. The lumen areas enclosed between each pair of intestinal folds were defined manually. Pixel intensities were determined using the histogram/area function of the FluoView software, which assigned the gray levels (GL) within a 0–256 Gy scale. Data were presented as mean fluorescence intensity.

Statistical analysis

Campylobacter jejuni mean log10 CFU g−1 of caecal content was analysed with anova contrasts with Statistica (ver. 10, StatSoft Inc., Tulsa, OK, USA). Each chicken was considered the experimental unit, and differences were considered significant at P < 0·05.

Quantitative histochemical and immunohistochemical data were analysed with the mixed model anova, which included the fixed effects of treatment, and the random effect of each chicken (SAS ver. 9.2; SAS Inc., Cary, NC, USA). Values from each chicken were considered as the experimental unit of all response variables. The data are presented as least-square means ± SEM. Differences between means were considered significant at < 0·05.

Results

In vitro antimicrobial assays

The results of the in vitro antimicrobial assays are shown in Table 1 and 2. Generally, Campylobacter jejuni 33291 was more sensitive to botanicals than to OA. In particular, the most effective botanicals were eugenol, carvacrol, salicylaldehyde and thymol with MIC values of 0·12, 0·12, 0·38 and 0·47%, respectively (Table 1). Among OA, citric, fumaric, succinic, acetic, formic, butyric, valeric, lactic and tartaric failed to inhibit the growth of Camp. jejuni at the highest concentration tested (1 mol l−1, ranging from 4·6 to 19·2% w/v, depending on the acid). The most effective OA were benzoic and propionic with MIC values of 0·38 and 0·47% (31·25 and 62·5 mmol l−1, respectively; Table 1).

Table 1. Minimal inhibitory concentration (MIC) of organic acids and botanicals against Campylobacter jejuni 33291 (n = 3)
BotanicalsMICTCA-acidsaMICSCFAbMICOthersMIC
mmol l−1%mmol l−1%mmol l−1%mmol l−1%
  1. a

    Krebs cycle acids.

  2. b

    Short-chain fatty acids.

Camphor62·500·95Citric>1000>19·20Acetic>1000>6Benzoic31·250·38
Carvacrol7·810·12Fumaric>1000>11·60Butyric>1000>8·8Caprylic62·500·9
Eugenol7·810·12Malic2503·35Formic>1000>4·6Heptanoic31·250·41
Salicylaldehyde31·250·38Succinic>1000>11·8Propionic62·50·46Sorbic5005·6
Thymol31·250·47   Valeric>1000>10·2Tartaric>1000>15
Vanillin62·500·95      Lactic>1000>9
Table 2. Evaluation of the synergy among propionic, sorbic acids and thymol and eugenol against Campylobacter jejuni 33291 and 923/2010
 StrainPropionic, %SorbicEugenolThymol
  1. a

    MIC of the substance when tested alone (n = 3).

  2. b

    MIC of the substance when tested in combination (n = 3). The ratio of the compounds when tested in combination was 58, 19·5, 18·6 and 3·9% for propionic and sorbic acid, thymol and eugenol, respectively.

  3. c

    Synergy definition = ΣFIC ≤ 0·5.

Minimal inhibitory concentrations (MIC)a, %332910·465·60·120·47
923/20100·465·60·120·47
MICb, %332910·0230·010·0020·009
923/20100·0230·010·0020·009
FIC index (MICb/MICa) 0·050·0020·020·02
Σ FIC c 0·09   

When tested alone, propionic acid and sorbic acid, as well as botanicals, were less effective than in combination. In particular, the combination of propionic, sorbic, thymol and eugenol resulted in MIC values of 0·1% for both Camp. jejuni 33291 and 923/2010 strains (Table 2).

Experiment 1

Cloacal swabs at 22 day were all negative to Campjejuni, but by day 24 all reverted to positive indicating that infection was established.

At 35 days (14 days after the challenge), control animals had a mean Camp. jejuni number of 6·9 log10 CFU g−1 of caecal content, whereas all of the treated animals had lower caecal Camp. jejuni (P < 0·01). In particular, compared to control, animals treated with the additive at 0·1, 0·3, 0·5 and 1% had lower counts by 2·1, 2·8, 1·2 and 3·3 log10 CFU, respectively (Fig. 1A).

Figure 1.

(A) Mean log10 CFU of Campylobacter jejuni per gram of caecal content at each time point after infection (experiment 1). Black bars represent the control (chickens challenged and not treated); dotted bars represent chickens challenged and treated with 0·1% of in-feed microencapsulated organic acids (OA) and botanicals (Vetagro SpA); horizontally, vertically and diagonally striped bars represent chickens challenged and treated with 0·3, 0·5 and 1% of microencapsulated OA and botanicals, respectively. For each time point, values with different letters (a, b, c and d) are significantly different (< 0·01). (B) Mean log10 CFU of Camp. jejuni per gram of caecal content at each time point after infection (experiment 2). Black bars represent the control (chickens challenged and not treated); dotted and horizontally striped bars represent chickens challenged and treated with 0·1 and 0·3%, respectively, of in-feed microencapsulated OA and botanicals (Vetagro SpA) from day 0 to 21; vertically and diagonally striped bars represent chickens challenged and treated with 0·1 and 0·3%, respectively, of microencapsulated OA and botanicals from day 22 to 42. For each time point, values with different letters (a, b, c and d) are significantly different (< 0·01).

At 42 days (21 days postchallenge), control animals had an increased number of Camp. jejuni compared to 35 days by almost 2 log10 (8·5 vs 6·9 for control); also groups treated with 0·1% of the additive tended to have an increased caecal load by 1 log10, whereas animals treated with 0·2, 0·5 and 1% remained fairly stable compared to 35 day (Fig. 1A). Compared to control, all of the groups resulted to have lower counts by 3·4, 4·5, 3·3 and 5·2 log10 CFU for 0·1, 0·2, 0·5 and 1%, respectively (P < 0·01; Fig. 1A).

Experiment 2

At 22 day, cloacal swabs were all negative but for control group, and at day 24 all groups were positive. At 35 days, compared to control, all of the treated groups had lower Campjejuni caecal counts, and groups having received the additive from day 22 to 42 at both 0·1 and 0·3% had the lowest shedding. In particular, caecal counts were 6·7, 5·9, 5·1, 5·0 and 3·4 log10 CFU g−1, for control, groups treated from 0 to 21 day at 0·1 and 0·3% (group 1 and 2), and groups treated from day 22 to 42 at 0·1 and 0·3% (group 3 and 4), respectively (P < 0·01; Fig. 1B).

At 42 days, the same trend observed at 35 days was maintained, and group 1 and 2 had 1·3 and 1·7 log10 CFU lower Camp. jejuni than control, respectively, whereas groups 3 and 4, receiving the additive at 0·1 and 0·3% in the last 3 weeks (day 22–42), had 2·8 and 4·3 log10 CFU lower Camp. jejuni than control, respectively (P < 0·01; Fig. 1B).

Histochemistry and Histometry

The AB/PAS sequential histochemical stain showed that mucous cells were rather abundant in CTR (Fig. 2A) and in group 3 chickens (fed 0·1% of the additive from day 22 to 42), but were less abundant in group 4 animals (receiving the additive at 0·3% from day 22 to 42; Fig. 2B). In addition, the mucous cells demonstrated a lower glycoconjugate content in group 4 than in the other two groups. The statistical analysis confirmed the morphological evaluation, because the number of goblet cells was lower in ceca of group 4 animals than in the other groups (P = 0·04; Table 3). The mucous cells containing acid, AB-reactive glycoconjugates (predominantly of the sulphated type, data not shown), were prevalent in the ceca of both treated animals and control ones (P < 0·01). On the contrary, the ceca of CTR animals showed a greater number of either AB- or PAS-reactive mucous cells (mixed type) than 0·1 and 0·3% fed animals (P < 0·01). No difference was observed among the experimental groups concerning the mucous cells containing neutral glycoconjugates.

Table 3. Effect of dietary microencapsulated organic acids and pure botanicals fed from day 22 to 42, on histometrical analyses related to histochemistry, immuno-histochemistry and immunofluorescence of ceca (Values are means ± SE; n = 360 for goblet cells number, acid glycoconjugates, neutral glycoconjugates, mixed glycoconjugates and peaks of Campylobacter-IR intestinal folds; n = 144 for Campylobacter-IR intestinal folds)
 0%0·1%0·3%P-valuea
  1. AB-PAS, Alcian blue 8GX pH 2·5-periodic acid Schiff.

  2. a

    Values with different superscripts within the same row are significantly different.

Goblet cells number28·33 ± 1·71a28·75 ± 2·28a22·94 ± 1·55b0·04
Goblet cells number (acid, AB+ glycoconjugates)6·22 ± 1·18b16·51 ± 2·23a18·31 ± 1·46a<0·01
Goblet cells number (neutral, PAS+ glycoconjugates)3·35 ± 0·804·29 ± 1·034·46 ± 0·550·53
Goblet cells number (mixed, AB/PAS+ glycoconjugates)16·71 ± 1·78a5·49 ± 1·14b3·53 ± 0·33b<0·01
Campylobacter-IR intestinal folds (%)92·31 ± 1·75a53·97 ± 3·28b57·37 ± 4·49b<0·01
Peaks of Campylobacter-immunofluorescence (gray levels)807·75 ± 52·39a314·50 ± 25·99b58·9 ± 23·69c<0·01
Figure 2.

Alcian blue 8GX pH 2·5-periodic acid Schiff (AB-PAS) stain for caecal mucous cells. (A) Mucous cells are abundant (arrows) in a control animal. (B) Mucous cells are lower and have a lower glycoconjugate content (arrows) in animals treated with the additive at 0·3% from day 22 to 42 compared to control. Scale bar: 50 μm.

Immunohistochemistry, immunofluorescence and histometry

Immuno-staining to detect Campylobacter spp bacteria revealed the presence of a variable number of micro-organisms as mainly adherent to the epithelial layer of several caecal intestinal folds (Fig. 3A). Limited to the ceca of CTR animals, their presence was detected embedded in enterocytes, too (translocation; Fig. 3B). CTR animals displayed a greater number of Campylobacter-IR intestinal folds than all of the other animals (P < 0·01; Table 3). Immunofluorescence peaks linked to the presence of Campylobacter-IR micro-organisms decreased progressively with the increase in concentration of the additive in the feed (P < 0·01; Table 3).

Figure 3.

Campylobacter-immunoreactivity. (A) Several Campylobacter-immunoreactive micro-organisms are detected in the lumen of intestinal folds (arrow) and adherent to the epithelial layer (arrowheads) in a control animal. Scale bar: 50 μm. (B) Campylobacter-immunofluorescence seen either in the lumen of intestinal folds (arrows) or in translocation across enterocytes (arrowheads) in a control animal. Scale bar: 10 μm.

Discussion

Organic acids and pure botanicals are currently included in-feed to improve feed digestibility and animal growth efficiency, and this practice is becoming increasingly popular especially in those countries where the use of antibiotic growth promoters is no longer allowed. More recently, there has been an increasing interest towards the use of OA and botanicals also from a food-safety perspective. The aim of such use is to take advantage of their ‘natural’ antimicrobial power and target it to prevent or treat intestinal colonization by specific foodborne pathogens, such as Salmonella, Campylobacter, Escherichia coli (Grilli and Piva 2012). While this hypothesis seems attractive and promising, results are not yet conclusive (Grilli and Piva 2012). The aim of this study was to develop a feed additive comprising OA and pure botanicals to reduce Campylobacter jejuni intestinal load in broilers, to decrease the risk of carcass contamination at slaughter. Moreover, we wanted to describe a stepwise scientific approach starting from in vitro evaluations and ending up with in vivo studies that would eventually provide a practical solution for in-field feed additive uses and applications.

In vitro antimicrobial assays demonstrated that Camp. jejuni is more sensitive to botanicals than to OA. Many authors have tested antimicrobial activity of essential oils, or their constituents, against foodborne pathogens (Friedman et al. 2002; Lahlou 2004; Peñalver et al. 2005), but results are not always comparable because essential oils, flavours and phytochemicals are a complex family of compounds, with different structures and compositions. Pure botanicals, instead, are chemically defined substances that constitute plant essential oils, and they comprise a wide range of molecules including phenolic compounds, such as carvacrol, thymol and eugenol, whose antimicrobial activity seems directed at cell membrane permeability (Ultee et al. 1999; Davidson 1997). In particular, through their hydrophobic structure, phenolic compounds are able to disrupt the bacterial membrane and to change its permeability (Ultee et al. 2000, 2002) causing an ion efflux from the inner cell to the external medium (Helander et al. 1998; Lambert et al. 2001). The ion leakage is usually coupled with leakage of other cytoplasmic constituents and can be tolerated by the bacterial cell without loss of viability up to a certain point, but, if the efflux is prolonged, it will cause the cell to collapse. Moreover, the presence of the hydroxyl group appears to be fundamental in determining the antimicrobial power, because it has been found that phenolic compounds have the highest antimicrobial activity (Dorman and Deans 2000; Ultee et al. 2002).

In this study, OA were generally less effective than botanicals: in fact, short-chain fatty acids, with the exception of propionic, failed to inhibit Camp. jejuni growth, whereas benzoic and heptanoic acids had a strong antimicrobial activity that was similar to that observed for some botanicals (salycilaldehyde and thymol). The reason for the acids being less effective than botanicals is probably the pH of the medium used to perform the experiment. OA, in fact, exploit their antimicrobial action in their undissociated form, whose concentration is strongly dependent on external pH and pKa values. Chaveerach et al. (2002) tested formic, acetic and propionic acids, alone or combined, and found little or no antimicrobial activity against Campjejuni at pH 5·5 and 5·0, whereas the activity was very high at lower pH (4·0 and 4·5). In fact, at pH values around neutrality, the degree of dissociation of an organic acid will depend on its pKa. As the pKa rises, the extent of dissociation lowers causing increased inhibition. In our experiment, the acids that resulted in the lowest MIC at pH 6·5 were those acids having the highest pKa values like benzoic, heptanoic, caprylic, propionic and sorbic (pKa values of 4·20, 4·89, 4·89, 4·88 and 4·76, respectively).

The second part of the in vitro study was designed to find whether there was a synergy among OA and botanicals to reduce their inclusion rate in the feed. Synergy is generally reported when a combination of two or more substances is more effective than the substance alone. Nazer et al. (2005) studied the synergy between OA, and carvacrol, thymol and eugenol, and although he did not find a real synergy, as described above, the addition of aromatic compounds to OA allowed to substantially reduce the inhibitory concentration for Salmonella thyphimurium. In another experiment conducted on Salmonella typhimurium by our research group (Grilli and Piva 2012), we evaluated the MIC of citric or sorbic acid in presence of either carvacrol or thymol. The study was conducted by measuring optical densities of Salmonella grown with the substances at pH 6·5. After 24 h of incubation, both citric and sorbic acid were only bacteriostatic at 50 mmol l−1, the highest concentration tested, but the addition of one of the two botanicals resulted in an increased antimicrobial power of both acids (Grilli and Piva 2012).

In this study, propionic and sorbic acids, and thymol and eugenol were combined together and tested on two strains of Camp. jejuni, and the combination allowed to decrease the MIC by 20, 560, 60 and 52 times for propionic, sorbic, eugenol and thymol, respectively (Table 2). To evaluate the presence of a synergism among the substances, the fractional inhibitory index (FIC) was calculated (Ohran et al. 2005). The following formulas were used to calculate the FIC index: ΣFIC = FIC pr + FIC sor + FIC eug + FIC thy, where FICpr was the ratio between the MIC of propionic acid in combination and MIC of propionic alone, and FICsor was the ratio between the MIC of sorbic acid in combination and MIC of sorbic alone, etc. FIC index of this experiment was 0·09, which complies with the definition of synergy by Ohran et al. (2005) (Table 2).

Although speculative, the proposed synergistic mechanism of action is the permeabilizing effect of aromatic compounds (soluble in fat, can dissolve in the phospholipid layer of the bacterial membrane), which facilitates the entrance of the undissociated acid, which, in turn, would dissociate in the cytoplasm of the bacterial cell eventually (Grilli et al. 2011).

The third and fourth steps of the protocol were the two in vivo experiments designed to 1) identify a dose of the additive effective in reducing Campjejuni in 42-day-old broilers and 2) establish a mode of employment of the additive in practical conditions. Results were encouraging as in both experiments, each supplementation programme was able to consistently reduce Camp. jejuni caecal contamination by 1·5 log10 minimum. In the first study, increasing doses of the additive were added to the feed and significant reductions in Camp. jejuni were achieved starting at 0·1%, the lowest dose, after 35 and 42 days. In the second study, the most effective supplementation plan was to feed the additive at 22 day, at the time of infection. In fact, there was only a small preventive effect when the additive was administered during the first 3 weeks, before Campjejuni infection occurred and colonization established: animals treated in the first 3 weeks had only a 1·5 log10 CFU reduction in Camp. jejuni intestinal load at 42 days despite the dose fed, whereas starting the administration of the additive at 22 days appears to be more effective to reduce carcass contamination, at both 0·1 and 0·3% (3–4 log10 CFU reduction). Moreover, histochemical results from the second experiment showed that the treatment given at 0·3% during the last 3 weeks was able to reduce the goblet cells number and to increase the acidic sulphated glycoconjugate portion of mucins, providing evidence that the additive was able to lead to significant changes in goblet cells expression of glycoconjugates. These host-derived results, along with a reduced number of Campjejuni, confirmed by both microbial counts and immunohistochemical reactions, support the highly protective role exerted by acidic glycoconjugates contents of the intestinal mucus against pathogenic micro-organisms (Fernandez et al. 2000; Forder et al. 2007). Whether these results were determined by a direct effect of the additive on the mucosa or were mediated by a reduction in Camp. jejuni, which in turn affects mucin expression, is unclear. Nevertheless, the parallel reduction in mucous secreting cells and the number of micro-organisms adherent to the epithelial layer caused by adding the additive at 0·3% suggest a positive effect of the additive as it is known that mucins are a growth substrate for a large number of micro-organisms, pathogenic and not (Fernandez et al. 2000; Forder et al. 2007 and Van Deun et al. 2008).

Many OA-based strategies have been tested to address the problem of Camp. jejuni. Currently, medium chain fatty acids (MCFA) appear to be one of the most promising solutions even if results are not always consistent. Hermans et al. (2010) tested the antimicrobial activity of caproic, caprylic and capric acids against Campjejuni both in vitro and in vivo. While in vitro results confirmed ten-fold lower MIC values for MCFA acids when compared to short-chain fatty acids, in vivo, none of the acids, as salts or in their coated form, included in the diet up to 1% 3 days before euthanization, produced a reduction in caecal Campjejuni. These results are in contrast to the work conducted by Solis de los Santos et al. (2008a), who found a significant reduction in caecal Campylobacter when 0·7% of caprylic acid was administered via the feed to newly hatched chicks for 10 days. These results were confirmed over four separated experiments, even though higher doses of the same acid proved to be inconsistently effective (Solis de los Santos et al. 2008a). In another experiment by the same authors, 0·7 and 1·4% caprylic acid supplementation to the feed of newly hatched chicks over a period of 15 days reduced caecal Campjejuni colonization by 3–4 log10 CFU in three separated trials, although the lowest and the highest dose, 0·35 and 2·8%, respectively, failed to exert any effect in two of three trials (Solis de los Santos et al. 2008b).

Most of these studies were conducted on newly hatched chicks, and fewer attempts have been made to evaluate strategies at slaughter age, which is epidemiologically more relevant. Again, Solis de los Santos et al. (2009) investigated the therapeutic supplementation of caprylic acid in 42-day-old chickens feed for 3 or 7 days before slaughter. Both the 3-day and 7-day supplementation with 0·7% of caprylic acid consistently reduced caecal Camp. jejuni counts by 2–3 log10, whereas the supplementation of 1·4% for 3 days before euthanasia was effective only in one trial. The dose 0·35% resulted to be effective over a 7-d supplementation period. Molatová et al. (2010) found that 0·25% of a mixture of caprilic and caproic acids reduced caecal Camp. jejuni by 2 log10 after 10 days of supplementation in both the coated and noncoated form (Molatová et al. 2010).

Interestingly enough, most of the studies using MCFA, fed either free or microencapsulated, were conducted with doses ≥0·25% and mostly failed to have an effect when fed at doses lower than 0·7%.

Skånseng et al. (2010), demonstrated the efficacy of feeding a combination of free formic acid and sorbate (used at 2 and 0·1%, respectively) to prevent completely Camp. jejuni colonization in chickens, but, when used alone, none of the acid was effective, regardless of the concentration used. These data support the synergistic effect of feeding different types of compounds to achieve better results; in particular, in our study, we demonstrated that adding pure botanicals to acids and microencapsulating them allowed a significant reduction in the inclusion level from 2·1% (Skånseng et al. 2010) to as little as 0·1% (20 times lower inclusion rate). Synergy allows to achieve better results at intestinal pH when most of the acids would be mostly dissociated and less efficacious, the membrane pore forming activity of botanicals making easier the passage of the acid through the bacterial cell. Moreover, microencapsulation enhances the antimicrobial activity of both OA and botanicals in the caecum by preventing their complete disappearance in the stomach and in the fore intestine. Nonetheless, despite the promising preliminary results, further studies are necessary to evaluate the impact of this additive in in-field conditions, especially with respect to palatability of the product and its influence on performance, as well as to establish an economical return on the investment.

Acknowledgements

The authors gratefully acknowledge the Premio Montana, granted by Montana Alimentari, and Vetagro SpA for the financial support to the study.

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