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Research Article
 

Ability of Select Probiotics to Reduce Enteric Campylobacter Colonization in Broiler Chickens



S. Shrestha, K. Arsi, B.R. Wagle, A.M. Donoghue and D.J. Donoghue
 
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ABSTRACT

Background and Objective: Campylobacter is the leading cause of foodborne enteritis worldwide and is primarily caused by consumption/mishandling of contaminated poultry. Probiotic use in poultry has been an effective strategy in reducing many enteric pathogens, but has not demonstrated consistent reduction against Campylobacter. This study was conducted to screen probiotic isolates that could eliminate or reduce cecal Campylobacter counts in poultry. Materials and Methods: As Campylobacter resides and utilizes intestinal mucin for growth, isolates selected on the basis of mucin utilization might be a strategy to screen for probiotic candidates with efficacy against Campylobacter . In this study, bacterial isolates demonstrating increased growth rates in the presence of mucin in media, in vitro were selected for their ability to reduce Campylobacter colonization in 14 day old broiler chickens. In replicate trials, 90 days-of-hatch chicks were randomly divided into 9 treatment groups (n = 10 chicks/treatment) and treated individually with one of four bacterial isolates (Bacillus spp.) grown in media with or without mucin prior to inoculation or a Campylobacter control (Campylobacter , no isolate). In both the trials, all the birds except control were orally gavaged with individual isolates at day-of-hatch. On day 7, all the birds were orally challenged with a four strain mixture of C. jejuni and ceca were collected on day 14 for Campylobacter enumeration. Results: Results from these two trials demonstrated two individual isolates, one isolate incubated with mucin in the media and another isolate incubated without mucin prior to inoculation, consistently reduced cecal Campylobacter counts (1.5-4 log reduction) compared to controls. Conclusion: These results support the potential use of mucin to pre-select isolates for their ability to reduce enteric colonization of Campylobacter in broiler chickens.

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  How to cite this article:

S. Shrestha, K. Arsi, B.R. Wagle, A.M. Donoghue and D.J. Donoghue, 2017. Ability of Select Probiotics to Reduce Enteric Campylobacter Colonization in Broiler Chickens. International Journal of Poultry Science, 16: 37-42.

DOI: 10.3923/ijps.2017.37.42

URL: https://scialert.net/abstract/?doi=ijps.2017.37.42
 
Received: December 02, 2016; Accepted: January 02, 2017; Published: January 15, 2017


Copyright: © 2017. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Campylobacter is one of the leading causes of bacterial gastroenteritis in humans worldwide1,2. In the United States alone, 1.3 million cases of human Campylobacter infections have been reported annually3. More than 17 Campylobacter spp. have been identified4,5, of which, Campylobacter jejuni is responsible for approximately 95-99% of cases of human campylobacteriosis6-8. Most of the human campylobacteriosis cases are self-limiting; however, some severe post-infectious sequelae such as Guillain-Barré syndrome and reactive arthritis have been reported9. Various sources of Campylobacter have been identified; among them, poultry is regarded as the principal source of infection for humans3,10-12. It has been reported that more than 90% of the US poultry flocks are contaminated with C. jejuni 13, which potentially present a serious threat for humans14,15. Hence, reduction or elimination of Campylobacter in poultry flocks would significantly reduce the human incidence of campylobacteriosis12. Several pre-harvest intervention strategies have been evaluated to eliminate/reduce Campylobacter prevalence in poultry flocks with varying degrees of success16-22. Unfortunately, none of them are successful in completely eliminating Campylobacter from poultry23. Application of probiotic bacteria is one strategy that may potentially inhibit or reduce Campylobacter colonization in poultry. Probiotics are "Live micro-organisms which when administered in adequate amounts can confer beneficial effects on host health24". Probiotics have effectively reduced foodborne pathogens such as Salmonella, E. coli, Listeria, Clostridium, etc., in vivo25-30. Although effective in vitro, administration of probiotics produced inconsistent reductions in Campylobacter colonization in broiler chickens21,31,32. Such inconsistent results against Campylobacter colonization suggest the need of better screening methods of probiotic bacteria. It has been reported that supplementation of porcine intestinal mucin in broth media induces the cell surface proteins in Lactobacillus reuteri strains and improve the mucus-binding properties in vitro33. Since Campylobacter colonizes in intestinal mucus and uses mucin as a source of carbon and energy34-36, selection of probiotic isolates which utilize mucin could be an effective approach to competitively inhibit the enteric colonization of Campylobacter.

The objective of this study was to screen probiotic isolates that could eliminate or reduce cecal Campylobacter counts in poultry. In this study we used selected bacterial isolates that are Generally Regarded as Safe (GRAS) and possess efficacy against Campylobacter in vitro. These isolates were further screened for their ability to utilize mucin. Isolates which demonstrated increased growth in the presence of mucin in vitro were selected and tested in vivo.

MATERIALS AND METHODS

Probiotic isolates: In this study, we used previously isolated GRAS bacterial isolates (Bacillus spp.) with efficacy against Campylobacter in vitro21,22.

Screening of mucin utilizing probiotic bacteria: A total of 38 isolates were screened for increased growth in the presence of mucin. The procedure involved growing selected bacterial isolates separately in Tryptic Soy Broth (TSB, Becton Dickinson and Company, Sparks, MD, USA) and in TSB supplemented with 3% porcine gastric mucin (Sigma-aldrich, St. Louis, MO, USA). The isolates were incubated aerobically at 37°C for 24 h. The cultures were then serially diluted with Butterfield's Phosphate Diluent (BPD, Becton Dickinson and Company, Sparks, MD, USA) and plated on Tryptic Soy Agar (TSA, Becton Dickinson and Company, Sparks, MD, USA) for enumeration of each bacterial isolate. Bacterial counts were logarithmically transformed (log10 CFU mL–1). The four isolates which demonstrated greatest increase in counts in the presence of mucin were selected and evaluated for their efficacy to reduce Campylobacter colonization in broiler chickens.

Experimental animals and housing: For all the in vivo trials, day of hatch broiler male chicks were procured from a local commercial hatchery. Chicks were weighed at the beginning and at the end of each trial. Birds were raised in floor pens with pine shavings, with ad libitum access to feed and water throughout the 14 days trial period.

Experimental design: A total of 2 birds trials were conducted at the University of Arkansas Poultry Research Farm and all the experiments were approved by the Institutional Animal Care and Use Committee of the University of Arkansas. Four probiotic isolates which had demonstrated increased growth in the presence of mucin in the broth media were selected for in vivo studies. In each trial, a total of 90 male chicks were randomly divided into 9 treatment groups (n = 10 chicks/treatment). The treatment groups included a Campylobacter control (Campylobacter, no isolate) and 8 treatment groups each receiving a separate bacterial isolate grown in the presence or absence of mucin prior to oral administration.

Bacterial dosing in chicks: In each trial, at day of hatch, chicks from all the treatment groups except Campylobacter control were orally gavaged individually with 0.25 mL of specific probiotic isolate containing approximately 106-108 CFU mL–1 as previously described21. On day 7, all the chicks were orally gavaged with a cocktail of 4 strains of wild type C. jejuni containing approximately 107 CFU mL–1 organisms as previously described37. Briefly, four-strains of wild type C. jejuni were successively sub-cultured twice at 42°C for 48 h under microaerophilic conditions. The strains were then pooled, centrifuged at 3000 rpm for 10 min and re-suspended in appropriate volume of BPD for oral challenge. On day 14, ceca were aseptically collected, cecal contents were serially diluted with BPD and each dilution were plated on Campylobacter Line Agar38 for direct enumeration.

Statistical analysis: To achieve homogeneity of variance, cecal Campylobacter counts were logarithmically transformed (log10 CFU g–1 of cecal material) before analysis of data39. Data were analyzed using the PROC GLM procedure of SAS40. Treatment means were partitioned by least square means (LSMEANS) analysis and a probability of p<0.05 was required for statistical significance.

RESULTS

A total of 38 GRAS isolates were tested in vitro in this study and the four isolates (Isolate 1, 2, 3 and 4) which showed a greatest increase in counts when grown in media supplemented with mucin compared with the unsupplemented media (Fig. 1) were selected for the in vivo studies.

Table 1:
Effect of selected bacterial isolates on cecal Campylobacter counts (log10 CFU g–1 of cecal contents) in 14-day old broiler chicks (Mean±SEM)*
a-dMeans within columns with no common superscript differ significantly (p<0.05), *Chicks were orally challenged on day 7 with 0.25 mL of approximately 1×107 CFU mL–1 of a 4 strain mixture of wild type Campylobacter jejuni in each trial (n = 10/treatment group), #Isolates incubated with mucin prior to oral challenge in chicks. All Campylobacter data were log10 CFU g–1 transformed for statistical analysis

Fig. 1:
Probiotic isolates demonstrating increased growth in the presence of media supplemented with porcine mucin. Values represents average of percentage increased growth of select probiotic isolates in mucin supplemented media from two separate replicate trials. Isolates were ranked in decreasing order of growth from highest to lowest. Isolates 1 through 4 were selected for in vivo trials 1 and 2

In trial 1, isolate 1 and isolate 4 grown in media without mucin prior to inoculation reduced cecal Campylobacter counts (approximately 2-3 log CFU g–1 ) whereas isolates 2, 3 and 4 incubated with mucin prior to inoculation reduced Campylobacter counts (approximately 2-3 log CFU g–1, Table 1) when compared with the controls. In trial 2, isolates 1, 2 and 3 grown without mucin reduced Campylobacter counts by approximately 1.5-4 log CFU g–1 in the ceca whereas only isolate 4 incubated with mucin reduced Campylobacter counts compared to controls (Table 1). When compared across trials, isolate 1 grown without mucin or isolate 4 incubated with mucin prior to inoculation consistently reduced Campylobacter counts in two separate trials (Table 1).

DISCUSSION

Campylobacter is a flagellated, highly motile, microaerophilic bacterium able to colonize heavily in cecal crypt mucus35,36. One theory of why probiotics are ineffective against enteric Campylobacter colonization is because Campylobacters are sequestered in the intestinal mucus laden crypts and the probiotic bacteria are not able to penetrate and inhibit their colonization in these locations41. In an effort to overcome this issue, four bacterial isolates demonstrating the ability to inhibit Campylobacter and increased growth in the presence of mucin, in vitro were selected to evaluate their ability to inhibit Campylobacter colonization in chickens (Fig. 1). Each of these isolates were separately grown in media with or without mucin prior to inoculation to determine if this would enhance efficacy, possibly due to changes in gene expression associated with mucin co-incubation42. In the first bird trial, two out of four isolates grown without mucin prior to inoculation reduced cecal Campylobacter counts (approximately 2-3 log CFU g–1) whereas three out of four of these isolates (isolates 2, 3 and 4) incubated with mucin prior to inoculation reduced Campylobacter counts (approximately 2-3 log CFU g–1, Table 1). In trial 2, many of these isolates also reduced Campylobacter counts by approximately 1.5-4 log CFU g–1 in the ceca. When compared across trials, two isolates consistently reduced Campylobacter counts in two separate trials (Table 1). Isolate 4 was more efficacious when grown in mucin prior to inoculation with an approximate 1.5-2.5 log reduction in Campylobacter counts whereas isolate 1 produced a greater reduction when not incubated with mucin prior to inoculation with an approximate 2-4 log reduction in Campylobacter counts. None of these isolates adversely affected body weight gains at 14 days of age when compared with controls. Some of the isolates grown in mucin prior to inoculation demonstrated a significant reduction in trial 1, however, these isolates (isolates 2 and 3) did not reduce Campylobacter colonization in trial 2. Previous study conducted in our laboratory demonstrates that probiotic isolates can maintain their efficacy when administered directly into the lower intestinal tract, as they bypass the acidic environment in the upper intestinal tract22. The gastrointestinal tract also contains a large, dynamic and complex microflora43, which makes the gut an extremely competitive environment. The interaction between the various types of bacteria in gut lumen is complex44 and these interactions may also inhibit or reduce the efficacy of probiotic isolates within the GI tract. The pre-selected bacterial isolates administered in the current study did not eliminate Campylobacter colonization in chickens possibly due to a reduction in the number of isolates reaching or penetrating the cecal crypts containing Campylobacter. Results from these trials suggest the need of additional isolates to be tested to verify the utility of this strategy. Also, the efficacy of probiotic bacteria can be enhanced by adhesion to GI tract, which may increase the residence time in vivo45 and understanding the molecular mechanisms behind probiotic adhesion in the mucus could help determining the efficacy of the probiotic isolates. In addition to the current strategy presented in this study, screening of probiotic isolates on the basis of their adhesive potential in mucus may also be considered as Ouwehand et al.46 demonstrated a significant variation (3-43%) in adhesion between the lactobacillus strains. Even though these isolates did not eliminate Campylobacter colonization, they did reduce Campylobacter counts by 1.5-4 log. Risk assessment studies conducted by Rosenquist et al.12 predicted that a 2 log reduction of the Campylobacter on chicken carcasses can reduce the human incidence by 30 times. Therefore, bacterial isolates demonstrating the reduction in counts produced in the current study could significantly reduce the incidence of this disease in humans.

CONCLUSION

In conclusion, this study supports the use of probiotic bacteria in reducing/eliminating Campylobacter in broiler chickens. However, detailed knowledge on Campylobacter colonization characteristics in the chicken gut should be explored, which would be helpful in selecting an effective strategy in controlling Campylobacter in broiler chickens.

Results from these trials demonstrated one isolate grown in mucin prior to inoculation consistently reduced cecal Campylobacter counts (1.5-3 log reduction). These results support this screening method could be part of a multifaceted approach in evaluating bacterial isolates with the ability to reduce enteric Campylobacter colonization. However, more isolates need to be tested to verify this screening strategy. Further study in probiotics is warranted to reduce or eliminate Campylobacter colonization in broiler chickens.

SIGNIFICANCE STATEMENTS

In the present study, specific probiotic isolates consistently reduced cecal Campylobacter counts up to 4 log10 CFU g–1 when compared to controls
Combining the probiotic isolates with a prebiotic supplementation in feed or protecting the isolates and facilitating targeted release of the probiotic isolates (e.g., encapsulation) may be effective in reducing cecal colonization of Campylobacter
The presented strategy could be used as part of a multi-faceted approach to reduce enteric Campylobacter counts in broiler chickens

ACKNOWLEDGMENT

This study was funded in part by the USDA-NIFA-OREI 2011-01955.

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