Characterization of probiotic strains: An application as feed additives in poultry against Campylobacter jejuni

https://doi.org/10.1016/j.ijfoodmicro.2010.03.039Get rights and content

Abstract

Campylobacteriosis is at present the most frequent zoonosis in humans and the main source is poultry meat contaminated by Campylobacter jejuni. An alternative and effective approach to antibiotic administration to livestock to reduce bacterial contamination is the use of probiotics, which can help to improve the natural defence of animals against pathogenic bacteria. In this study 55 lactic acid bacteria and bifidobacteria were screened for desirable properties for their application as probiotics against Campylobacter in poultry. All bacteria were examined for their antimicrobial activity against three C. jejuni strains. Strains exhibiting the highest anti-Campylobacter activity were examined for their survival in the gastro intestinal tract (low pH and presence of bile salts) and food/feed processing conditions (high temperature, high NaCl concentration and starvation) and basic safety aspects such as antibiotic susceptibility and hemolytic activity were studied. On the basis of these activities, two strains, namely Lactobacillus plantarum PCS 20 and Bifidobacterium longum PCB 133, were chosen for an in vivo trial in poultry. They were separately administered to healthy chickens in order to evaluate their capability of colonizing the GI tract of poultry and to estimate their effect on C. jejuni population. The results evidenced that L. plantarum PCS 20 was not present in poultry feces at detectable concentration, whereas B. longum PCB 133 significantly increased after two weeks of daily administration and its amount was still high after a wash-out period of 6 days. In the same period, C. jejuni concentration in poultry feces was significantly reduced in chickens administered with B. longum PCB 133. Therefore, B. longum PCB 133, possessing interesting probiotic properties and a marked anti-Campylobacter activity both in vitro and in vivo, is an excellent candidate for being employed as additives to feed for poultry for the reduction of food-borne campylobacteriosis in humans.

Introduction

Food safety is of fundamental importance to the consumer, food industry and economy. Despite significant investment in this field, the incidence of food-borne diseases is still on the rise in the European Union (EU) (Hugas et al., 2009, Smulders et al., 2008). Several food-borne diseases are caused by the growth of pathogenic microorganisms in the food; among them, zoonoses are infections which are transmitted from animals to humans either directly or via the food chain. In order to protect human health, it is important to identify which animals and foods are the main sources of these infections and to develop appropriate intervention strategies to prevent zoonotic diseases from occurring.

Campylobacteriosis is the most frequently reported zoonotic disease in humans in the EU in recent years (Hugas et al., 2009, Westrell et al., 2009) and the bacterial species most frequently implicated is Campylobacter jejuni (Humphrey et al., 2007, Keener et al., 2004). Epidemiological data obtained by the European Food Safety Authority (EFSA) (EFSA, 2005) have concluded that poultry meat is the major source of sporadic campylobacteriosis. It has been reported that 26% of samples of fresh broiler meat and 25% of broiler flocks tested in the EU in 2007 were found positive for Campylobacter (Westrell et al., 2009). Campylobacter is considered to be a commensal organism in many avian species, including those grown commercially, and the spread of Campylobacter spp. among chickens is very rapid (Keener et al., 2004). Reducing the proportion of Campylobacter infected poultry flocks and/or reducing the number of Campylobacter in live poultry will lower the risk to consumers considerably (Keener et al., 2004, Westrell et al., 2009). A possible way to reduce Campylobacter contamination in poultry is to develop new actions at the primary production level.

The increasing concern about the spreading of antibiotic resistance in humans has determined the elimination of antibiotics as growth promoters in livestock (Feed Additives Regulation 1831/2003/EC; Schwartz et al., 2001). Therefore, feed companies and researchers have been looking for alternative products and strategies that can help to maintain animal gut health in order to prevent or reduce the prevalence of pathogens in the food chain. An alternative and effective approach to antibiotic administration to livestock is the use of probiotics, which can help to improve gut microbial balance and therefore the natural defence of the animal against pathogenic bacteria (Modesto et al., 2009, Patterson and Burkholder, 2003).

The use of probiotics in the farm industry dates back to the 1960s (Fuller, 1999), but much of the information on their use on farm animals was initially derived from in-house experiments which had several weak points, considering that the origin of the strain was rarely given and the real ability of the probiotic to colonize the gut was often not assessed (Fuller, 1999, Nahashon et al., 1996). Study performed later devoted more attention to the capability of the administered strains of colonizing the gut and reducing pathogen incidence, as reviewed by Musa et al. (2009). In the last years several efforts have been dedicated to define targeted microbial mixtures that could have preventive activity in poultry against Salmonella infections. Different Lactobacillus strains have demonstrated to protect chickens from this pathogen (Pascual et al., 1999, Van Coillie et al., 2007, Vicente et al., 2007) and to have a protective effect on raw chicken meat against Listeria monocytogenes and Salmonella enteriditis (Maragkoudakis et al., 2009). A reduction of necrotic enteritis due to Clostridium perfringens was evidenced upon administration of L. johnsonii FI9785 (La Ragione et al., 2004). The use of bifidobacteria in poultry feeding is, to our knowledge, less widespread with respect to lactobacilli administration, although bifidobacteria are an important component of the chicken gut microbiota (Amit-Romach et al., 2004) and have shown to exert positive effect when administered to other animals such as piglets (Modesto et al., 2009, Shu et al., 2001). In vivo trials have regarded the use of a probiotic mixed preparation also containing a Bifidobacterium strain (Montzouris et al., 2007) and the administration of a synbiotic mixture containing galacto-oligosaccharides and B. lactis (Jung et al., 2008), evidencing an increase of bifidobacteria in the poultry gut; the only experiment with a selected strain focused on the administration of a commercial B. bifidum strain to poultry, resulting in a reduction of cellulitis in broiler chickens (Estrada et al., 2001).

To date, only a few studies have evidenced a possible role of probiotics in preventing the shedding of C. jejuni at the level of primary production, although in vitro studies reported a strong antimicrobial activity of several probiotic strains towards this pathogen (Chaveerach et al., 2004, Fooks and Gibson, 2002). Morishita et al. (1997) reported a 70% reduction in the frequency of C. jejuni in chicks with the use of a commercial probiotic containing L. acidophilus and Enterococcus faecium. Willis and Reid (2008) showed that C. jejuni was present at a lower lever in broiler chickens fed with a standard diet supplemented with a probiotic formulation containing L. acidophilus, L. casei, B. thermophilus, and E. faecium (108 cfu/g) with respect to the control, although the real colonization of the administered strains was not studied.

Therefore, more research is needed in finding new probiotic strains with inhibiting activity against Campylobacter and capable of colonizing the guts of poultry animals with the final aim of reducing the contamination of the intestinal pathogen at the farm level and in the chicken meat.

In this work a number of lactic acid bacteria (LAB) and bifidobacteria were screened for desirable functional properties for their application as probiotics against Campylobacter in poultry. In particular, their antimicrobial activity against three C. jejuni strains, their survival in the gastro intestinal (GI) tract and food processing conditions were studied in addition to the determination of basic safety aspects such as antibiotic susceptibility and hemolytic activity. The two strains showing the best probiotic performances were administered to poultry in order to evaluate their capability to colonize the GI tract of poultry animals and to estimate their effect on C. jejuni population.

Section snippets

Microorganisms and culture conditions

55 strains of LAB and bifidobacteria were used in this study. They were obtained by the collections of the Agricultural University of Athens (strains designed with PCA), Danisco A/S (strains designed with PCD), the Max Rubner Institute in Kalsruhe, Germany (strains designed with PCK), the University of Maribor-Faculty of Agriculture and life sciences (strains designed with PCS), and the Bologna University Scardovi collection of bifidobacteria (BUSCoB) (strains designed with PCB) (Table 1). The

Antimicrobial activity against Campylobacter

The results obtained with the spot agar test with cell cultures evidenced that 16 strains out of the 55 tested showed antimicrobial activity against all the 3 strains of C. jejuni (Table 1). In particular, the strains which showed clear inhibitory activity against at least one of the Campylobacter strains were 8 Lactobacillus strains mainly isolated from milk derivatives (PCA 236, PCA 259, PCA 275, PCA 293, PCA 227, PCA 306, PCK 161 and PCS20), a L. pseudomesenteroides strain (PCK 18), and 7

Discussion

The beneficial effects of probiotics on animal health and on the reduction of pathogens in the food chain have increasingly been highlighted in the past years (Modesto et al., 2009, Patterson and Burkholder, 2003, Tannock, 1995). The aim of the present work was to perform in vitro studies according to FAO and WHO guidelines (FAO/WHO, 2002) to evaluate the probiotic potential of selected LAB and bifidobacteria strains and to assess the capability of the most promising strains to colonize the GI

Acknowledgement

This work was performed in the framework of the EU funded project entitled “Control and prevention of emerging and future pathogens at cellular and molecular level throughout the food chain” (PathogenCombat FP6-007081).

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