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Beneficial Effect of Bacteriocin-Producing Strain Enterococcus Durans ED 26E/7 in Model Experiment Using Broiler Rabbits

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Beneficial Effect of Bacteriocin-Producing Strain Enterococcus Durans ED 26E/7 in Model Experiment Using Broiler Rabbits Original Paper Czech J. Anim. Sci., 62, 2017 (4): 168–177 doi: 10.17221/21/2016-CJAS Beneficial Effect of Bacteriocin-producing Strain Enterococcus durans ED 26E/7 in Model Experiment Using Broiler Rabbits Andrea Lauková1*, Monika Pogány Simonová1, Ľubica Chrastinová2, Anna Kandričáková1, Jana Ščerbová1, Iveta Plachá1, Klaudia Čobanová1, Zuzana Formelová2, Ľubomír Ondruška2, Gabriela Štrkolcová3, Viola Strompfová1 1Institute of Animal Physiology, Slovak Academy of Sciences, Košice, Slovak Republic 2National Agricultural and Food Centre, Lužianky, Slovak Republic 3University of Veterinary Medicine and Pharmacy, Košice, Slovak Republic *Correspoding author: [email protected] ABSTRACT Lauková A., Pogány Simonová M., Chrastinová Ľ., Kandričáková A., Ščerbová J., Plachá I., Čobanová K., Formelová Z., Ondruška Ľ., Štrkolcová G., Strompfová V. (2017): Beneficial effect of bacteriocin-producing strain Enterococcus durans ED 26E/7 in model experiment using broiler rabbits. Czech J. Anim. Sci., 62, 168–177. From the aspect of probiotic properties and bacteriocins, Enterococcus faecium belongs to the most frequently studied species among enterococci. This study deals with testing the strain of the species Enterococcus durans ED 26E/7 in broiler rabbits. The strain ED 26E/7 isolated from ewes lump cheese produces an antimicrobial sub- stance durancin. Forty-eight post-weaned rabbits (aged 5 weeks) of both sexes were divided into experimental group (EG) and control group (CG) per 24 animals each, and kept in standard cages, two animals per cage. EG group rabbits were additionally administered the ED 26E/7 strain (500 µl/animal/day) into water for 21 days. CG group rabbits were fed a commercial feed. The experiment lasted 42 days. Faeces and blood samples were taken on days 0–1 (experiment onset), 21 (after a 3-week application), and 42 (3 weeks after ED 26E/7 strain cessation). On days 21 and 42, rabbits were slaughtered and caeca and appendix were sampled. The rabbits’ digestive tract was found to be sufficiently colonized by the strain ED 26E/7; the antimicrobial effect was demonstrated in caecum and appen- dix (e.g. decrease in coliforms). Reduction of Eimeria sp. oocysts in EG compared to CG rabbits was detected on day 21, when also a significant (P < 0.05) increase of phagocytic activity in EG was registered. Values of glutathione- peroxidase were lower in EG than in CG rabbits on day 21 implying that the ED 26E/7 application had not evoked oxidative stress. Biochemical blood parameters and quality of meat were not negatively influenced. First time tested in animals, E. durans ED 26E/7 seems to be a new candidate for use in rabbits husbandry. Keywords: enterococcal species; influence; rabbit; gut microbiota; physiological parameters; meat quality Enterococci have constituted a unique taxonomic separation into a new bacterial genus Enterococcus entity since the mid-90s when results of DNA– from the former genus Streptococcus (Schleifer DNA hybridization experiments suggested their and Kilpper-Balz 1987; Werner et al. 2013). These Supported by the Scientific Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Acad- emy of Sciences (Projects VEGA No. 2/0006/17 and No. 2/0004/14). 168 Czech J. Anim. Sci., 62, 2017 (4): 168–177 Original Paper doi: 10.17221/21/2016-CJAS Gram-positive bacteria form the third largest MATERIAL AND METHODS genus as lactic acid bacteria (LAB) following the genera Lactobacillus and Streptococcus (Franz Enterococcus durans ED 26/7 (marked by ri- et al. 2011). Some enterococci are also known to fampicin, concentration 109 CFU/ml) at a dose of produce bacteriocins – proteinaceous substances 500 μl per animal per day was used in our experi- with antimicrobial effect (Laukova et al. 1993; Franz ment prepared as previously described by Laukova et al. 2007) which can influence the intestinal mi- et al. (2012a). RifR variant (to distinguish it from crobiota (Pogany Simonova et al. 2009; Laukova et the other enterococci) was prepared as follows: al. 2012a). Enterococci belong to the phylum Fir- 0.1% inoculum in MRS broth (Merck, Germany) micutes, Class Cocci, Order Lactobacillales, Family was grown for 18 h at 37°C. Overnight culture was Enterococcaceae, Genus Enterococcus (De Vos et centrifuged at 10 000 g for 30 min. Supernatant al. 2009). Up to now, 54 enterococcal species have was removed and the cells were resuspended in been validly described, most of them falling into Ringer solution (Merck) to reach the final cell con- seven species groups on the basis of 16S rRNA gene centration 109 CFU/ml. The counts were checked similarity (Franz et al. 2011). Following this clas- by the spreading of the dilutions in Ringer solu- sification, the species Enterococcus durans belongs tion onto M-Enterococcus agar (Difco, USA) and to the E. faecium group (Tanasupawat et al. 2008). incubated at 37°C for 24–48 h. Application doses The species E. durans is most frequently detected (500 μl) for each animal and a 21-day treatment in food products, e.g. cheese (Pieniz et al. 2014), were prepared and stored at 4°C. but it can be isolated also from the digestive tract The experiment was performed in co-operation of animals, including food-producing animals and with our colleagues from the National Agricultural human (Devriese et al. 2002). Similarly as some and Food Centre, Nitra, Slovakia. Forty-eight E. faecium strains, also some E. durans strains post-weaned rabbits (aged 5 weeks) of both sexes are able to produce bacteriocins (Yanagida et al. were divided into the experimental group (EG) 2005). Because our laboratory has been focused and the control group (CG) (n = 24 animals per on bacteriocin-producing and probiotic entero- each group). Animals involved in the experiment cocci and their use in protecting animal health for represented maternal albinotic line (crossbreed years, in this study we attempted to apply E. durans New Zealand White × Buskat Rabbit × French ED 26E/7 in broiler rabbits. ED 26E/7 was isolated Silver) and paternal acromalictic line (crossbreed from ewes cheese lump. Its identity was confirmed Nitra’s Rabbit × Californian Rabbit × Big Light by MALDI-TOF spectrometry. It is a lactic acid and Silver). Rabbits were kept in standard cages sizing bacteriocin-producing strain possessing probiotic 0.61 m × 0.34 m × 0.33 m, type D-KV-72 (Kovo- properties (Laukova et al. 2012b). bel, Czech Republic), two animals per cage. The Rabbits represent not only a suitable animal cages allowed faeces separation. A lighting cycle model, but after weaning they have still problems of 16 h light/8 h darkness was used throughout the with digestive disorders such as colibacillosis or experiment. The temperature and humidity were clostridial enteritis. Therefore breeders search for recorded continuously with a digital thermograph different (innovative) ways to eliminate/prevent positioned at the same level as the cages. The this problem. Following our previous studies, this heating and forced ventilation systems allowed study was focused on testing E. durans ED 26E/7 the building air temperature to be maintained in broiler rabbits to find a new strain to protect within 16 ± 4°C throughout the experiment. Rela- husbandry, but also to compare its effect with tive humidity was about 70 ± 5%. All care and other promising additives. Colonization of the experimental procedures involving animals fol- strain was checked and its influence on microbiota, lowed the guidelines stated in the Guide for the biochemical parameters, immunity, meat parame- Care and Use of Laboratory Animals approved by ters (total water content, water holding capacity, the Slovak Veterinary and Food Administration energy value, total fat and proteins content, pH24) and by the Ethic Commissions of both institutes. was analyzed. Moreover, the effect of ED 26E/7 on The rabbits were fed a commercial feed mixture Eimeria oocysts in faeces of broiler rabbits was for broiler rabbits (pelets 3.5 mm in average size) verified. To our knowledge, this is the first study containing crude protein in the volume 177.99 g/kg on the effect of E. durans strain in animals. of feed mixture/diet, fibre content was 146.97 g/kg, 169 Original Paper Czech J. Anim. Sci., 62, 2017 (4): 168–177 doi: 10.17221/21/2016-CJAS fat content was 36.08 g/kg, starch 129.05 g/kg, ash Blood samples (n = 6) were taken from rabbits’ content 97.32 g/kg, organic matter 847.49 g/kg. marginal ear vein (vena auricularis). On day 0–1 Samples of individual feeds and complete granu- mixed blood samples including all groups were lated mixture were analyzed for the content of taken, on days 21 and 42 we took mixed samples nutrients according to the Slovak Technical Norm separately for each group. Phagocytic activity (PA) (STN 46 7092 from 2010). The rabbits had ad was measured using the direct counting procedure libitum access to daily checked feed and water with microspheric hydrophilic particles (MSHP). consumption. The EG group rabbits were more- Ingestion of MSHP by polymorphonuclear cells over administered Enterococcus durans ED 26E/7 (PMNs) was determined using a modified test strain (marked by rifampicin 109 CFU/ml) at a described by Vetvicka et al. (1982): 50 ml of MSH dose of 500 μl/animal/day in water for 21 days. particle suspension (ARTIM, Czech Republic) was The CG rabbits were fed commercial feed. The mixed with 100 ml of blood in an Eppendorf-type duration of the experiment was 42 days. test tube and incubated at 37°C for 1 h. Blood Faecal sampling was carried out on day 0–1 (the smears were then prepared and stained by May- start of experiment; ten mixture samples from Grünwald and Giemsa-Romanowski (Merck). In 48 rabbits – initial microbial background), on each smear, 100 cells were observed to determine day 21 (the end of E.
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