Scholarly article on topic 'Can enterocins affect phagocytosis and glutathione-peroxidase in rabbits?'

Can enterocins affect phagocytosis and glutathione-peroxidase in rabbits? Academic research paper on "Veterinary science"

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Academic research paper on topic "Can enterocins affect phagocytosis and glutathione-peroxidase in rabbits?"


Eur. J. Biol. • 8(8) • 2013 • 730-734 DOI: 10.2478/s11535-013-0198-x

Central European Journal of Biology

Can enterocins affect phagocytosis and glutathione-peroxidase in rabbits?

Rapid Communication

Monika Pogany Simonova1*, Andrea Laukova1, Iveta Placha1, Klaudia Cobanova1, Viola Strompfova1, Renata Szaboova1, Lubica Chrastinova2

Institute of Animal Physiology, Slovak Academy of Sciences, 04001 Kosice, Slovakia

2Animal Production Research Centre, Department of Animal Nutrition, 95141 Nitra-Luzianky, Slovakia

Received 27 February 2013; Accepted 29 April 2013

Abstract: The gastrointestinal microbiota in rabbits play an important role in protection against potential pathogens via the development of the mucosal immune system. The gut health, including the microbial and immunological stability, is often influenced by exogenous factors, mainly around the weaning period. Therefore, alternative strategies are required to improve the animal's health. In this study, the diet of rabbits was supplemented with the semi-purified enterocins Ent 2019, Ent M and Ent 4231, produced by bacteriocinogenic strains with probiotic properties, rabbit-derived Enterococcus faecium CCM7420 and non rabbit-derived E. faecium strains AL41 and CCM4231. The phagocytic activity, index of phagocytic activity and the gluthatione-peroxidase enzyme activity in blood were determined during the Ents consumption and also 3 weeks after their cessation. At 21 days into the experiment the highest phagocytic activity was observed in rabbits receiving Ent M. A significant increase in phagocytosis was noted in rabbits with Ent 2019 over the entire experiment. Moreover, a lower gluthatione-peroxidase activity was measured in rabbits receiving Ent M and Ent 2019. No effect of Ent 4231 application on the tested parameters was recorded. The Ent M and Ent 2019 improved the digestive immunity and the host's defense capacities by stimulating leucocyte phagocytosis, without oxidative stress induction in rabbits.

Keywords: Bacteriocins • Blood • Phagocytic activity © Versita Sp. z o.o.

1. Introduction

Knowledge of the immune response and homeostasis in farm animals is important to protect animals from diseases (especially bacterial diseases) and/or to improve the health and/or productivity of animals which are bred for consumption - „food animals". In broiler rabbits, the overall organisation of the digestive immune - lymphoid system is similar to that of other mammalian species with the exception of two additional structures: the sacculus rotundus and the vermiform apendix. These have been identified only in this species and generally act synergistically [1]. The vermiform appendix is an additional place of lymphoid cell differentiation and maturation. Other primary lymphoid organs are the bone marrow, thymus,

foetal liver. After differentiation and migration to the secondary lymphoid organs - spleen, lymph nodes, gut associated lymphoid tissues (GALT) - the lymphoid cells are stimulated and proliferated [2]. Several studies have reviewed the effects of therapeutical agents, both synthetic and natural, on the general immune response in humans and animals [3,4]. However, only a few studies have been published concerning the influence of natural substances - probiotics, prebiotics, plant extracts, and bacteriocins- on the immune profile of rabbits [5,6].

There is also an interaction between gut microflora and health status of the host organism. Microbiota protect against pathogens as a constituent of the intestine's defense barrier and are also implicated in the development and maturation of the digestive mucosal

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M. Pogany Simonova et al.

immunity. Application of natural antimicrobials, mainly probiotics, modulates and balances the intestinal microbiota favoring the growth of the beneficial lactic acid bacteria, enhancing immune response [2].

Manipulation, i.e. animal handling, caging, etc., as well as environmental and dietary changes changes in rabbit husbandries often induce physiological or pathological oxidative stress. To avoid these reactions, an antioxidant defense system has been developed by aerobic organisms for free radical elimination. The glutathione peroxidase (GPx) enzyme family is a prominent member of this defense system. There are several factors affecting the GPx activity, the most essential among them are the nutritional factors [7]. The influence of natural antimicrobials, mainly probiotics and plant extracts, on GPx activity in rabbit blood samples was also recorded [6,8,9]. However, it is necessary to supplement these data concerning the application of new natural substances, e.g. enterocins (bacteriocin-like substance of proteinaceous character produced by some strains of enterococci, and active against both Gram-positive and Gram-negative microorganisms) for rabbit husbandries [10].

In this study, the diet for rabbits was supplemented with semi-purified enterocins (Ents) Ent 2019, Ent M and Ent 4231, produced by bacteriocinogenic strains with probiotic properties : rabbit-derived strain Enterococcus faecium CCM7420 (previously EF2019) and non rabbit-derived strains E. faecium AL41 and CCM4231. During their application, the stimulation of leucocyte phagocytosis and glutathione-peroxidase activity in rabbit blood were determined and compared based upon the strain's origin.

2. Experimental Procedures

A total of 96 rabbits (Hycole breed, male), weaned at 5 weeks of age, were randomly divided into 3 experimental groups (EG1 - Ent 4231; EG2 - Ent M; EG3 - Ent 2019) and one control group (CG), each containing 24 rabbits (age 5 weeks at the beginning of the experiment, day 0-1). Rabbits were housed in standard cages (0.61x0.34x0.33 m, the type D-KV-72 supplied by Kovobel company, Czech Republic), with two animals per cage. The cages allowed the separation of faeces. A cycle of 16 h of light and 8 h of dark was used throughout the experiment. Temperature and humidity were recorded continuously by a digital thermograph positioned at the same level as the cages. Heating and forced ventilation systems allowed the building air temperature to be maintained within 16±4°C throughout the experiment. Relative humidity was approximately 70±5%. The rabbits were fed the commercial diet for growing rabbits (Table 1) and had access to water ad libitum. The diet was granulated andnon-supplemented by coccidiostat. For twenty-one days, at the same time in the morning, the rabbits in each treament group were adminitstered the appropriate enterocin intotheir drinking water: EG1 rabbits were administered semi-purified Ent 4231 (produced by the strain Enterococcus faecium CCM4231, 6400 AU/ml activity; 50 pl/animal/day; [11]), in EG2, Ent M was administered (produced by the strain E. faecium AL41, 6400 AU/ml activity; 50 pl/animal/day; [12]) and in EG3, Ent 2019(produced by the strain E. faecium CCM7420, previously EF2019, 25600 AU/ml activity; 50 pl/animal/day; [13]). The

Ingredients: (%) (g kg-1)

Extracted clover (grass) meal 27.00 Mineral and vitamin premix1 3.00

Extracted sugar beel 10.00 Crude protein 197.00

Oats 13.00 Crude fibre 166.50

Wheat bran 6.00 Fat 39.00

Soybean meal 7.50 Ash 80.00

Sunflower meal 14.00 Organic compounds 921.00

Monocalcium phosphate 0.60 Starch 178.00

Dicalcium carbonate 0.90 Lysin 7.50

Salt 0.30 Methionine+cysteine 6.50

Carob 2.50 Cholinchloride 0.80

DL-Methionine+wheat meal 0.10+0.10 ME 10 MJ

Table 1 . Ingredients and chemical composition and nutritive value of diets.1Premix provided per kg diet: vitamin A, 10,000 IU; vitamin D3, 2,000 IU;

vitamin E acetate, 30 mg; vitamin B2, 5 mg; vitamin B6, 2 mg; vitamin B12, 8 mg; Ca, 9.25 g; P 6.2 g; Na, 1.6 g; Mg, 1.0 g; K, 10.8 g; Fe, 327.5 mg; Mn, 80 mg; Zn, 0.7 mg.

animals were not given additives the following twenty-one days. All care and experimental procedures involving animals followed the guidelines stated in the Guide for the Care and Use of Laboratory Animals [14] and the trials were approved by the Ethical Commission of Institute of Animal Physiology in Kosice and the State Veterinary and Food Administration.

Blood samples were obtained from the marginal ear vein (Vena auricularis) into a dry heparinized glass tube on days 0-1, 21 and 42 (PA, IPA: n=5; GPx: n=8). The activity of blood GPx was determined by the colorimetric method (Spectrophotometer UV-2550 Shimadzu, Japan) using the commercial kit Randox RS 504 (Randox Laboratories Ltd., UK). Phagocytic activity (PA) was tested microscopically by a direct counting procedure using microspheric hydrophilic particles (MSHP). Ingestion of MSH particles by polymorphonuclear cells (PMNs) was determined by a modified test [15]: 50 ml of MSH particle suspension (ARTIM, Prague, Czech Republic) was mixed with 100 ml of blood in an Eppendorf-type test tube and incubated at 37°C for 1 h. Blood smears were then prepared by Pappenheim staining according to Steruska [16]. For each smear, 100 cells were observed to determine the relative number of white cells containing at least three engulfed particles (phagocytic activity), and the index of phagocytic activity (number of engulfed particles/total number of neutrophils and monocytes observed). The percentage of phagocytic cells was evaluated using an optical microscope, by counting 100 PMN. Body weight and feed consumption of rabbits were measured every week of the experiment. Mortality and morbidity were also recorded in groups daily, over the entire period of the experiment.

The results are expressed as the mean + standard error (PA, IPA) and as the mean + standard deviation (GPx). Data were analysed for the effects of treatment and time (the results were compared between the days 0-1, 21 and 42, and the sample collections' dates to check the changes during the experiment within individual experimentally groups). Statistical analysis of the results was performed by repeated measures analysis of variance (ANOVA, Dunnett post-test) with the level of significance set at P<0.05.

3. Results and Discussion

A significant increase in PA and IPA was noted in EG3 (Ent 2019, P<0.01) during the entire experiment. On day 21, the highest PA was observed in EG2 (Ent M) and showed a tendency to increase until the end of the experiment (P<0.01, day 42, Table 2). The PA and IPA values are in accordance with those previously reported by Szaboova et al. [6] using Ent 4231 and sage extract. Deptula et al. [5] recorded higher IPA values, ranging between 6.5 and 7.8. The continuous increase in phagocytosis and the prolonged immuno-stimulative effect were observed at the end of the experiment (day 42) in rabbits receiving Ents 2019 and M, independently of the strain's origin. It is well known that probiotics are able to stimulate immunity by improving the intestinal barrier and mucosal immune system via modulation of intestinal microbiota, and by production of antibacterial compounds (organic acids, hydrogen peroxide, carbon dioxide, bacteriocins). Higgins et al. [17] e.g. presented the activation of innate immunity through phagocytic


day 0-1 day 21 day 42

Ent 4231 - day 0-1 21.6 ± 4.2 1.1 ± 0.3 227.7 ± 37.5

day 21 18.7 ± 0.4 0.8 ± 0.1 240.0 ± 56.5

day 42 25.2 ± 0.8 1.0 ± 0.1 259.7 ± 34.5

Ent M - day 0-1 22.8 ± 3.4 2.2 ± 0.6 119.4 ± 49.1

day 21 32.2 ± 0.9a 2.9 ± 0.1a 106.9 ± 48.4

day 42 68.8 ± 0.9b 4.9 ± 0.1b 100.6 ± 53.6

Ent 2019 - day 0-1 16.7 ± 0.7 1.8 ± 0.5 159.0 ± 40.6

day 21 26.0 ± 1.8b 2.2 ± 0.3b 141.6 ± 20.7

day 42 34.6 ± 1.7b 2.4 ± 0.1b 179.9 ± 41.9

Control group - day 0-1 21.6 ± 4.2 1.1 ± 0.3 227.7 ± 37.5

day 21 22.5 ± 0.9 2.2 ± 0.1 241.0 ± 30.6

day 42 20.4 ± 0.5 2.0 ± 0.1 226.3 ± 24.4

Table 2. Phagocytic activity (PA; %; n=5), index of phagocytic activity (IPA; n=5) and glutathione peroxidase (GPx; U/g Hb) enzyme activity (n= Values are means ± SEM (PA, IPA) and means ± SD (GPx). Significant results: a P<0.05; b P<0.01, Ent - enterocin.

M. Pogany Simonova et al

cells by Lactobacillus-based probiotic treatment in chickens. The stimulating and protective effect of the probiotic treatment was associated with an increased macrophage PA in mice [18] and also in a piglet model, where higher blood leukocyte phagocytic responses were attained [19]. While many studies have dealt with the subject ofimmunity stimulation - both cellular and humoral, including the stimulation of GALT - by natural substances (probiotics and plant extracts), only a few studies have described the effect of bacteriocins on the non-specific immunity as well as their mechanism of action. For example, Villamil et al. [20] also described the immunomodulatory effect of nisin on non-specific immunity in turbot - their hypotheses were that receptor competition or an excessive accumulation of competent cells were factors in this documented response. We supposed that our enterocins were able to stimulate the immune system through the modulation of gut microbiota favoring lactic acid bacteria and via supporting/improving the GALT by the stimulation of the IgA system. These findings are also supported with our previous results from several in vitro and in vivo experiments concerning the antimicrobial activity of enterocins and enterocin-producing strains with probiotic properties [6,8,9]. On the other hand, the prolonged immuno-stimulative effect (even 21 days after ceasing the administration of enterocins) should be also explained by the stimulation of GALT and IgA secretion.

The lowest GPx activity was observed in rabbits of EG2 (Ent M); the values had the tendency to decrease till the end of the experiment (Table 2). In rabbits of EG3 (Ent 2019), a lower GPx activity was measured in the blood only at the end of Ent 2019 application (day 21), compared to the initial value. The activity of GPx in blood can be a stress marker, induced by the factors such as diet changes and manipulation. The GPx activity values in rabbits are in accordance with our previous results [9]. No effect of Ent 4231 on the PA (IPA) and GPx activity was noted; the GPx activity in rabbits of EG2 and EG3 (Ent M, Ent 2019) decreased during their application.

We suppose that the application of Ents does not evoke oxidative stress in rabbits.

All animals were found in good health conditions throughout the trial. The average daily weight gain was higher in all experimental groups comparing to the control group: EG1 (Ent 4231): 38.65 g, EG2 (Ent M): 39.09 g, EG3 (Ent 2019): 38.65 g, CG: 37.95 g). These findings are in agreement with our previous results [6,8,9]. The feed conversion ratio also decreased in groups administering enterocins: EG1 (Ent 4231): 2.81, EG2 (Ent M): 2.74, EG3 (Ent 2019): 2.87, CG: 3.01.

In conclusion, the indigenous intestinal microbiota play an important role in the regulation of intestinal development and in supporting the host against colonization with potential pathogens. However, the eventual effects of exogenous factors - diet changes, manipulation and stressors (weaning period), are also very important factors that influence this stability. Feeding strategies, for example the use of Ents 2019, M and 4231 (produced by bacteriocinogenic enterococci with probiotic characters), could have partially improved the digestive immunity and the host's defense capacities by stimulating leucocyte phagocytosis, without the induction of oxidative stress in rabbits.


This work was partially supported by the project VEGA 2/0002/11. The authors would like to thank Mrs. M. Bodnarova for her excellent technical assistance, Ing L Ondruska, Dr. R. Jurcik and Dr. V. Parkanyi from Animal Production Research Centre in Nitra for blood sampling. The part of the results concerning the feed conversion ratio and average daily gain was already reported in Proceedings of National Conferences about rabbits (in Slovak, Simonova et al., Laukova et al., Szaboova et al., in Proceedings from National Conferences in Slovak language - Actual ways in broiler rabbits breedings, Nitra, Slovakia, 2008, 2012.


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