Enterococcus faecium EF2019(CCM7420): Comparison
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Weaning is the most important and critical period in rabbits breeding, when only small dietary and/or environmental changes can disturb the stable microbial population/fermentation and gut health, leading to digestive dysbiosis, increased morbidity/mortality and big economic losses. Control of the microbiota, prevention of digestive disturbances and improving gut health and immunity can be achieved through the natural substances application in rabbit nutrition. Probiotics are frequently used in rabbits farms, however, the most of them are non-autochtonous (have a different origin than the rabbits ecosystem), while the “in vivo” administration of bacteriocins (antimicrobial substances produced by bacteria, which usually possess also probiotic properties) in these animals are often limited and has become an area of research activity. Therefore, we focused on improving rabbits health using the autochtonous strain Enterococcus faecium EF2019(CCM7420; fresh culture, lyophilized form) and its enterocin (Ent7420) in broiler rabbits. During these experiments, the following effects of the strain and its enterocin were observed: improved average daily weight gain and feed conversion ratio, good colonization ability of the tested strain (maximum counts in first 2-3 weeks of application), increased lactic acid bacteria and reduced coagulase-positive staphylococi including S. aureus, coliforms and clostridia population as well as the Eimeria sp. oocysts counts in the rabbits gut. Both additives showed a tendency to modulate the serum biochemistry parameters (increased level of total proteins, glucose, triglycerids) and to stimulate the blood phagocytic activity and improved jejunal morphology (enlargement of the absorption surface in jejunum, higher villi height:crypt depth (VH:CD) ratio). The physico-chemical properties of rabbit meat was not negatively affected by the CCM7420 strain, while the meat iron content significantly increased during its application, which improves the rabbit meat quality. It could be also emphasized that knowing the probiotic properties and the ability of Enterococcus faecium CCM7420 to produce enterocin Ent7420 with antimicrobial effect is of great interest mainly in the case of several disease/pathologies, such as Epizootic Rabbit Enteropathy, which are difficult to prevent and combat because their etiology is not known and there are no vaccine. This strain is the main component of the Prorabbit probiotic preparation, which is often used in Slovak rabbit farms (at dosage 1-2 g/animal/day for 21 days as prevention and 3 g/animal/day with therapeutic effect; resolved in water or mixed into feed).

  • Enterococcus faecium
  • enterocin
  • rabbit
  • immunity
  • meat quality
  • microbiota
  • intestinal morphology
  • weight gain

1. Definition

E. faecium CCM7420 strain was able to colonize the gastrointestinal tract (caecum) of rabbits (in the range < 1.0–6.7 log cycle, respectively 3.66 log cycle on average), to change the composition of intestinal microbiota (increased lactic acid bacteria, reduced counts of coliforms, clostridia and staphylococci), to modulate the immunity (significant increase of phagocytic activity), morphometry (enlargement absorption surface in jejunum, higher villi height:crypt depth (VH:CD) ratio), physiological (serum biochemistry; altered total proteins, glucose and triglycerides levels) and parasitological (Eimeria sp. oocysts) parameters and to improve weight gains (in the range 4.8–22.0%, respectively 11.2% on average), feed conversion ratio and meat quality (physicochemical traits and mineral content).

2. Introduction

Rabbit breeding has a great potential because of the small body size, short generation interval, rapid growth rate, high productive capacity and healthy, easily digestible meat of rabbits [1][2]. Moreover, rabbits can convert a higher amount (20%) of the protein they eat into edible meat, compared with pigs (16–18%) and cattle (8–12%; [3]). In several European countries, in which the rabbit breeding has a long history and high production efficiency, presently is regressing, whereas in the developing countries of the world rabbit farming has become to an important emerging enterprise. In growing rabbits, the most critical period is the weaning, when the kits are separated from mother and the milk is substituted with solid feed [4]. During these environmental and physiological changes/stresses, the rabbits are very sensitive to digestive disturbances, also called non specific enteritis (usually caused by dietary stresses, parasites—Coccidia and bacteria—Clostridia sp. and enteropathogenic Escherichia coli) and gastrointestinal infections (epizootic rabbit enteropathy—ERE, a multifactorial gastrointestinal syndrome, [5]). These dietary and bacterial changes are the main reason of morbidity and mortality and have negative effects on feed consumption, growth performance and health status of animals in this period and also on the economic aspects of rabbit farming.

To overcome this period, to reduce economic losses and to improve and stabilize the health status and gastrointestinal tract development, antibiotic growth promoters (AGPs) have been widely used for years. Although, these synthetic drugs showed good effects on production indicators, on the other hand, there was a risk of increasing resistance to antibiotics and transferring of antibiotic resistance genes from animal to human, which also threatened the human health and quality of meat and food [6]. For this reason, AGPs have been banned by the European Union (began in 1986 in Sweden and completely banned in January 2006, when the last four antibiotics have been permitted as feed additives was no longer allowed to be marketed or used from this date; IP/03/1058; [7][8]). As a result of the ban, researchers had to substitute AGPs and to find new feed additives that were supposed to be safer, without leaving residues and spreading resistance to themselves, but also improving health and productivity of rabbits. Therefore, the antibiotics have been replaced with new, naturally based supplements: probiotics, prebiotics, synbiotics, enzymes, bacteriocins, organic acids, herbs and their extracts, which are well-tried tools for disease prevention and therapy in various animal species, including rabbits [9].

The last two decades have seen a substantial increase in the use natural supplements and/or additives in animal nutrition, in which their antimicrobial activity has been highlighted many times. The EU in its Regulation EC 1831/2003 defined the terms “feed additives” as “substances, microorganisms or preparations, other than feed material and premixtures, which are intentionally added to feed or water” and the “antimicrobials” as “substances produced either synthetically or naturally, used to kill or inhibit the growth of microorganisms, including bacteria, viruses or fungi, or of parasites, in particular protozoa” [10]. According to the World Health Organization (WHO) probiotics are defined as “live microorganisms which, when administered in adequate amount, confer a health benefit on the host”. Although this definition is widely accepted, a 2007 guidance document from the European Commission on Regulation EC 1924/2006 on nutrition and health claims (NHCR) categorizes the term ‘probiotic’ as a health claim on the basis that it implies a health benefit. For this reason, the term “beneficial microbes” is used more often instead of a “probiotic microorganism” [11].

The use of several natural feed additives has already been reviewed in rabbit breeding [10][12][13][14][15]. The positive effects of probiotics and their antibacterial products—bacteriocins on health, growth performance, nutrient utilization and metabolism changes, microbial composition [14][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35], blood serum biochemistry, oxidative stress, immune response, intestinal morphology [21][24][28][29][30][31][34][36][37][38] and meat quality of rabbits [32][34][39][40][41][42][43] was described. However, in spite of the achieved results, there are still few declared probiotic preparations based (Lactina, Toyocerin; [44][45]; Prorabbit—declared in Slovakia; [25]) and detailed studies of microorganisms with beneficial properties that have also the ability to produce antimicrobial substances, enterocins.

3. Enterococcus faecium CCM7420 (EF2019) and Its Bacteriocin-Enterocin (Ent7420)

Enterococcus faecium EF2019 (CCM7420) is a bacteriocin-producing strain [47], which was isolated from the rabbit feces and genetically confirmed by the PCR method and subsequently by MALDI-TOF mass spectrophotometry as well as the sequencing procedure of this strain was provided (Dr. Kopčáková, IAP CBs SAS). This strain produces lactic acid, tolerates low pH (3.0; 63% surviving of cells) and is able to grow even in 5% oxgall—bile (80% surviving of cells), shows sensitivity to antibiotics, including vancomycin [25][48] and possess lipolytic activity [49]. Other unpublished data suggests that the CCM7420 does not produce biogenic amines and enzymes such as β-glucuronidase, β-galactosidase or N-acetyl-β-glucosaminidase (enzymes produced by unfriendly gut bacteria; their increased levels are usually the indicators of colon cancer), and it does not show any gelatinase (absence of the gelE gene) or hemolytic activities with low ability to form biofilm (0.092). The strain was deponed into Czech Collection of Microorganisms in Brno, Czech Republic to have number CCM7420. This strain showed the broadest inhibitory activity from all tested rabbits enterococcal strains against the indicators E. avium EA5, Listeria innocua LMG13568 and L. monocytogenes CCM4699 and against other tested enterococci and staphylococci tested such as clostridia, pseudomonads, enterobacteria and coliform bacteria [48]. The presence of the structural genes for enterocins (ent) A, P and L50B was detected; however, the CCM7420 did not possessed gene for ent B [47]. The molecular mass of its bacteriocin-like substance ranged from 3 to 10 kDa. Proteinaceous substance produced by CCM7420 strain was partially purified (partially purified bacteriocin (PPB) or enterocin (Ent) 2019 =7420). It is thermostable substance as well as stable at pH 4.0, 7.0 and 9.0. Its production starts in early logarithmic growth phase and it culminates in the late logarithmic phase of CCM7420 strain growth. By its properties, it can probably be included in the II. classification group of bacteriocins. Ent2019 or Ent7420 added to the growing strain L. innocua LMG13568 (after 4 h) inhibited its growth already at 1 h after enterocin addition with a difference of 1.5 log cycles (5 h of cultivation). This effect was prolonged up to 24 h. The Ent7420 was tested against more than 300 strains of enterococci, staphylococci, clostridia, pseudomonads, enterobacteria and coliforms [48]. The inhibitory activity of this substance was preserved after 24 months of storage at −20 °C (6400 AU/mL; [48]) and also after lyophilization (freeze dried) and redissolution in PBS buffer (25600 AU/mL; not published data). The CCM7420 strain is currently available in the ProRabbit, probiotic product for rabbits and other rodents, made by the International Probiotic Company Košice (Slovakia).

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