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Introduction
Trichinellosis is a serious food-borne parasitic zoonosis caused by the nematode of the genus Trichinella, which is characterized by an extremely wide host range and worldwide distribution (Bruschi,2012;Goździket al., 2017). The pathology of trichinellosis is associated with initial inflammatory response during the intestinal phase, and subsequent allergic and inflammatory responses during larval migration and invasion of the host muscles (Bruschi & Chiumiento, 2011; Hasby Saad et al.,2018).The efficacy of classic therapy with benzimidazoles is limited due to weak activity against encapsulated larvae, low water solubility, anthelmintic resistance, contraindication in children and pregnancy (Yadav & Temjenmongla, 2012). The anti-parasitic potential of alternative therapeutic approaches is therefore increasing in recent years. The need for development of a new methods to control this disease is necessary where the utilisation of the beneficial – probiotic bacteria hasbeen proposed. In general, probiotics may be useful in prevention and treatment in various health conditions and diseases (Singh et al.,2013). It is supposed that similar benefits will occur during the treatment of parasitic infections affecting gastrointestinal tract. The identification of factors mediating beneficial effects of probiotics presents an opportunity not only to understand their fine mechanisms of action, but also to develop the effective pharmacological strategies that could participate in treatments for various pathogens. Positive effects of probiotic bacteria reducing the parasite burden and pathological changes in experimental trichinellosis were previously described (Bautista-Garfiaset al., 1999, 2001; Martínez-Gómez et al., 2009, 2011; El Temsahy et al., 2015; Dvorožňákováet al.,2016;Buckováet al., 2018).
Gut microbiota plays a crucial role in completing the life cycle of the parasite in the intestine, in both enabling the development into adults and their reproduction and modulating the host immune response. Probiotic bacteria, which confer a health benefit on the host (FAO/WHO, 2002), may strongly interfere with the pathophysiology of parasitic infections, and determine the parasite survival along with the outcome of parasitic infections (Berrilli et al., 2012). Nematode Trichinella spiralis causes an intestinal and tissue disease – trichinellosis (Bruschi, 2002) characterized by the enteritis (induced by adult worms) and the inflammation with degenerative changes in the skeletal muscles (induced by larvae). Probiotic bacteria can provide an indirect protection to the host, probably by modulation the effect on newborn and muscle T. spiralis larvae (Travers et al., 2011; Dvorožňáková et al., 2016; Bucková et al., 2018). The main mechanisms of probiotic actions include enhancement of the gut epithelial barrier, increase of adhesion to the intestinal mucosa and simultaneous inhibition of pathogen adhesion, competitive elimination of pathogens, production of anti-microbial molecules, and modulation of the immune system (Goudarzi et al., 2014). They are able to modulate both, the innate and adaptive immune responses. In the context of non-specific immunity, their immunomodulatory activity is performed through modulation of inflammatory response, stimulation of phagocytic activity and activation of antigen-presenting cells (Tsai et al., 2012).
Macrophages, as part of the innate immune response, play a key role in resistance to a helminth Trichinella spiralis (Wing et al., 1979; Kołodziej-Sobocińska et al., 2007; Bruschi & Chiumiento, 2011). Macrophages belong to the antigen-presenting cells and are essential in initiation and modulation of the host immune response (phagocytosis, release cytokines, and inflammation) to parasite infection (Bai et al., 2012a; Franken et al., 2016). Excretory-secretory products of each T. spiralis stage modulate macrophage function through toll-like receptors expression and signalling pathways in macrophages (Han et al., 2018; 2019). Macrophages prevent the invasion of pathogens by release of cytotoxic molecules such as reactive oxygen species (ROS) and by secretion of proinflammatory cytokines (Franken et al., 2016). Macrophages express conserved pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs), which recognize microbe-associated molecular patterns (MAMPs) expressed on the cell surface of probiotic bacteria and thus can mediate the host immune response (Lebeer et al., 2010). The interaction between beneficial microbiota, macrophages and parasite in the host represent very active system where all components play a relevant role in modulating each other and in the maintenance of homeostasis important for the health of the host. Immunomodulatory activity of probiotic bacteria on macrophage’s function can uncover their potential role in control of trichinellosis.
The present study was designed to examine the effect of probiotic and bacteriocin-producing bacterial strains on the production of superoxide anion (O2-) in the peritoneal macrophages of mice infected with T. spiralis.
Materials and Methods
Probiotic strains
The effects of the following bacteria were tested: bacteriocin-producing strains with probiotic properties (Enterococcus faecium EF55, Enterococcus faecium 2019 = CCM7420, Enterococcus faecium AL41 = CCM8558, Enterococcus durans ED26E/7, and Lactobacillus plantarum 17L/1) and probiotic strain Lactobacillus fermentum AD1 = CCM7421. All used strains are original isolates (Bucková et al., 2018), not previously used for this purpose.
Enterococcus faecium EF55 was isolated from the chicken crop and characterized at the Institute of Animal Physiology CBs SAS – IAP CBs SAS, Košice, Slovakia. The strain produces a thermo-stable bacteriocin EF55.
Enterococcus faecium 2019 = CCM7420 is a rabbit-derived strain with probiotic properties, which produces enterocin 2019 (Ent 2019). It was isolated and characterized at IAP CBs SAS, Košice, Slovakia and deposited in the Czech Culture Collection of Microorganisms, Brno, Czech Republic – CCM7420.
Enterococcus faecium AL41 = CCM8558 (isolated and characterized at IAP CBs SAS, Košice, Slovakia and deposited in the Czech Culture Collection of Microorganisms, Brno, Czech Republic – CCM8558) is an environment-derived strain. The strain produces enterocin M with a wide antimicrobial inhibitory spectrum and possesses probiotic properties.
Enterococcus durans ED26E/7 was isolated from traditional ewes milk lump cheese at the Research Dairy Institute, Žilina – RDI, Žilina, Slovakia; but identified, characterized and prepared for experiment at IAP CBs SAS, Košice, Slovakia.
Lactobacillus plantarum 17L/1 was isolated from stored ewes cheese (RDI, Žilina, Slovakia) but identified, characterized and prepared for experiment at IAP CBs SAS, Košice, Slovakia.
Lactobacillus fermentum AD1 = CCM7421 was isolated and characterized at IAP CBs SAS, Košice, Slovakia and deposited in the Czech Culture Collection of Microorganisms, Brno, Czech Republic – CCM7421. It is a canine-derived strain possessing probiotic properties.
All used strains were evaluated according to the EFSA rules (Piskoríková, 2010). For the experiment they were cultivated in MRS broth (Merck, Eppelheim, Germany) at 37 °C for 24 h. Broth cultures were centrifuged (30 min at 10,000 x g) and the cells were resuspended in Ringer solution (Merck, pH 7.0) to a concentration of 109 colony forming units per ml (CFU/ml). The purity of the strains was checked by the standard microbiological method (ISO-International Organization for Standardization) by spreading dilutions in Ringer solution (pH 7.0, Merck, Eppelheim, Germany) onto the selective medium M-Enterococcus agar (ISO-15214, Difco, Detroit, USA) and/or MRS agar (Merck, Eppelheim, Germany). The cultures for application were stored at 4 °C.
Parasite
The reference isolate of Trichinella spiralis (ISS 004) (obtained and assigned codes from the Trichinella Reference Centre in Rome) was maintained by serial passages on ICR mice at the Institute of Parasitology SAS, and used for the infection. Larvae were released by artificial digestion (1 % pepsin, 1 % HCl for 4 h at 37 °C; both from Sigma-Aldrich, Hamburg, Germany) of tissue following the Kapel and Gamble protocol (2000) and kept in saline solution until inoculation of experimental mice.
Experimental design
The experiment was performed on pathogen-free eight week old male BALB/c mice (VELAZ, Prague, Czech Republic; n = 147) weighting 18 – 20 g. Mice were kept under a 12-h light/dark regime at room temperature (22 – 24 °C) with 56 % humidity and kept on a commercial diet and water available without restrictions. Animals were divided randomly into 7 groups: Control (n = 21) – T. spiralis infection without the administration of bacterial strains; Group 1 (n = 21) – E. faecium EF55 + T. spiralis; Group 2 (n = 21) – E. faecium CCM7420 + T. spiralis; Group 3 (n = 21) – E. faecium CCM8558 + T. spiralis; Group 4 (n = 21) – E. durans ED26E/7 + T. spiralis; Group 5 (n = 21) – L. plantarum 17L/1 + T. spiralis; Group 6 (n = 21) – L. fermentum CCM7421 + T. spiralis. Probiotic strains were administered per os daily at a dose of 109 CFU/ml in a total volume of 100 μl. Mice were infected per os with 400 T. spiralis larvae/mouse on day 7 of treatment. Samples of the peritoneal macrophages were obtained on days: -7, 0, 5, 11, 18, 25 and 32 post infection (p.i.).
Intestinal worm burdens
The intestinal phase of infection was investigated on days 5, 11 and 18 p.i. The small intestine was cut into 5 – 10 cm long pieces, placed into a sieve and incubated in conical pilsner glasses in 37 °C NaCl (0.9 % saline) overnight. After incubation, gut pieces were discarded and the worms in the sediment were counted under stereomicroscope at 60 x magnification (Leica S8APO, Leica Microsystems, Germany).
Isolation of muscle larvae
The muscle phase of infection was examined on days 18, 25 and 32 p.i. Whole eviscerated carcasses were minced and artificially digested (1 % pepsin HCl for 4h at 37 °C; both from Sigma-Aldrich, Germany) following the protocol of Kapel and Gamble (2000). Samples were allowed to settle for 20 min before the supernatant was discarded and the sediment was poured through a 180 μm sieve into a conical glass and washed with tap water. The sediment was finally transferred to a gridded Petri dish and counted under stereomicroscope at 40 x magnification (Leica S8APO, Leica Microsystems, Germany). Depending on the density of larvae either a sub-sample or the whole sample was counted.
Superoxide anion assay
Generation of extracellular superoxide anion (O2-) from peritoneal macrophages was assayed as superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome C with or without stimulation with phorbol myristate acetate (PMA) (Dvorožňáková et al., 2008). Peritoneal cells were aseptically harvested in RPMI 1640 (Sigma-Aldrich, Hamburg, Germany) to a final concentration of 1x106 cells/ml. One ml of cell suspension was adhered to each well using 24-well plates (Falcon, France) and incubated at 37 oC in 5 % CO2 and 85 % humidity for 2 h. The reaction was carried out in 0.3 ml/well of 160 μM ferricytochrome C (Sigma-Aldrich, Hamburg, Germany) in Earl’s balanced salt solution (EBSS) (pH 7.2). For control the reaction was blocked by 300 μg SOD/10 μl in EBSS (Sigma-Aldrich, Hamburg, Germany). 10 μl of PMA (Sigma-Aldrich, Hamburg, Germany) in ethanol was used for the stimulation of cells for respiratory burst. Cells were incubated at 37 oC in 5 % CO2 and 85 % humidity for 2 h and the optical density (OD) of supernatant was measured at 550 nm. The amount of O2- produced was calculated from the difference between OD in reactions blocked by SOD and without SOD. As the resulting value, the nanomols (nmol) of produced O2- were calculated according to the formula: nmol O2- = (OD/6.3) x 100 and determinated for 1 mg of cell proteins.
Statistical evaluation
Statistical differences were assessed using one-way ANOVA, followed by post hoc Tukey’s test (a value of P<0.05 was considered significant), which allowed comparison between each two groups at each time point. The analyses were performed using the Statistica 6.O (Stat Soft, Tulsa, USA) statistical package.
Ethical Approval and/or Informed Consent
The research related to animals has been complied with all the relevant national regulations and institutional policies for the care and use of animals. The experimental protocol was in compliance with current Slovak ethical rules for animal handling and it was approved by the Animal Care Committee of the Institute of Parasitology SAS and the State Veterinary and Food Administration of the Slovak Republic (Ro-3184/14-221).
Results
Parasite burden – numbers of adults and muscle larvae
A significant reduction of parasites in the gut occurred on day 11 p.i. (Table 1) in mice treated with strains E. faecium CCM8558 (53 %), E. faecium CCM7420(51 %), E. durans ED26E/7 (38 %), and E. faecium EF55 (30 %). Mice with this probiotic treatment absolutely eliminated adults from the small intestine till day 18 p.i. Lactobacilli did not affect the presence of adults in the intestine.
The highest numbers of muscle larvae were detected in untreated mice (Table 1) on days 25 and 32 p.i. The probiotic treatment resulted in a significant larval count reduction on day 25 p.i., as follows: E. faecium EF55 (44 %), E. faecium CCM8558 (74 %), E. faecium CCM7420 (26 %), E. durans ED26E/7 (54 %), L. fermentum CCM7421(64 %) and L. plantarum 17L/1(69 %).
Parasite burden in mice treated with probiotic bacteria and infected with T. spiralis.
Intestinal phase (numbers of adults)
Days post infection
5
11
18
(mean ± S.D. )
reduction (%)
(mean ± S.D.)
reduction (%)
(mean ± S.D.)
reduction (%)
T.spiralis (control)
295 ± 24
229 ± 37
2±2
E.faecium EF55 + T.spiralis
293 ± 38
0.7
160 ±39
30.1
0±0
100
E.faecium CCM8558 + T.spiralis
244 ± 25
17.3
*107 ±25
53.3
0±0
100
E.faecium CCM7420 + T.spiralis
235 ± 35
20.3
*112±14
51.1
0±0
100
E.durans ED26E/7 + T.spiralis
256 ±7
13.2
*142 ±29
38
1±2
50
L.fermentum CCM7421 + T.spiralis
209 ± 21
29.2
210 ±27
8.3
40 ±12
0
L.plantarum 17L/L1 + T.spiralis
326 ± 48
0
192 ±8
16.2
1±2
0
Muscle phase (numbers of larvae)
Days post infection
18
25
32
(mean ±S.D.)
reduction (%)
(mean ± S.D.)
reduction (%)
(mean ± S.D.)
reduction (%)
T.spiralis (control)
2±4
50,080 ± 4,931
54,069 ± 8,020
E.faecium EF55 + T.spiralis
40 ±9
0
37,060 ±4,150
26
42,580 ± 7,750
21.2
E.faecium CCM8558 + T.spiralis
47 ±2
0
**13,220 ± 1,842
73.6
*23,810 ±799
56
E.faecium CCM7420 + T.spiralis
24 ±6
0
27,970 ±7,212
44.1
46,950 ±3,818
13.2
E.durans ED26E/7 + T.spiralis
28 ±16
0
*23,250 ± 5,938
53.6
**29,080 ± 2,204
46.2
L.fermentum CCM7421 + T.spiralis
4±2
0
**18,380 ± 2,039
63.7
26,272 ± 2,566
51.4
L.plantarum 17L/L1 + T.spiralis
65 ±6
0
**15,540± 2,191
69
32,070 ± 6,463
40.7
*P<0.05; **P<0.01 - statistically significant differences from T. spiralis infected group without treatment
Superoxide anion production
T. spiralis infection in untreated mice (control) induced a great suppression of the metabolic activity of peritoneal macrophages on day 5 p.i. (Fig. 1). The generation of O2- was restored and stimulated on days 11 and 18 p.i. During the muscle phase of trichinellosis the O2- production in untreated and infected mice (control) was decreasing till the end of the experiment (day 32 p.i.).
Fig. 1
Production of superoxide anion by peritoneal macrophages from mice treated with probiotic bacteria (enterococci) and infected with T. spiralis. *P<0.05; **P<0.01– statistically significant differences from T. spiralis infected group without probiotic treatment (control).
The administration of E. faecium EF55 and E. faecium CCM8558 significantly (P<0.05; P<0.01) increased metabolic activity of macrophages before the parasite infection. This happened on day 0, e.i. after one week of the treatment (Fig. 1). All enterococci prevented the strong inhibition of the O2- production on day 5 p.i. caused by T. spiralis, and significantly (P<0.05; P<0.01) stimulated its generation. The increased metabolic activity induced by enterococci was also recorded in the muscle phase on days 25 (E. faecium CCM8558) and 32 p.i. (P<0.01; E. faecium EF55, E. faecium CCM7420, E. faecium CCM8558, and E. durans ED26E/7).
Similarly, lactobacilli stimulated the O2- production during the acute phase of trichinellosis, on day 5 p.i. (P<0.05), and prevented the strong inhibitory effect of T. spiralis infection (Fig. 2). The stimulative effect on macrophage’s metabolic activity after treatment with L. plantarum 17L/1 and L. fermentum CCM7421 lasted throughout the entire experiment, where a significant increase on days 11, 25 and 32 p.i. (P<0.05; P<0.01) was observed.
Fig. 2
Production of superoxide anion by peritoneal macrophages from mice treated with probiotic bacteria (lactobacilli) and infected with T. spiralis. *P<0.05; **P<0.01– statistically significant differences from T. spiralis infected group without probiotic treatment (control).
Discussion
Our study revealed that the examined probiotic strains may influence macrophage’s oxidative metabolism in T. spiralis infection and their effect is strain-specific. Macrophages are important cells involved in initiation and modulation of the host immune response to the helminth parasite. After a contact with the antigen they stimulate and produce reactive oxygen species such as superoxide anion (O2-), hydroxyl radical (OH), hydrogen peroxide (H2O2), nitric oxide (NO), peroxynitrite (ONOO-) that are highly toxic to the parasites (James, 1995; Martinez et al., 2004).
In our study, T. spiralis infection in mice without probiotic treatment induced a significant suppression of superoxide anion (O2-) production in peritoneal macrophages in the first week of infection, what was followed by an increase in the third week of infection, i.e. at the time of newborn larvae migration into the host muscles. Trichinella adults and muscle larvae are more resistant to the superoxide anion because they contain 3 to 5 times higher amount of antagonist enzyme superoxide dismutase (SOD) when compared to newborn larvae that are highly susceptible to the oxygen radicals (Kazura & Meshnick, 1984). The SOD plays an important role in parasite-host interaction by modulating the reactions of these host cells. This enzyme may act as a necessary defensive mechanism of the parasite against the highly destructive superoxide radical of the host’s immune system.
In the muscle phase of trichinellosis, the O- generation was de2creased, probably because of the Th2 response dominance. An induction of the host immunosuppression represents an important parasite evasive strategy, in context of which Trichinella spp. is characterized by an extremely strong ability to induce suppression of the host immune system (Gruden-Movsesijan et al., 2011; Aranzamendi et al., 2012). Trichinella excretory-secretory proteins (ESP) induce strong immunosuppression during the first 2 weeks of infection, what is followed by a Th2 polarized immune response with alternatively activated M2 macrophages (Ilic et al., 2012). Larval ESP significantly inhibit the activity of M1 macrophages, which play a key role in the host immune response against various pathogens (Bai et al., 2012b). Within this scope, Zhao et al. (2017) found that recombinant T. spiralis calreticulin (the surface protein produced by adults and muscle larvae) binding complement C1q reduced monocyte and macrophage adhesion to NBL mediated by complement, and also reduced the release of reactive oxygen products in the macrophages. Hence, the above studies demonstrated that larval ES products affect both classical and alternative activation of macrophages.
Wang et al. (2019) summarized the ability of probiotic bacteria to regulate macrophage polarization. Probiotic bacteria may induce different macrophage phenotypes depending on physiological and pathological conditions. Some probiotic strains activate the M1 macrophages to exert pro-inflammatory effect (Th1 immunity, increased oxidative metabolism), whereas a variety of the other probiotics can induce anti-inflammatory M2 macrophages. The interactions between probiotics, macrophages and pathogens are described particularly on bacterial and protozoan infections (Vitetta et al., 2016; Wang et al., 2019). Nevertheless, data from helminth infections are missing. In our experiment, the probiotic strains E. faecium EF55 and E. faecium CCM8558 increased the production of the superoxide anion prior the infection on day 0 of the experiment (i.e. after 7 days of application). Similarly, Lactobacillus paracasei isolated from fermented milk stimulated the formation of reactive nitrogen in macrophages (Kapila et al., 2013). A significant increase in the production of oxygen radicals in blood leukocytes has also been reported in humans with probiotic diets of Enterococcus faecium M-74 or Lactobacillus johnsonii (Mikeš et al., 1995; Donnet-Hughes et al., 1999). In our study, enterococcal strains stimulated the O2- synthesis during the acute phase of T. spiralis infection on day 5 p.i. Lactobacilli showed a weaker stimulating effect on macrophage’s activity on day 5 p.i. in comparison with enterococci, but prevented the suppression caused by parasite in untreated mice. However, in the developed intestinal phase of the infection (from day 11 p.i.) lactobacilli increased the O2- production with greater intensity than enterococci. Probably as a result of antiparasitic defence against longer persisting worms in the gut of infected mice treated with lactobacilli (till day 18 p.i.). In our experiment, the application of Enterococcus strains resulted in a significant reduction of the number of adults on day 11 p.i. (30 – 53 %), which might be associated with the early activation of proinflammatory M1 macrophages induced by bacterial products (Rolot & Dewals, 2018). The M1 macrophages are involved in mucin hyperproduction (Martinez & Gordon, 2014) that helps to remove the parasite from the intestine (Miller, 1987). In addition, as compared to lactobacilli, enterococci are better adhered to the intestinal mucosa (Lauková et al., 2004), thus preventing the parasite penetration into the intestinal willi and crypts. L. plantarum 17L/1 and L. fermentum CCM7421 did not affected the number of adults in the intestine. Probiotic treatment by all strains did not have a significant effect on peritoneal macrophages on day 18 p.i. Assuming that the parasites were not present for a longer time in the host gut. E. durans ED26E/7 has even significantly reduced the formation of this oxygen radical on day 18 p.i. This might be explained by the fact that mice modulated by this strain showed low numbers of muscle larvae on day 18 p.i. This was due to the significantly increased enzymatic activity of blood phagocytes (Dvorožňáková et al., 2016), which could contribute to the elimination of newborn larvae (NBL) and prevent their migration into the muscles. During the muscular phase, the “nurse cell” is an immunologically protected site for T. spiralis where larval E/S products maintain long-term communication with the host (Sofronic-Milosavljevic et al., 2015). The E/S products of muscle larvae contain various proteases and protease inhibitors including serine, cysteine, metalloproteases, serpine and cystatin that can modulate the immune response of the host leading to chronic, long-term survival of Trichinella larvae in muscle cells (Ashour, 2013). In the present study, we have detected a significant reduction of larval burden in muscles of T. spiralis-infected mice and treated with probiotic strains on day 25 p.i. (26 – 74 %). Nevertheless, the production of superoxide anion in peritoneal macrophages was also stimulated during the muscle phase. In mice with probiotic treatment, despite the significant reduction compared to control on day 32 p.i. (13 – 56 %), an increase of 5,000 – 19,000 larvae/mouse between days 25 and 32 p.i was observed. This growth points to the fact that probiotic strains have slowed down migration of NBL to the target tissues - muscles. Oxygen radicals from macrophages, production of which was stimulated and peaked on day 25 p.i. after the treatment with strains L. fermentum CCM7421, L. plantarum 17L/1, E. faecium CCM8558, and on day 32 p.i. after the administration of E. faecium E55 and E. faecium CCM7420, could be involved in the damage and reduction of larval infectivity. E. durans ED26E/7 induced the balanced production of O2- during the muscle phase. Our results suggest that probiotic strains induced the classical activation of M1 macrophages during the NBL migration. Despite alternative macrophage phenotype induced by the Th2 immune response is being activated in helminthoses (Rodríguez-Sosa et al., 2002), the work of Ilic et al. (2011) documented that the soluble NBL antigen induces the mixed Th1/Th2 polarization. Alternatively activated macrophages (AAM) are identified as regulatory cells able to act against proinflammatory and cellular immune effector mechanisms (Martinez & Gordon, 2014). They also play an important role in the regeneration of skeletal muscle in vivo and have beneficial effects on myogenic growth and cell differentiation (Arnold et al., 2007). We assume that alternatively activated macrophages are abundantly present in muscle tissue and blood in the course of trichinellosis. In addition to AAM, NBL are also able to induce classical activation of pulmonary macrophages during migration (Falduto et al., 2016). It is noteworthy that in our study the metabolic activity of peritoneal macrophages, which are associated with NBL migration from the intestine to other host tissues, was induced by probiotic therapy, that might have contributed to the NBL damage and reduced the intensity of infection in the host muscles. Probiotic treatment has also stimulated the oxidative metabolism of peritoneal macrophages during the muscle phase, which can result from the activation of immune memory cells but also can be influenced by low levels of the antagonistic enzyme superoxide dismutase due to the low number of its producers – muscle larvae.
Our experimental model study showed that probiotic strains have the ability to decrease the intensity of parasitic infection by affecting important components of the innate immune system such as phagocytosis (Dvorožňáková et al., 2016) or macrophage’s production of cytotoxic superoxide anion. The best protective effect against T. spiralis infection associated with the increased oxidative metabolism of peritoneal macrophages was exhibited by L. fermentum CCM7421 and L. plantarum 17L/1, which activated the metabolic activity of macrophages during the migration of newborn larvae (from day 5 to 25 p.i.). Enterococci efficiently supported the metabolic activity of macrophages in the anti-parasitic defence at the early phase of trichinellosis (day 5 p.i.). The obtained results confirmed the impact of the used probiotic strains as effective mediators to regulate macrophage´s oxidative metabolism in T. spiralis infection. These are promising data which link use of probiotic with their prophylactic and therapeutic properties in trichinellosis treatment and prevention.
