Antiparasitic Action of Lactobacillus casei ATCC 393 and Lactobacillus paracasei CNCM Strains in CD-1 Mice Experimentally Infected with Trichinella britovi

Nematodes of the genus Trichinella are among the most widespread parasites of domestic and wild omnivores and predatory animals. The present study aimed to evaluate the antiparasitic effect of Lactobacillus casei ATCC 393 (original) and L. paracasei CNCM in CD-1 mice experimentally infected with Trichinella britovi. Four groups of 20 mice (10 females and 10 males/group) were used, with two control (C) groups and two experimental (E) groups, in which each animal received a daily oral dose of 100 µL of 105 CFU/mL probiotics in Ringer’s solution. On day 7, all mice (except the negative control group) were infected orally with Trichinella (100 larvae/animal) as well as the two probiotics. On day 9 post-infection (p.i.), 10 mice/group were euthanized, and the presence of adult parasites in the intestinal content and wall was tested. On day 32 p.i., 10 mice/group were euthanized, then trichinoscopy and artificial digestion were performed to assess the muscle infection with T. britovi. On day 9 p.i., the experimental group pretreated with L. casei ATCC 393 (6.3 ± 3.03) showed a significantly lower number of adult parasites in the intestinal wall compared with the positive control group (24.6 ± 4.78). Additionally, a significantly lower adult parasite count in the intestinal wall was registered in female mice pretreated with L. paracasei CNCM (7.4 ± 4.71) compared to female mice from the positive control (29.0 ± 5.17). No statistically relevant results were obtained concerning the male mice or the data obtained at 32 days p.i., irrespective of mice gender.


Introduction
Trichinellosis is a zoonotic disease caused by nematodes of the genus Trichinella, which are among the most widespread parasites of domestic and wild omnivores and predatory animals [1]. Hosts can acquire infection by the consumption of raw or undercooked infected meat. The adults and larvae of Trichinella spp. reside in the same host, causing various adverse effects [2]. To date, two species of Trichinella have been reported in Romanianamely, T. spiralis (in domestic mammals) and T. britovi (in wild animals), but co-infections can also occur [3]. Trichinellosis is still present in Romania due to the local traditions and culinary customs that favor the transmission to humans. Humans can become infected through the consumption of untested raw meat, especially pork and wild boar [4,5], which has led to multiple outbreaks over the years [6,7]. The treatment of trichinellosis includes the use of benzimidazole derivates, such as albendazole or mebendazole [8,9]. Recently, the interest in developing different anthelmintic treatments that are both efficacious and safe has been increasing. Particular attention has been given to the potential anti-parasitic effect of probiotics [10].
Probiotics are live microorganisms that can provide a health benefit to the host when administered in adequate amounts [11]. Among them, lactobacilli are the most commonly used probiotics [12]. The Lactobacillus genus contains over 180 Gram-positive, anaerobic or microaerophilic, rod-shaped, non-spore-forming bacteria species [13].
Several studies suggest that probiotics can decrease the pathogenicity of parasites and, consequently, influence the course of parasitic infections [14]. In this regard, probiotics' main mechanisms of action are related to their ability to compete with pathogens for adhesion sites, enhance intestinal mucosal barrier activity, produce antimicrobial agents, and regulate the host's immune responses [15,16]. A good example, in this case, is an in vivo study conducted on gerbils (Meriones unguiculatus) that were experimentally infected with Gardia lamblia trophozoites and treated with 10 8 CFU of Lactobacillus johnsonii La1 for seven days prior to parasite inoculation. During the study, no morphological damage to the intestinal epithelium was observed, but a reduction in active trophozoites and infection length was noticed [17]. Regarding nematodes, oral supplementation with Lactobacillus rhamnosus (JB-1) at a dose of 1 × 10 9 CFU/day significantly accelerated Trichuris muris larvae removal in mice. This was accompanied by the upregulation of anti-inflammatory cytokine IL-10 levels and mucus-secreting epithelial cell numbers [18]. The positive effects of probiotic bacteria, such as reducing the parasite burden and pathological changes in experimental trichinellosis, have been previously described in several studies [19][20][21][22][23].
The most efficient manner of keeping the human population safe against trichinellosis is to eliminate the positive animals from human consumption. The use of probiotics in this parasitic infection is still in its infancy and they can only be used in experimental models. However, little is known concerning the effect of probiotics on species of Trichinella other than T. spiralis. Therefore, the present study aimed to evaluate the antiparasitic effect of two different probiotic strains of lactobacilli, Lactobacillus casei ATCC 393 (original) and L. paracasei CNCM (commercial product), in CD-1 mice experimentally infected with T. britovi.

Results
The mean number of adults (9 days p.i.) and larvae (32 days p.i.) of T. britovi in the experimental groups can be seen in Table 1 (for all mice), Table 2 (for female mice), and  Table 3 (for male mice). On the 9th and 32nd days after T. britovi infection (p.i.), the number of adult parasites and larvae found in the intestines (content and walls) and muscle (by trichinoscopy and artificial digestion) of CD-1 mice was higher in the positive control group than in the experimental groups that were pretreated with one of the two probiotic strains (Table 1, Table 2, Table 3). However, significant differences were registered for the number of adult parasites in the intestinal wall only in mice pretreated with L. casei (per total, and in female mice) and in female mice pretreated with L. paracasei ( Table 1, Table 2).

Discussion
Experimental studies regarding the use of probiotics against helminthic infections are scarce [24]. To the best of our knowledge, this is the first study that aimed to evaluate the antiparasitic effect of Lactobacillus strains against T. britovi in a murine experimental model. The current study emphasizes the antiparasitic effect of L. casei and L. paracasei against T. britovi (adults) infection in mice. The mice treated with these two probiotic strains showcased an overall lower parasite count (both adults and larvae) but with statistically significant results found only in the case of adult parasites compared to the control groups. The observed differences in adult worm count could be linked to the effect of the used probiotics on the development of the larvae to the adult stage, yet not affecting the reproductive capability of the adult worms. This effect was more pronounced in female mice, which might indicate that hormonal changes that appear during their sexual cycle could influence the efficacy of some probiotics. Different mechanisms can explain this outcome-mainly, the ability of probiotics to compete with pathogens for adhesion sites, increase intestinal mucosal barrier activity, synthesize antimicrobial agents, and modulate the host's immune system [14][15][16].
The mechanism behind the immunomodulation exhibited by probiotics involves interactions between these microorganisms and the gut-associated lymphoid tissue (GALT), which is an important local immune compartment. Probiotics can modulate the activity of several cells that compose GALT, such as enterocytes, dendritic cells, and T-cells, therefore increasing the protection against intestinal infections [25][26][27][28]. Furthermore, evidence regarding the role of gastrointestinal bacteria on nematode infections can be found in the correlation between microbiota composition and parasite species infections [28].
The results of the current study showed only a moderate inhibition capacity of L. casei and L. paracasei on T. britovi larvae and a stronger one against adult parasites. Considering the lack of previous research on the effects of probiotics on T. britovi, the interpretation of the obtained results is somewhat difficult. Therefore, several studies conducted on T. spiralis will be further used for comparison.
In the current study, 10 5 CFU/mL/animal in 100 µL of Ringer's solution was administered to each mouse. In contrast, other studies used different doses. For example, in one study, 1.9 × 10 9 CFU/mL of viable L. casei was orally administered to mice seven days before T. spiralis infection. This latter dose induced a significantly protective response against trichinellosis [29].
The current experimental protocol used two strains of Lactobacillus (L. casei and L. paracasei) and tested the mice on days 9 and 32 p.i., whereas other similar studies included a higher number of Lactobacillus strains and tested the animals during other time frames. In the study by El Temsahy et al. [21], among L. plantarum, L. casei, and L. acidophilus, the most effective in reducing the number of T. spiralis larvae in male Swiss mice, on day 28 p.i., was L. plantarum (87%), followed by L. casei (75%), and finally L. acidophilus (61%). Dvoroznáková et al. [22] found that on day 18 p.i., the number of T. spiralis larvae in BALB/c male mice was under 10/mouse, as assessed by artificial digestion, in the groups treated with L. fermentum, L. plantarum, E. faecium, and E. durans. Likewise, in another study, a significant decrease in the number of muscle larvae was detected on days 25 and 32 p.i. in the groups of BALB/c male mice that received L. fermentum AD1-CCM7420 and L. plantarum 17L/1 probiotic strain, and E. faecium AL41 and E. durans ED26E/7 bacteriocinproducing strains, respectively [23]. In the case of this latter study, the production of larvae was evaluated by the reproductive capacity index (RCI). The administration of E. faecium CCM8558, E. durans ED26E/7, L. fermentum CCM7421, and L. plantarum 17L/1 strains in male BALB/c mice resulted in a significant T. spiralis larval reduction, with a higher efficacy seen on day 25 p.i. (13,220-23,250 larvae/mice) [23].
The present study also suggests that the probiotics used might influence the local immune response of the host at the intestinal level, influencing the adults' reproductive capability. This theory is supported by other similar experimental protocols. Vargová et al. [9] showed that the administration of E. faecium EF55, E. faecium CCM7420, E. faecium CCM8558, E. durans ED26E/7, L. fermentum CCM7421, and L. plantarum 17L/1 influenced T. spiralis in male BALB/c mice during the intestinal and muscular phase of infection, while it may also influence the host's macrophagic activity. Both lactobacilli stimulated the metabolic activity of macrophages during the intestinal and early muscular phase of the infection (days 5 to 25 p.i). However, in the same study, enterococci increased the O 2 -production in the intestinal phase (day 5 p.i.) and during the muscular phase (days 25 and 32 p.i.) of this parasitic infection. The detection of helper CD4, cytotoxic CD8 T lymphocytes, and B lymphocytes in the small intestine of mice treated with probiotic strains and infected with T. spiralis was observed in a similar experiment [30]. Lactobacillus fermentum CCM7421 and L. plantarum 17L / 1 increased the numbers of helper CD4 T cells in the epithelium and cytotoxic CD8 T cells in the lamina propria on the 7th day of administration (before parasitic infection) [30]. Moreover, an 83.3% reduction in the burden of T. spiralis muscle larvae was observed on day 28 p.i. in female BALB/c mice which were immune-stimulated with NC8-pSIP409-pgsA-mIL-4 (IL-4 co-expressed with pgsA anchor system of Lactobacillus plantarum NC8 and delivered by live Lactobacillus plantarum NC8) [31].
Similar studies involving the use of probiotics have been conducted on other parasitic infections. A good example is an experimental study in mice (30 Swiss female mice) infected with Toxocara canis (100 eggs) and inoculated with Saccharomyces boulardii (10 7 CFU/g). The probiotic used promoted a reduction in the intensity of T. canis infection and modulated cytokine mRNA expression [32]. Another study investigated the effect of Bifidobacterium lactis subspecies animalis (3.5 × 10 10 CFU) on the immune and intestinal function of young pigs subsequently infected with Ascaris suum eggs (100/animal). The probiotic treatment significantly increased mRNA expression of genes associated with enhanced protection against parasitic infection, including IL-25, RETNLB, and SOCS3, and did not interfere with the normal expulsion of L4 from the jejunum [33].

Animals
Male (n = 40) and female (n = 40) white CD-1 mice, six to eight weeks old, weighing 20-30 g each, were used for the present study. The decision to add female mice to the experimental protocol was taken to observe if the hormonal changes that occur during their sexual cycle would influence the efficiency of the chosen probiotics. The mice were kept in the rodent facility of the University of Agricultural Sciences and Veterinary Medicine (UASVM) Cluj-Napoca (Romania) with the approval of the Ethics Committee of the same university, through permit number 265/12.07.2021. Mice were housed in clean polypropylene-covered cages, in a well-ventilated room (24 ± 2 • C) with relative humidity (42 ± 2%) and a 12:12 h light: dark cycle. The mice were fed with standard pellet food with free access to drinking water, under strict hygiene conditions. The bedding was changed weekly. Before the experiment, the mice were given five days to adapt to the laboratory environment.

Probiotic Strains and Parasites
Lactobacillus casei ATCC 393 strain was obtained from the Department of Food Science, UASVM Cluj-Napoca, while L. paracasei CNCM was purchased from a local pharmaceutical company (Sofar, Bucharest, Romania).
The T. britovi larvae that were used during the present experiment were previously isolated from a wolf (Canis lupus), with the species confirmed by molecular biology (Multiplex Polymerase Chain Reaction) at UASVM Cluj-Napoca and kept alive in the Parasitology Department, Faculty of Veterinary Medicine, UASVM Cluj-Napoca (Romania), until the experiment [34].

Experimental Design
The experimental design and protocol were approved by the Veterinary Sanitary and Food Safety Department in Cluj-Napoca, with the permit number 277, and by the Ethics Committee of the UASVM Cluj-Napoca (Romania) and respected the national legislation (Law 43/2014) and European legislation (EU Directive 63/2010), respectively.
Animals were randomly divided into four groups, each with two female and two male replicates at five mice/cage (n = 20) (Table 4). Probiotic strains were administered orally, in a daily dose of 10 5 CFU/mL/animal, in 100 µL of Ringer's solution. The mice were infected with 100 larvae/animal of T. britovi, on the 7 th day of treatment. On day 9 p.i., 10 mice (5 females and 5 males) from each group were euthanized (cervical dislocation), then their intestinal walls and intestinal content were examined. On day 32 p.i., the remaining 10 mice (5 females and 5 males) from each group were euthanized (cervical dislocation), and muscle samples were evaluated by trichinoscopy and artificial digestion.

Collection of Adult Parasites from the Intestinal Contents and Walls of Mice
To obtain the intestinal content, 10 mice (5 females and 5 males) were euthanized from each of the 4 groups on day 9 p.i. Afterward, the intestines were collected and cut transversally in segments of 5-10 cm. Each obtained segment was then cut longitudinally to gain access to the content and placed in a sieve in a conical container with saline (0.9% NaCl) solution. The samples were incubated at 37 • C for 4-5 h. Finally, the obtained sediment was examined with a 60× stereomicroscope (Olympus Bx61, Olympus Europe Holding, Hamburg, Germany) to count the adult parasites. The intestinal segments used in the previous step were collected and digested using the artificial digestion method as described in previously published articles and EU regulations [35], following the official protocol [34], to count the adults (females) in the intestinal mucosa.

Trichinoscopy and Artificial Digestion
The presence and distribution of Trichinella larvae in various muscle groups were determined through trichinoscopy. From each mouse, one compressor with 28 sections was examined as follows: masseter muscle −3 positions, diaphragm muscle −5 positions, forearm muscle −10 positions (5 left, 5 right), and hind leg muscles −10 positions (5 left, 5 right). The samples were examined at 4× magnification [3]. The mice carcasses (without fur, skin, and internal organs) used in the previous step were collected and digested individually with the artificial digestion method [35], following the official protocol [34], to count the larvae.

Statistical Analysis
Arithmetic mean and standard error of the mean were calculated for adults and larvae of T. britovi obtained after experimental infection in mice at 9 and 32 days p.i. by different techniques. These were computed by the gender of mice (females and males) and, in total, in the experimentally infected groups using MedCalc®Statistical Software version 20.014 [36]. Then, all data were analyzed by one-way ANOVA and Tukey-Kramer HSD test using online software. The level of significance was set at 0.05 [37].

Conclusions
The current experimental study in a murine model showed that L. casei ATCC 393 and L. paracasei CNCM are two probiotics that could potentially impact the development of the intestinal stage of T. britovi, although the exact mechanism behind this process needs further research. The results of the present study also indicated that L. casei ATCC 393 may be more efficient in reducing the number of T. britovi adults in female mice than L. paracasei CNCM.