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PROTECTIVE EFFECTS OF PROBIOTIC BACTERIA AG AINST EXPERIMENTAL TRICHINELLOSIS
DvorozAakova E,1, Buckova B.1, Hurnikova Z.1, Laukova A.2, Revajovd V.3
'institute of Parasitology, Slovak Academy of Sciences, Kosice, Slovakia institute of Animal Physiology - Centre of Biosciences, Slovak Academy of Sciences, Kosice, Slovakia 3University of Veterinary Medicine and Pharmacy in Kosice, Slovakia
Introduction. Gut microbiota represents a relevant factor that may strongly interfere with the pathophysiology of parasitic infections, determine the parasite survival and the outcome of parasitic infections. Therefore probiotics can play an important role in reducing the pathogenicity of many parasites (Berrilli et al., 2012). During the last decade, probiotics as means for the control of parasite infections were recorded mainly in intestinal diseases either in nongut infections (Travers et al., 2011). Nematode Trichinella spiralis causes an intestinal and tissue disease -trichinellosis characterized by the enteritis (induced by adult worms) and the inflammation with degenerative changes in the skeletal muscles (induced by larvae). The chemotherapy of trichinellosis with available anthelmintics is active only against adult worms, but not against muscle larvae. Therefore, the need for developing new methods for controlling this disease is living and anti-parasitic potential of probiotic bacteria could be successful. The present study was designed to study the antiparasitic effects of six different probiotic strains of lactobacilli and enterococci on the parasite burden in the host and on the fecundity of T. spiralis females.
Materials and methods. Animals (BALB/c mice) were divided into 7 groups: Control (n = 15) - T. spiralis infection without the administration of bacterial strains; Group 1 (n = 15) - Enterococcus faecium EF55 + T. spiralis; Group 2 (n = 15) - E. faecium CCM7420 + T. spiralis-, Group 3 (n = 15) - E. faecium CCM8558 + T. spiralis; Group 4 (n = 15) - E. durans ED26E/7 + T. spiralis; Group 5 (n = 15) -Lactobacillus fermentum CCM7421 + T. spiralis; Group 6 (n = 15) - L. plantarum 17L/1 + T. spiralis. Probiotic strains were administered per os daily at a dose of 109 CFU/ml in 100 (il and mice were infected per os with 400 T. spiralis larvae/mouse on day 7 of treatment.
Probiotic strains (Institute of Animal Physiology, Kosice): E. faecium EF55 was isolated from the chicken crop. E. faecium 2019 = CCM7420 is a rabbit-derived strain with probiotic properties, which produces enterocin 2019 (Ent 2019). E. faecium AL41 = CCM8558 is an environment-derived strain producing enterocin M. E. durans ED26E/7 was isolated from traditional ewes milk lump cheese. I. plantarum 17L/1 was isolated from stored ewes cheese. L. fermentum AD1 = CCM7421 is a canine-derived strain.
Parasite isolation: adults - intestinal washes and incubation; muscle larvae -digestive method. Fecundity test - females were isolated from the small intestine on day 5 p.i. and incubated for 20 h at 37 °C in 5% C02. Newborn larvae (NBL) were
counted per one female. A direct effect of probiotic strains examined in vitro -females were incubated with addition of the probiotic strain at different concentration (107, 105, 103and 101 CFU/ml) or without strains (control).
Results and discussion The highest numbers of T. spiralis adults (209 - 326) were found in the small intestine on day 5 post infection (p.i.) in all groups. A significant reduction of intestinal parasites occurred on day 11 p.i. in mice with therapy with E. faecium CCM8558 (107 ± 25), E. faecium CCM7420 (112 ± 14) and E. durans ED26E/7 (142 ± 29). Mice with the probiotic treatment absolutely eliminated adults from the small intestine till day 18 p.i.
In evaluation of muscle phase of infection, the occurrence of T. spiralis larvae was sporadic on day 18 p.i. (2 - 65). Numbers of muscle larvae reached the maximum in untreated mice on day 25 and 32 p.i. (50,080 ± 4,931 and 54,069 ± 8,020; respectively). Administration of strains E. faecium CCM8558, E. durans ED26E/7, I. fermentum CCM7421 and L. plantarum 17L/1 resulted in a significant larval count reduction with a higher efficacy on day 25 p.i. (13,220 - 23,250 larvae/mice).
The parasite infectivity is a result of the inteiplay of four components: the number of females that develop into adults, their fecundity, the length of their survival in the gut, and the period during which the muscle larvae remain viable (Dvoroznakova et ai, 2011). Female fecundity tested ex vivo was significantly decreased after the administration of enterococci and lactobacilli in comparison to T. spiralis infected group without treatment. The greatest inhibition was caused by strains E. faecium CCM8558 and E. durans ED26E/7 with 94% reduction of NBL. Similarly, the high reduction was recorded after treatment with L. fermentum CCM7421 and L. plantarum 17L/1 (78% and 83%). In contrast to non-affected numbers of adults presented in the gut of mice treated with Lactobacillus strains, their reproductive capacity was suppressed. These strains did not affect the maturation of T. spiralis larvae into adults or their expulsion from the gut, but they contributed to the decreased muscle parasite loads in the host by the control of NBL production. The application of strains E. faecium EF55 and E. faecium CCM7420 had only a modest inhibitory effect on the fecundity of females. In vitro fecundity test documented that the highest strain concentration of 107 CFU/ml was the most effective. The highest decrease in the number of NBL was recorded after incubation of females with L. fermentum CCM7421 (93%) followed by E. faecium CCM8558 (79%), L. plantarum 17L/1 (78%), E. faecium EF55 (76%), E. faecium CCM7420 (62%) and finally E. durans ED26E/7, which showed the 60% reduction. The production of NBL was increased in relation to a decreasing concentration of bacteria.
The resistance to T. spiralis infection is related to the ability of the host to prevent the development of infective larvae by removing adult worms from the small intestine, limiting the fecundity of adult females, and destroying NBL (Vasconi et al, 2015). Our study confirmed the anti-parasitic effect of six selected probiotic strains using an accelerated kinetics of worm expulsion from the gut (E. faecium CCM8558, E. faecium CCM7420 and E. durans ED26E/7), the reduction in female's
reproductive capacity (all examined strains), and by reduction of muscle larvae (L. fermentum CCM7421, L. plantarum 17L/1, E. faecium CCM8558 and E. durans ED26E/7). The inhibited female fecundity played an important role in infected mice treated with E. faecium CCM8558 and E. durans ED26E/7. However, in vitro conditions revealed a strong effect against NBL production in strains L. fermentum CCM7421, L. plantarum 17L/1, E. faecium CCM8558, and E. faecium EF55. This effect was suppressed in the host environment by interactions between bacterial strains, host immune response, and inflammatory processes.
Summary. The study was focused on the impact of six selected probiotic (bacteriocinogenic) strains (Enterococcus faecium EF55, E. faecium 2019 = CCM7420, E. faecium AL41 = CCM8558, E. durans ED26E/7, Lactobacillus fermentum AD1 = CCM7421, L. plantarum 17L/1) on the intensity of T. spiralis infection and female fertility ex vivo and in vitro. Strains E. faecium CCM8558, E. faecium CCM7420 and E. durans ED26E/7 strains significantly reduced the number of adults in the intestine (reducing effects 38-53%). The application of L. fermentum CCM7421, L. plantarum 17L/1, E. faecium CCM8558 and E. durans ED26E/7 caused a significant decrease in the number of muscle larvae (at average 65%). The treatment with E. faecium CCM8558 and E. durans ED26E/7 showed the highest inhibitory effect on female fecundity ex vivo (94%), followed by L. fermentum CCM7421 and L. plantarum 17L/1 (both 80%). A direct impact of probiotic strains examined in vitro manifested as follows: L. fermentum CCM7421 (93%), E. faecium CCM8558,L. plantarum 17L/1, E. faecium EF55 (about 80%), E. faecium CCM7420 and E. durans ED26E/7 (about 60%).
Literature: 1. Berrilli F., Di Cave D., Cavallero S., DAmelio S. // Front. Cell. Infect. Microbiol. - 2012. - 2(Article 141). - p.1-6. 2. Travers M.A., Florent I., Kohl L„ Grellier P.// J. Parasitol. Res. - 2011. - 2011 (610769). 3. Dvoroznakova E., Hurnikova Z., Kolodziej-Sobocinska M. // Parasitol. Res. - 2011- Vol.108.- p.169 -176. 4. Vasconi M.D., Bertorini G., Codina A.V., Indelman P., Di Masso R.J., Hinrichsen L.I.11 Open J. Vet. Med. - 2015. - Vol. 5. - p.ll -122.
Протективный эффект пробиотических бактерий при экспериментальном тихинеллезе. Дворожнакова Е., Буккова Б., Хурникова 3., Лаукова А., Ревайова В. Институт паразитологии Словацкой АН, Кошице, Словакия, Институт физиологии животных - Центр биологических наук Словацкой Академии ветеринарной медицины и фармации в Кошице, Словакия.
Резюме. Исследования посвящены изучению влияния 6 селективных штаммов (Enterococcus faecium EF55, Е. faecium 2019=ССМ7420, Е. faecium AL41=CCM8558, Е. durans ED26E/7, Lactobacillus fermentum AD1=CCM7421, L. plantarum 17L/1) на интенсивность заражения Т. spiralis и фертильность самок в условиях in vivo и in vitro. Штаммы Е. faecium ССМ8558, Е. faecium ССМ7420 и Е. durans ED26E/7 значительно снижали число имагинальных особей в
кишечнике (снижение на 38-53%). Применение L. fermentum ССМ7421, L. plantarum 17L/1, Е. faecium ССМ8558 и Е. durans ED26E/7 вызывало значительное снижение числа мышечных личинок (в среднем на 65%). Использование Е. faecium ССМ8558 и Е. durans ED26E/7 показало высокий ингибиторный эффект на плодовитость самок in vivo (94%), а после L. fermentum ССМ7421 и!, plantarum 17L/1 наблюдали снижение на 80%. Прямое влияние пробиотических штаммов, исследованных in vitro, было следующим: L. fermentum ССМ7421 (93%), Е. faecium ССМ8558, L. plantarum 17L/1, Е. faecium EF55 (около 80%), Е. faecium ССМ7420 и Е. durans ED26E/7 (около 60%).
