الفهرس 
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Abstract
Since the mid nineteenth century, trichinellosis has been recognized as a meat-borne zoonosis. In the international ranking of foodborne parasites, Trichinella spiralis (T. spiralis), was ranked among the top ten. As a result of the growing evidence of the poor effect of drugs against larvae and the difficulties in producing vaccines, the need for new alternatives to control trichinellosis aroused. The potential use of probiotics in controlling enteric infections has received tremendous interest in the last decade. Thus, the present study was carried out to test the in vivo protective efficacy of two newly isolated Egyptian probiotic strains; Lactobacillus acidophilus P110 (L. acidophilus) and Lactobacillus plantarum P164 (L. plantarum) versus Lactobacillus casei ATCC 7469 (L. casei) whose efficacy was previously documented against intestinal trichinellosis.
This study was carried out on Swiss strain albino mice. For evaluating the in vivo safety and colonization of the tested Lactobacillus strains, 40 mice were divided into four equal groups: L. casei-fed, L. acidophilus-fed, L. plantarum-fed and broth-fed groups. In the three probiotic-fed groups, mice were orally fed with 1.9x109 CFU/ml of viable Lactobacilli for seven days. While mice in the broth-fed group received the same daily volume of broth as the probiotic-fed groups. Both physical and biochemical safety of the probiotics studied were performed. Firstly, physical safety was assessed by the daily monitoring of the mortality rate, general health, changes in behaviour, as well as body weight per mouse in each group along the feeding period (seven days and one day later). Secondly, biochemical safety was assessed one day P.F. by measuring SGOT, SGPT, serum urea, serum creatinine as well as total serum cholesterol. Bacterial colonization in mice of the three probiotic-fed groups was performed both qualitatively and quantitatively in Gram-stained jejunal sections and smears respectively one day P.F.
Three hundred and seventy mice were allocated into two main groups: non infected group (I) and infected group (II). Group (I) constituted of 170 mice and was further subdivided into three subgroups; Ia: (10 non infected non fed mice), Ib: (150 non infected probiotic-fed mice) and Ic: (10 non infected broth-fed mice). The mice in subgroup Ib were further equally subdivided into three subgroups (Ib1, Ib2, Ib3); each was fed with one of the tested probiotics; L. casei, L. acidophilus and L. plantarum, respectively. The infected group (II) constituted of 200 mice which were subdivided into two subgroups; IIa: (50 non fed infected mice) and IIb: (150 probiotic-fed infected mice). The latter subgroup was further equally subdivided into three subgroups (IIb1, IIb2, IIb3); each was fed with one of the tested probiotics. The dose of infection was 300 T. spiralis larvae. The mice in subgroup (IIb) were infected one day post feeding. from each infected subgroup ten mice were sacrificed at five different dates; namely the zero, fifth, 12th, 17th and 28th day P.I. to undergo assessments criteria via parasitological, immunological and histopathological studies.
The parasitological study included assessment of adult T. spiralis count on the fifth, 12th and 17th day P.I. as well as larval count on the 28th day P.I. As regards the immunological assessment, sera from each group were pooled and the level of IFN-γ was measured by an ELISA kit at five different dates. In the non infected group (Group I), the IFN-γ level was measured one day P.F. (zero day), on the fifth, 12th, 17th and 28th day P.F., while in the infected group (Group II) IFN-γ was measured on the day of infection (zero day), fifth, 12th, 17th and 28th days P.I. While the histopathological study included assessment of inflammatory cells infiltration, tissue damage, measurement of villus heights, goblet cells number estimation in the jejunal tissue stained with H&E one day P.F. in the non infected subgroups and on the fifth day P.I. in the infected subgroups. Results were tabulated and statistical analysis was performed.
Concerning the safety results; no deaths or changes in general behaviour of mice of neither in the three probiotic-fed groups nor the broth-fed group were noted. As for the body weight, only the L. acidophilus-fed group and the L. plantarum-fed group showed a statistically significant weight gain. Meanwhile, the biochemical analysis regarding liver enzymes (SGOT, SGPT), renal function tests (blood urea and creatinine) and total serum cholesterol levels; showed that all values were within the normal range in the four groups. As regards the colonization results, the jejunal sections stained with Gram stain in the three probiotic-fed groups revealed colonized and luminal gram positive bacilli. In addition, the difference in mean Lactobacilli count per oil field in the jejunal smears of the three probiotic-fed groups was statistically non significant.
Parasitologically, the highest percentage reduction in the mean adult count was observed in L. plantarum-fed infected subgroup (56.98%, 65.42% and 69.02%) on the fifth, 12th and 17th day post infection (P.I.), respectively). Lesser percentage reductions were recorded in both the L. casei-fed infected subgroup (36.19%, 23.68% and 31.58% respectively) and L. acidophilus-fed infected subgroup (36.50%, 11.8% and 7.61% respectively). The comparison between the two newly isolated strains was in favour of L. plantarum in the three dates.
On the 28th day after challenge, the highest reduction in the mean larval count was in L. plantarum-fed infected subgroup (87.92%). While lower percentage reduction was observed in the L. casei-fed infected (74.88%) and L. acidophilus-fed infected subgroups (60.98%). When comparing the effect of L. casei with that of L. acidophilus and L. plantarum on the larval count, it was found that only L. plantarum was superior to L. casei with a statistically significant difference.
Immunologically, one day P.F., the IFN-γ level was higher in the probiotic-fed non infected subgroups (Ib) than the broth-fed subgroup (Ic), then showed a gradual decrease along the durations studied. On the 17th day P.F. IFN-γ was only detectable in L. plantarum-fed subgroup (Ib3). While on the 28th day P.F. the IFN-γ was below the detectable limit in all non infected subgroups. On the other hand, in the infected control subgroup (IIa) the IFN-γ level was detected on the day of infection (zero) and fifth day P.I., while its level on the 12th, 17th and 28th day P.I. was below the detectable limit. Regarding the probiotic-fed infected subgroups (IIb), the levels of IFN-γ started at higher levels than the infected control subgroup (IIa), then decreased gradually until becoming non detectable on the 17th and 28th day P.I.
Histopathologically, the examination of the H&E-stained jejunal sections of the probiotic-fed non infected subgroups (Ib1, Ib2, Ib3) as well as the broth-fed subgroup (Ic) has reflected neither inflammation nor tissue damage. A statistically significant increase in the mean villus height as well as goblet cells hyperplasia were noticed in the three probiotic-fed subgroups.
All jejunal sections of the probiotic-fed infected subgroups showed amelioration of the inflammation and damage resulting from T. spiralis infection as well as goblet cell hyperplasia and a significant restoration of villus heights. Noticeably, the mean villus height measured in the L. plantarum-fed infected subgroup (IIb3) was significantly higher than both; L. casei-fed infected (IIb1) and L. acidophilus-fed infected (IIb2) subgroups. Thus histopathological parameters showed that L. plantarum was noticeably superior to the other two strains which gave almost comparable results.
The results of the present study indicate that L. plantarum P164, through mechanical and immunological protective mechanisms, verified a remarkable protective superiority over both L. casei ATCC 7469 and L. acidophilus P110 against T. spiralis infection observed in both parasitological and histopathological parameters. Thus this promising probiotic strain which is a safe, natural protective agent provides a future preventive scope against T. spiralis infection.