Protective and therapeutic effects of Lactobacillus brevis PQ214320 and Bacillus subtilis PQ198038 Against experimental Trichinella Infection

Abstract

One of the most significant lactic acid bacteria genera is Lactobacillus, which is known to generate compounds such as bacteriocins that can stop the growth of harmful bacteria. The current study investigated the protective and therapeutic effects of two novel probiotic strains, Lactobacillus brevis PQ214320, and Bacillus subtilis PQ198038, on parasitological, histopathological, and immunological responses in mice infected with Trichinella spiralis. A total of 120 mice were divided into six groups, including a positive control group (C) which was infected with Trichinella infection and not treated, mice treated orally with albendazole (ALB) at a dose of 5 mg/kg bw for 3 days after infection, and mice treated with probiotics (109 Colony-Forming Unit (CFU)/mL/animal, in 100 µL of Ringer's solution) either pre- and post-infection with L. brevis PQ214320 (LP) or B. subtilis PQ198038 (BSP), or only post-infection with L. brevis PQ214320 (L) and B. subtilis PQ198038 (BS). Infection was induced by oral inoculation of 400 T. spiralis larvae. Parasite burden and, histopathological, and immune responses were assessed at 5 and 19 days post-infection. The results showed that the LP group had significantly reduced adult worm and muscle larval counts compared with the positive control group. In contrast, BSP reduced the parasite burden, but to a lesser extent. The immune response was characterized by elevated levels of IL 12 and IFN-γ in the LP group at 5 days -post-infection (dpi), indicating a strong Th1 response, which declined but remained significantly higher than in the control infected group at 19 dpi. Serum IgG responses were higher in the LP group at 19 dpi, suggesting that a more robust adaptive immune response was triggered by L. brevis. Pre- and post-treatment with B. subtilis PQ198038 and L. brevis PQ214320 significantly improved the histopathological abnormalities and collagen deposition in the small intestinal and diaphragm muscular tissues caused Trichinella infection and restored claudin 1 content in the same tissues. These findings suggest that L. brevis PQ214320 offers a stronger protective effect against T. spiralis infection, potentially through enhanced immune modulation and parasite reduction, whereas B. subtilis PQ198038 provides beneficial but less potent responses. This study highlights the potential of novel probiotics strains as adjunct protective agents and therapies against T. spiralis infection.

Fig 1. Infographic to show timings, sample collection, and processing (https://app.diagrams.net/).

Fig 2. (A) T. spiralis adult worms’ numbers in the small intestine of mice (n = 5) treated with probiotics.

(B) Numbers of muscle larvae isolated from mice (n = 5) with probiotic treatment and T. spiralis infection. The significant (P < 0.01) differences are (A) compared to the C group, (B) against the ALB group, (C) compared with the LP group, (d) compared with the BSP group, (E) compared with L group, and (F) compared with BS group. One-way ANOVA, post hoc Tukey’s test for multiple comparisons was used.

Fig 3. Levels of IL 12(a, b), IFN-γ (c,d), and IL 17 (e,f) from different groups of mice.

The data are shown as the mean ± SD of 5 mice per group. The cytokines were quantified using an ELISA. One-way ANOVA, post hoc Tukey’s test for multiple comparisons was used. * Indicate significance difference (P < 0.05) in comparison with control infected non-treated group (C). (ns) indicate non-significant differences in comparison with the C group.

Fig 4. Detection of anti-Trichinella IgG in mice tested groups at 5 dpi and 19 dpi by ELISA.

All serum samples were tested in triplicate. The data are presented as the mean OD values ± standard deviation (SD) of 5 mice per group of anti-Trichinella IgG level. The anti-Trichinella IgG level was detected using an indirect ELISA. One-way ANOVA, post hoc Tukey’s test for multiple comparisons was used. * Mean significance difference (P < 0.05) in comparison with control infected non-treated group (C). (ns) indicate non-significance difference in comparison with the C group.

Fig 5. Representative histopathological photomicrographs of small intestine in the different experimental mice groups (n = 5).

C: Positive Trichinella untreated control mice showing thickening and flat of the villi (V), degeneration, atrophy, necrosis with sloughing of the upper tips of the villi (S), multiple T. spiralis larvae (arrow) in the villus cryptus junctions and at muscularis (M) associated with severe infiltration of inflammatory cells in submucosa (Sm) and lamina propria (star), hyperplasia of goblet cells (green arrow), dilatation of the blood capillary (red arrow), and edema in different intestinal layers (arrowhead) (H&E, low × 200 and high ×400 Magnifications). ALB: Infected mice treated with Albendazole, ALB group displaying sub-epithelial cell edema (arrowhead), atrophy, and sloughing (S) of the upper tips of some villi (V) with infiltration of inflammatory cells (star), muscular layer (M) (H&E, low × 200 and high ×400 Magnifications). BS: Infected mice post-treated with B. subtilis PQ198038, BS group exhibiting degeneration of the villi (V) with infiltration of inflammatory cells (star), and sub-mucosal edema (arrowhead) in-between crypts of Lieberkühn (CL), muscular layer (M) (H&E, low × 200 and high ×400 Magnifications). BSP: Infected mice pre-treated with B. subtilis PQ198038, before infection and continued after infection, BSP group displaying a sloughing (S) of the upper tips of some villi (V) with infiltration of inflammatory cells (star), degenerated crypts of Lieberkühn (blue arrow), hyperplasia of goblet cells (green arrow), and dilatation of the blood capillary (red arrow), muscular layer (M) (H&E, low × 200 and high ×400 Magnifications). L: Infected mice post-treated with L. brevis PQ214320, L group showing an improvement of villi (V) with a slight sloughing (S), crypts of Lieberkühn (CL), muscular layer (M) (H&E, low × 200 and high ×400 Magnifications). LP: Infected mice pre-treated with L. brevis PQ214320, before infection and continued after infection, LP group exhibiting a noticeable improvement with slight signs of edema (arrowhead), crypts of Lieberkühn (CL), muscular layer (M) (H&E, low × 200 and high ×400 Magnifications).

Fig 6. Representative histopathological photomicrographs of diaphragm muscle in the different experimental mice groups (n = 5).

C: Positive Trichinella untreated control mice showing multiple T. spiralis larvae (black arrow) associated with a severe inflammatory cell infiltrate (green arrow), and congestion of blood vessels (BV) (H&E, low × 200 and high ×400 Magnifications). ALB: Infected mice treated with Albendazole, ALB group displaying damaged T. spiralis larvae (black arrow) associated with moderate infiltration of inflammatory cells (green arrow) (H&E, low × 200 and high ×400 Magnifications). BS: Infected mice post-treated with B. subtilis PQ198038, BS group exhibiting some T. spiralis larvae (black arrow) associated with a mild inflammatory cell aggregation (green arrow) (H&E, low × 200 and high ×400 Magnifications). BSP: Infected mice pre-treated with B. subtilis PQ198038, before infection and continued after infection, BSP group displaying a small number of T. spiralis larvae (black arrow) associated with a mild inflammatory cellular infiltration (green arrow), and congested blood vessels (BV) (H&E, low × 200 and high ×400 Magnifications). L: Infected mice post-treated with L. brevis PQ214320, 19 days after infection, L group showing a small number of T. spiralis larvae (black arrow) associated with a mild aggregation of inflammatory cells (green arrow) (H&E, low × 200 and high ×400 Magnifications). LP: Infected mice pre-treated with L. brevis PQ214320, before infection and continued to 19 days after infection, LP group exhibiting a noticeable improvement without any larvae (Star), with minimal aggregation of inflammatory cells (arrow) (H&E, low × 200 and high ×400 Magnifications).

Fig 7. Representative photomicrographs of intestinal and diaphragm muscular sections stained by Picrosirius red distinguishing the organization of collagen fibers (red color, arrows), cytoplasm and muscle fibers appeared as a yellow color in the different experimental mice groups (n = 5).

C: Positive Trichinella untreated control mice, ALB: Infected mice treated with Albendazole, BS: Infected mice post-treated with B. subtilis PQ198038, BSP: Infected mice pre-treated with B. subtilis PQ198038 before infection and after infection, L: Infected mice post-treated with L. brevis PQ214320, LP: Infected mice pre-treated with L. brevis PQ214320 before infection and continued after infection, (SR × 200).

Fig 8. All the intestinal (a,b) and muscular (c,d) Picrosirius red and Claudin 1 expression values are expressed as the mean ± SD (n = 5, 10 random fields per each one).

C: Positive Trichinella untreated control mice, ALB: Infected mice treated with albendazole, BS: Infected mice post-treated with B. subtilis PQ198038, BSP: Infected mice pre-treated with B. subtilis PQ198038 before of infection and continued after infection, L: Infected mice post-treated with L. brevis PQ214320, LP: Infected mice pre-treated with L. brevis PQ214320 before of infection and continued after infection. The significant (P < 0.01) differences are (a) compared to the C group, (b) against the ALB group, (c) compared with the BS group, (d) compared with the BSP group, and (e) compared with L group. The significant (P < 0.05) difference is (^f) compared to the C group. The remaining groups representing insignificant difference relative to each other’s.

Fig 9. Representative photomicrographs of the immunohistochemical membranous Claudin-1 reactions (brown color, arrows) lining the total borders in the intestinal and muscular tissues of the different experimental mice groups (n = 5).

C: Positive Trichinella untreated control mice, ALB: Infected mice treated with Albendazole, BS: Infected mice post-treated with B. subtilis PQ198038, BSP: Infected mice pre-treated with B. subtilis PQ198038 before infection and continued after infection, L: Infected mice post-treated with L. brevis PQ214320, LP: Infected mice pre-treated with L. brevis PQ214320 before 7 days of infection and continued to 5 days after infection, (IHC, × 200).