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Probiotic microorganisms can stimulate an immune response and increase the efficiency of vaccines. For example, Bacillus toyonensis is a nonpathogenic, Gram-positive bacterium that has been used as a probiotic in animal supplementation. It induces immunomodulatory effects and increases the vaccine response in several species. This study aimed to evaluate the effect of B. toyonensis supplementation on the modulation of the immune response in horses vaccinated with recombinant Clostridium tetani toxin. Twenty horses were vaccinated twice, with an interval of 21 days between doses, and equally divided into two groups: the first group was supplemented orally for 42 days with feed containing viable spores of B. toyonensis (1 × 108) mixed with molasses (40 ml), starting 7 days before the first vaccination; the second (control) group received only feed mixed with molasses, starting 7 days before the first vaccination. Serum samples were collected to evaluate the humoral immune response using an in-house indirect enzyme-linked immunosorbent assay (ELISA), and peripheral blood mononuclear cells (PBMCs) were collected to evaluate cytokine transcription (qPCR). For the specific IgG-anti-rTENT titer, the supplemented group had ELISA values that were four times higher than those of the control group (p < 0.05). The supplemented group also showed higher ELISA values for the IgGa and IgGT sub-isotypes compared to the control group. In PBMCs stimulated with B. toyonensis, relative cytokine transcription of the supplemented group showed 15-, 8-, 7-, and 6-fold increases for IL1, TNFα, IL10 and IL4, respectively. When stimulated with a vaccine antigen, the supplemented group showed 1.6-, 1.8-, and 0.5-fold increases in IL1, TNFα, and IL4, respectively, compared to the control group. Horses supplemented with B. toyonensis had a significantly improved vaccine immune response compared to those in the control group, which suggests a promising approach for improving vaccine efficacy with probiotics.

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The bacterium Clostridium chauvoei is the causative agent of blackleg in livestock, and vaccination is the most effective means of prevention. The aim of this study was to assess the effect of short-term supplementation with Bacillus toyonensis and Saccharomyces boulardii on the immune response to a C. chauvoei vaccine in sheep. Sheep were vaccinated subcutaneously on day 0 and received a booster dose on day 21, with 2 mL of a commercial vaccine formulated with inactivated C. chauvoei bacterin adsorbed on aluminum hydroxide. Probiotics were orally administered B. toyonensis (3 × 108 cfu) and S. boulardii (3 × 108 cfu) over five days prior to the first and second doses of the vaccine. Sheep supplemented with B. toyonensis and S. boulardii showed significantly higher specific IgG, IgG1, and IgG2 titers (P<0.05), with approximately 24- and 14-fold increases in total IgG levels, respectively, than the nonsupplemented group. Peripheral blood mononuclear cells from the supplemented group had increased mRNA transcription levels of the IFN-γ, IL2, and Bcl6 genes. These results demonstrate an adjuvant effect of short-term supplementation with B. toyonensis and S. boulardii on the immune response against the C. chauvoei vaccine in sheep.

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Bacterial spores of the genus Bacillus are being evaluated as adjuvant molecules capable of improving the immune response to vaccines. In this study, we investigate whether subcutaneously administered spores of B. toyonensis BCT-7112T could enhance a vaccine immune response in mice. Three groups of mice were subcutaneously vaccinated on day 0 and received a booster on day 21 of the experiment, with the following vaccine formulations: 40 µg of recombinant glycoprotein D (rgD) from bovine herpesvirus type 5 (BoHV-5) adsorbed in 10% aluminum hydroxide (alum) without B. toyonensis spores (group 1) and B. toyonensis (1 × 106 viable spores) + 40 µg of rgD adsorbed in 10% alum (group 2); and B. toyonensis (1 × 106 viable spores) without rgD (group 3). Group 2 showed significantly higher titers (P < 0.05) of total specific serum IgG, IgG2a, and neutralizing antibodies, when compared with the groups 1 and 3. A significantly higher (P < 0.05) transcription level of cytokines IL-4, IL-12, and IFN-γ was observed in splenocytes from mice that received the B. toyonensis spores in the vaccine formulation. In addition, stimulation of the macrophage-like cell line RAW264.7 with spores of B. toyonensis markedly enhanced the cell proliferation and mRNA transcription levels of IL-4, and IL-12 cytokines in these cells. Our findings indicated that the subcutaneous administration of B. toyonensis BCT-7112T spores enhanced the humoral and cellular immune response against BoHV-5 in mice.

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There have been significant efforts toward the development of more efficient vaccines for animal health. A strategy that may be used to improve vaccine efficacy is the use of probiotics. Bovine herpesvirus 5 (BoHV-5) is an example of an important animal pathogen for which vaccines have provided only limited protection. In this study, we examined the use of the probiotics Bacillus toyonensis and Saccharomyces boulardii as a potential immune modulator to improve vaccine efficiency. Thirty, 5-month-old lambs were randomly grouped in three lots of 10 each and vaccinated at days 0, 21 and 42 of the experiment. They grazed on the same pasture and were fed ad libitum twice a day with commercial sheep feed supplemented with either B. toyonensis (1×106CFU/g of feed) or S. boulardii (1×107CFU/g of feed), or non-supplemented feed. The probiotic supplementation was suspended day 28; thereafter, the next 35days, they were fed with the same commercial feed as control group. Animals supplemented with probiotics showed a significant (p>0.001) increased seroconversions against BoHV-5, and higher neutralizing antibodies titres (p>0.05) to BoHV-5 than non-supplemented animals. At 63days of experiment, splenocytes from the supplemented sheep had higher mRNA transcription levels of cytokines IL-10 and IL-17A. These results suggest that these probiotics could provide a promising means of improving vaccine efficacy.

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Probiotics modulate the immune response and can increase the effectiveness of vaccines. Bacillus toyonensis is widely used as a probiotic in animal feed. The aim of this study was to assess the effects of B. toyonensis administration on the immune response to an experimental recombinant vaccine against bovine herpesvirus type 5 (BoHV-5) in mice. Mice were vaccinated with BoHV-5 recombinant glycoprotein D and supplemented with the probiotic B. toyonensis in two regimes: one group received the probiotic only during seven days prior to the initial vaccination while the second group was given the probiotic throughout the experimental period of seven weeks. Animals supplemented with probiotic B. toyonensis in two regimes showed an increase in total immunoglobulin (Ig)G, IgG1 and IgG2a levels in serum, in addition to higher titres of antibodies capable of neutralising the BoHV-5 virus than non-supplemented animals (P<0.05). Splenocytes from the supplemented mice had higher mRNA transcription levels of cytokines interleukin (IL)-4 and IL-12. These results show that the use of this probiotic may significantly contribute to the response elicited by recombinant vaccines, especially those that rely on increasing antibody and cell-mediated immune responses for efficacy. Further, the data support an immunomodulatory effect for probiotic B. toyonensis and imply that enhance effect on the immune response against a BoHV-5 recombinant vaccine in mice.

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This experiment was conducted to investigate the effects of Bacillus coagulans on the growth performance and immune functions of the intestinal mucosa of yellow broilers. Three hundred and sixty one-day-old yellow chicks were randomly allocated to four treatments groups with six replicates of 15 chicks each. The broilers were randomly subjected to one of the following treatments for 28 days: control group (group1, fed a basal diet) and three treatments (group 2, 3, 4) fed the basal diet supplemented with 100, 200, or 300 mg/kg Bacillus coagulans , respectively). The results showed that for 28 days, compared with the control diet, the dietary addition of 200 mg/kg Bacillus coagulans significantly decreased the feed/gain ratio (F/G) (p 0.05), improved the thymus index, spleen index and bursa index (p 0.05), increased the villus height to crypt depth ratio (V/C) in the duodenum (p 0.05), increased the number of secretory immunoglobulin (sIgA) positive cells ( p 0.05). The dietary addition of 200 mg/kg Bacillus coagulans promoted a significant increase in Lactobacillus spp. populations and suppressed Escherichia coli replication in cecum, compared with the control (p 0.05). Moreover, the dietary addition of 200 mg/kg Bacillus coagulans also significantly enhanced the levels of interferon alpha (IFN), toll-like receptor (TLR3), and melanoma differentiation-associated protein 5(MDA5) in the duodenum (p 0.05). In conclusion, the dietary addition of Bacillus coagulans significantly improved broiler performance, and enhanced the intestinal mucosal barrier and immune function. The optimal dosage of Bacillus coagulans for yellow broilers was determined as 2×108 cfu/kg.(AU)

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This experiment was conducted to investigate the effects of Bacillus coagulans on the growth performance and immune functions of the intestinal mucosa of yellow broilers. Three hundred and sixty one-day-old yellow chicks were randomly allocated to four treatments groups with six replicates of 15 chicks each. The broilers were randomly subjected to one of the following treatments for 28 days: control group (group1, fed a basal diet) and three treatments (group 2, 3, 4) fed the basal diet supplemented with 100, 200, or 300 mg/kg Bacillus coagulans , respectively). The results showed that for 28 days, compared with the control diet, the dietary addition of 200 mg/kg Bacillus coagulans significantly decreased the feed/gain ratio (F/G) (p 0.05), improved the thymus index, spleen index and bursa index (p 0.05), increased the villus height to crypt depth ratio (V/C) in the duodenum (p 0.05), increased the number of secretory immunoglobulin (sIgA) positive cells ( p 0.05). The dietary addition of 200 mg/kg Bacillus coagulans promoted a significant increase in Lactobacillus spp. populations and suppressed Escherichia coli replication in cecum, compared with the control (p 0.05). Moreover, the dietary addition of 200 mg/kg Bacillus coagulans also significantly enhanced the levels of interferon alpha (IFN), toll-like receptor (TLR3), and melanoma differentiation-associated protein 5(MDA5) in the duodenum (p 0.05). In conclusion, the dietary addition of Bacillus coagulans significantly improved broiler performance, and enhanced the intestinal mucosal barrier and immune function. The optimal dosage of Bacillus coagulans for yellow broilers was determined as 2×108 cfu/kg.

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The widespread overuse of antibiotics in aquaculture has led to the emergence of antibiotic-resistance shrimp pathogens, the negative impact on shrimp gut microbiota, and the presence of antimicrobial residues in aquaculture products, with negative consequences on human health. Alternatively, probiotics have positive effects on immunological responses and productive performance of aquatic animals. In this study, three probiotic bacteria, (Bacillus licheniformis MAt32, B. subtilis MAt43 and B. subtilis subsp. subtilis GAtB1), isolated from the Anadara tuberculosa were included in diets for juvenile shrimp, Litopenaeus vannamei, to evaluate their effects on growth, survival, disease prevalence, and immune-related gene expression. Shrimp naturally infected with WSSV and IHHNV were fed with the basal diet (control, T1) and diets supplemented with four levels of bacilli probiotic mix (1:1:1) at final concentration of (T2) 1 × 106, (T3) 2 × 106, (T4) 4 × 106, and (T5) 6 × 106 CFU g-1 of feed. The specific growth rate of shrimp was significantly higher in T2 than in T1 (control) treatment, and the final growth as well as the survival were similar among treated groups. The prevalence of WSSV and IHHNV infected shrimp was reduced in T2 and T4 treatments, respectively, compared with control. The mRNA expression of proPO gene was higher in treatment T4 than control. The LvToll1 gene was significantly up-regulated in treatments T4 and T5 compared to control. The SOD gene was up-regulated in treatment T5 compared to control. In contrast, the mRNA expression of the Hsp70 gene was down-regulated in treatments T4 and T5 respect to control, and the TGase gene remained unaffected by the level of bacillus probiotic mix. As conclusion, the bacilli probiotic mix (Bacillus spp.) enhanced immune-related gene expression in WSSV and IHHNV naturally infected shrimp. This is the first report of probiotic potential of bacteria isolated from A. tuberculosa on the immune response and viral prevalence in shrimp Litopenaeus vannamei.

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Anopheles aquasalis Curry es el principal vector de malaria de la región nororiental de Venezuela. Un estudio sobre el control biológico de la especie fue realizado en Rio Chiquito Abajo, municipio Mariño, estado Sucre. El objetivo del mismo fue evaluar en condiciones de campo, la eficacia y persistencia de tres formulaciones comerciales de Bacillus sphaericus contra larvas de An. aquasalis. Los criaderos típicos de esta especie son humedales de agua dulce con vegetación herbácea emergente y manglares salobres estacionales con vegetación arbórea (Avicennia germinans). El hábitat seleccionado para realizar los bioensayos fue un típico manglar de Avicennia germinans. En este criadero de An. aquasalis, se evaluaron estas formulaciones de B. sphaericus: dos formulaciones liquidas (GRISELESF-Cepa 2362 y VECTOLEX® WGD-51,2%) y una formulación granulada (VECTOLEX® CG-7,5%). En cada prueba, se aplicó un diseño completamente al azar con 4 repeticiones por tratamiento y por control. Los resultados fueron expresados como porcentajes de reducción larval, después de aplicar la formula de Mulla. De acuerdo con los resultados obtenidos, la formulación granulada VECTOLEX® CG-7,5% con dosis de 30 Kg/ha (3 g/m²), resultó ser altamente eficaz hasta 7 días post-aplicación con 92,3% de reducción, la misma fue menos efectiva después de 14 días post-aplicación con 74,8%. Al aumentar la dosis a 40 Kg/ha (4 g/m²), la eficacia después de 7 días de la aplicación fue de 93,9 %, es decir muy similar que a la dosis anterior. Ambas dosis presentaron la misma eficacia contra larvas de An. aquasalis. Por otra parte, la otra formulación VECTOLEX® WGD-51,2%, con la dosis de 2,0 Kg/ ha, resultó ser eficaz hasta 8 días post-aplicación con 85,2% de reducción larval; la misma produjo 13,3% de reducción a los 16 días post-tratamiento; sin embargo, esta formulación con la dosis de 2,5 Kg/ha, fue altamente eficaz hasta 16 días postaplicación con más de 90% de reducción larval. En cambio, en el mismo manglar el producto GRISELESF fue menos eficaz tanto a 2,0 Kg/ha como a 2,5 Kg/ha, obteniéndose porcentajes de reducción muy bajos a los 8 y 16 días post-aplicación.

Anopheles aquasalis Curry is the main vector of malaria in northeastern Venezuela. A study of biological control of An. aquasalis larvae was carried out in Río Chiquito Abajo, Sucre State, Venezuela. The objective of this study was to evaluate effectivity and persistence of three formulations of Bacillus sphaericus against larval populations of An. aquasalis. The main breeding-sites of An. aquasalis are seasonal brackish mangrove with Avicennia germinans and semi-permanent freshwater wetlands with herbaceous emergent vegetation. A typical seasonal brackish mangrove with Avicennia germinans was selected. In these breeding-sites of An. aquasalis, three commercial formulations of B. sphaericus: VECTOLEX® CG-7.5%, VECTOLEX® WGD-51.2% and GRISELESF were evaluated in field trials. According to the results obtained VECTOLEX® CG-7.5% was effective against An. aquasalis larvae during 7 days post-treatment at rates not lower than 30 Kg/ha (3 g/m²), producing a larval reduction greater than 92.3%. This granular formulation provided very good larval control during 7 days. The two dosages (30 and 40 Kg/ha) had the same efficacy against An. aquasalis larvae. The second commercial formulation VECTOLEX® WGD-51.2% was effective against An. aquasalis larvae at dose of 2.0 Kg/ ha, producing a larval reduction greater than 85 % during 8 days post-treatment, and only 13.3 % larval reduction at 16 days after treatment; but the same formulation with at dose of 2.5 Kg/ha was more effective, producing a larval reduction greater than 90 % during 16 days posttreatment. In the same brackish mangrove, GRISELESF at rates not lower than 2.0 and 2.5 Kg/ha, appears to be less effective against An. aquasalis larvae, producing very low larval reduction at 8 and 16 days post-treatment.

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Rabies is a viral encephalitis, nearly always fatal, but preventable through vaccines. Rabid animal bite is the prime transmission act, while veterinary vaccination is one of the best strategies for rabies general prevention. Aluminum compounds and saponin are the commercial adjuvants used for this vaccine nowadays. Nevertheless, aluminum compounds can provoke undesired side effects and saponin has a narrow activity range without toxicity. B. atrophaeus inactivated spores (BAIS), with or without saponin, were then used as an alternative to boost the inactivated rabies virus response. BAIS was as effective as saponin in augmenting antibody titers, but combination of both adjuvants doubled the titers raised by them individually. The combined adjuvant formulation maintained viability for 21 months when stored at 4-8°C. Overall, BAIS was demonstrated as a viable alternative to commercial adjuvants, while its combination with saponin resulted in even higher vaccine potency with good stability.

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