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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 cytokine microenvironment is crucial in generating and polarizing the immune response. A means of monitoring this environment would be of great value for better understanding Toxocara canis immune modulation. The aim of this study was to analyze the dynamics of cytokine transcription ex vivo, during early (24-48 hours) and late (15-30 days) times post-infection, in the mesenteric lymph nodes, spleen and intestinal mucosa of Balb/c mice experimentally infected with T. canis larvae. Mice in the treated group were infected with 100 third-stage larvae (L3), whereas mice in the control group were not infected. Analyses were performed at different times: 24-48 hours post-infection (HPI), 15-30 days post-infection (DPI). IL4, IL10, IL12 and Ym1 mRNA transcriptions were analyzed through qPCR. This study showed cytokine transcription mediated by migrating larvae in the mesenteric lymph nodes and spleen at 24-48 HPI, whereas cytokine transcription in the intestinal mucosa was observed only at late times (15-30 DPI). These results suggest that the T. canis larvae migration during infection might play a role in cytokine dynamics. Since the cytokine microenvironment is crucial in modulating immune response, knowledge of cytokine dynamics during T. canis infections pave the way to better understand its interaction with the host.

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The aim of the present study was to carry out a serological survey to identify the seroprevalence of Lawsonia intracellularis in six Thoroughbred farms in the Southern region of the state of Rio Grande do Sul, Brazil. During 2019 and 2020, blood samples from 686 Thoroughbred horses were obtained from six different breeding farms. Horses were divided into groups according to age: (1) broodmares (>5 years), (2) two-year-old foals, (3) yearlings, and (4) 0-6 months-old foals. Blood samples were collected by venipuncture of the external jugular vein. The detection of antibodies (IgG) against L. intracellularis was performed by Immunoperoxidase Monolayer Assay. The detection of specific antibodies (IgG) against L. intracellularis in the evaluated population was 51%. The highest detection (86.8%) of IgG was in the broodmares category, while the lowest (5.2%) was in foals of 0-6 months of age. Regarding the farms, the Farm 1 had the highest (67.4%) prevalence of seropositivity against L. intracellularis, while Farm 4 had the lowest (30.6%). There was no record of clinical manifestation of Equine Proliferative Enteropathy in the sampled animals. The results of this study show the high seroprevalence of L. intracellularis in Thoroughbred farms in the Southern of Rio Grande do Sul, suggesting a large and continuous exposure to the agent.

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Bacillus thuringiensis (Bt) Berliner has potential for use in insect management. Its use can be an alternative for the management of Bradysia aff. ocellaris (Comstock), considered one of the main strawberry pests in a soilless system. Therefore, the objective of this work was to evaluate the toxicity of different bacteria on B. aff. ocellaris in laboratory and greenhouse bioassays. The following isolates were used in the experiments: Bacillus circulans (Bc), B. thuringiensis var. oswaldo cruzi (Bto) or B. thuringiensis var. israelensis (Bti) and B. thuringiensis var. kurstaki (Btk) In the laboratory, B. aff. ocellaris larvae showed high susceptibility to Bti isolate (92 % mortality) 14 days after treatment exposure (DAET). In contrast, the isolates Bc, Bto, and Btk showed less than 32 % mortality, not differing from the control treatment (water ­ 22 % mortality). According to the concentration-response curves the values of lethal concentration LC50 and LC90 were 4 x 106 CFU.mL-1 and 4 x 1015 CFU.mL-1. By applying Bti (4 × 1012 CFU.mL-1) at the base of strawberry plants growing in plastic pots containing commercial plant substrate, a reduction of 26 % in the emergence of B. aff. ocellaris was observed. According to these results, the Bti isolate is considered promising for the formulation of bioinsecticides based on Bt for the management of B. aff. ocellaris in strawberry culture.

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Solid-state fermentation (SSF) is a type of fermentation process with potential to use agro-industrial by-products as a carbon source. Nonetheless, there are few studies evaluating SSF compared to submerged fermentation (SmF) to produce polyhydroxyalkanoates (PHAs). Different methodologies are available associating the two processes. In general, the studies employ a 1st step by SSF to hydrolyze the agro-industrial by-products used as a carbon source, and a 2nd step to produce PHA that can be carried out by SmF or SSF. This paper reviewed and compared the different methodologies described in the literature to assess their potential for use in PHA production. The studies evaluated showed that highest PHA yields (86.2% and 82.3%) were achieved by associating SSF and SmF by Cupriavidus necator. Meanwhile, in methodologies using only SSF, Bacillus produced the highest yields (62% and 56.8%). Since PHA (%) does not necessarily represent a higher production by biomass, the productivity parameter was also compared between studies. We observed that the highest productivity results did not necessarily represent the highest PHA (%). C. necator presented the highest PHA yields associating SSF and SmF, however, is not the most suitable microorganism for PHA production by SSF. Concomitant use of C. necator and Bacillus is suggested for future studies in SSF. Also, it discusses the lack of studies on the association of the two fermentation methodologies, and on the scaling of SSF process for PHA production. In addition to demonstrating the need for standardization of results, for comparison between different methodologies.

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Haemonchus contortus is the most pathogenic parasite for sheep. The objective was to evaluate immunomodulation of the probiotic Saccharomyces boulardii in sheep experimentally infected with H. contortus. Twenty-four sheep were divided into three groups: one infected with 500 H. contortus larvae/day for 26 days and supplemented with S. boulardii (40 ml with 1 × 108 CFU/ml/day); a control group only infected with H. contortus but not supplemented; and a naïve group that never came into contact with either parasites or S. boulardii. To assess the humoral immune response, production of specific serum IgG anti-somatic H. contortus antigen was evaluated through indirect ELISA. To assess the cellular immune response, cell populations and cytokine (IL-4, IL-5 and IL-10) production were evaluated through flow cytometry. For parasitological analyses, the counts of eggs per gram of faeces (EPG) and larvae per faecal culture were assessed. At all the study points, the concentration of IgG anti-H. contortus was higher (p < .05) in the S. boulardii group than in the other groups. The cell analysis revealed that there were significantly higher numbers (p < .05) of cells expressing MHC-II and significantly higher numbers (p < .05) of eosinophils in the mucosa in the S. boulardii group. Significant expression of IL-10 was observed only in the control infected group. There were significant reductions (p < .05) in EPG and larval counts in the S. boulardii supplemented group. These results show that S. boulardii supplementation modulated the immune response against H. contortus, thereby reducing its infection.

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Haemonchus contortus is one of the most important gastrointestinal nematodes infecting sheep, being a production-limiting factor in sheep herds. Biological control has been used for many years either in combination with traditional parasiticides or as an alternative treatment itself. Bacillus thuringiensis can be a promising tool for an integrate control of H. contortus in sheep herds by disrupting nematode's life cycle, thus decontaminating pasture. This study aims to evaluate the larvicidal efficacy of Bacillus thuringiensis var. oswaldocruzi (Bto) on the development of H. contortus larvae in fecal sheep cultures. Bto concentrations (~1010 colony forming units, CFU / mL) were orally administered to thirty-six ewes naturally infected with H. contortus in three different pharmaceutical forms: hard gelatin capsule, mucoadhesive gel or suspension. The different treatments were carried out in a single oral administration (SOA). All formulations were effective in inhibiting larvae development compared with control groups in all time points studied (p < 0.05). The mucoadhesive gel showed ~90% of efficacy on inhibiting larvae development during all the time points post SOA. The suspension had 85% efficacy on inhibiting larvae development at 24-48 h and 96% at 72-96 h. The hard gelatin capsule was 57%, 62%, 73% and 96% effective inhibiting larvae development at 24, 48, 72 and 96 h, respectively. This study highlights that disrupting the infective larval stage in the environment Bacillus thuringiensis var. oswaldocruzi is a promising prophylactic alternative in the H. contortus control.

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The objective of this work was to evaluate the early and late immunological modulation of an experimental infection of T. canis larvae in mice. Mice were infected with 100 infective larvae and euthanized at different period: 24, 48 hours post infection (HPI), 15- and 30 days post infection (DPI). The humoral response was evaluated by indirect ELISA. Quantitative RT-PCR (qPCR) was used to quantify the mRNA transcription of cytokines IL4, IL10, IL12 and Ym1 in the early and late infection periods. Infection with T. canis was able to generate specific total IgG at 15- and 30- DPI. Analyzing the IgG isotype revealed a significant differentiation for IgG1 compared with IgG2a, IgG2b and IgG3, characterizing a Th-2 response. Evaluating the gene transcription at the early phase of infection, higher transcription levels of IL10, IL4 and Ym1 and a downregulation of IL12 were observed. By the late phase, increased transcription levels of IL4, Ym1 and IL12 were observed, and downregulation of IL-10 transcription was observed. The data obtained suggest that during experimental infection with T. canis, the participation of the IL4, IL10, IL12 cytokines and Ym1 can play an important role in T. canis immunomodulation.(AU)

O objetivo deste trabalho foi avaliar a modulação imunológica precoce e tardia da infecção experimental de larvas de T. canis em camundongos. Estes foram infectados com 100 larvas infectantes e eutanasiados em diferentes períodos: 24 e 48 horas pós infecção (HPI), 15 e 30 dias após a infecção (DPI). A resposta humoral foi avaliada por ELISA indireto. RT-PCR quantitativo (qPCR) foi usado para quantificar a transcrição de mRNA das citocinas IL4, IL10, IL12 e Ym1 nos períodos de infecção precoce e tardia. A infecção por T. canis foi capaz de gerar IgG total específico aos 15 e 30 DPI. A análise do isótipo IgG revelou uma diferenciação significativa para IgG1 em comparação com IgG2a, IgG2b e IgG3, caracterizando uma resposta Th-2. Avaliando-se a transcrição gênica na fase inicial da infecção, foram observados níveis mais elevados de transcrição de IL10, IL4 e Ym1 e a regulação negativa de IL12. Na fase tardia, foram observados níveis aumentados de transcrição de IL4, Ym1 e IL12, e foi observada regulação negativa da transcrição de IL-10. Os dados obtidos sugerem, que durante a infecção experimental com T. canis, a participação das citocinas IL4, IL10, IL12 e Ym1 podem desempenhar um papel importante na imunomodulação de T. canis.(AU)

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The aims of this study were: 1) determine total specific IgG and subisotypes in serum and colostrum of pregnant mares vaccinated against Theileria equi and Streptococcus equi subspecies equi; 2) determine total specific IgG and subisotypes in foals born from these mares. In mares, the highest total serum IgG value for T. equi was at 335 days of gestation declining 30 days postpartum, while for S. equi was at 328 days of gestation remaining high up to 30 days postpartum. Transfer of passive immunity against both antigens was observed with specific IgG values in colostrum and foals' serum proportional to mares' values. The most detected specific IgG subisotypes were IgG3/5 and IgG4/7 for both antigens. Foals born from mares immunized with T. equi kept maternal IgG values until 2 months of age, while those born from mares immunized with S. equi kept maternal IgG values until 3-4 months of age. These results suggest that foals should be vaccinated after this period.

Os objetivos deste estudo foram: 1) determinar IgG total específica e seus subisotipos no soro e colostro de éguas vacinadas contra Theileria equi e Streptococcus equi subspécie equi; 2) determinar IgG total específica e seus subisotipos em potros provenientes dessas éguas. Em éguas vacinadas contra T. equi, o maior nível sérico de IgG total específico ocorreu aos 335 dias de gestação, decaindo 30 dias pós parto, enquanto nas vacinadas contra S. equi, ocorreu aos 328 dias de gestação e manteve-se pelo mesmo período. A avaliação de IgG total específica no colostro de éguas vacinadas demonstrou níveis de IgG proporcionais aos títulos do soro materno. Os isotipos de IgG mais detectados foram IgG3/5 e IgG4/7 para ambos antígenos avaliados. Potros provenientes de éguas vacinadas com antígeno de T. equi mantiveram os níveis de IgG específica até dois meses de idade, enquanto potros provenientes de éguas vacinadas com antígeno de S. equi mantiveram por três a quatro meses, sugerindo que esse seja o período ideal para início do esquema vacinal em potros.

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Equine theileriosis, caused by the Theileria equi protozoan, is a disease of worldwide importance. T. equi expresses surface proteins, of which the EMA-2 protein is a promising antigen for vaccine use. The aim of this study was to evaluate the immune response of adult horses, pregnant mares, and foals to an experimental EMA-2 protein of recombinant T. equi vaccine. A total of 46 horses were used in this study for vaccine trials and challenges. Twelve geldings, 14 pregnant mares, and 14 foals were divided into vaccinated and control groups. Total serum specific anti-rEMA-2 IgG, IgG subclasses, and transcription of cytokines related to the immune response were evaluated. For the vaccine challenge, six six-month-old foals were divided into vaccinated and control groups. For the challenge, blood from a horse with theileriosis was transfused to the foals. Geldings and pregnant mares maintained anti-rEMA-2 IgG levels at 130 and 140 days after vaccination, respectively. The most-detected IgG subclasses in vaccinated were IgG3/5, IgG4/7, and IgG1. IL2, IL10, IL12, IL17, IFN-γ, and TNF-α were the most-transcribed cytokines in PBMCs of vaccinated horses stimulated with rEMA-2. Challenge with T. equi demonstrated that vaccinated foals had an increase of 33% in total IgG four days after blood transfusion, while control foals had no significant response, suggesting that vaccine antibodies may have recognized EMA-2 protein of the native T. equi antigen. T. equi recombinant EMA-2 was shown to be a promising vaccine antigen by inducing humoral and cellular immunity similar to that observed in natural parasite infections.

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