RESUMO
INTRODUCTION: Producing commercial bacterins/toxoids against Clostridium spp. is laborious and hazardous. Conversely, developing prototype vaccines using purified recombinant toxoids, though safe and effective, is both laborious and costly for application in production animals. OBJECTIVE: Considering that inactivated recombinant Escherichiacoli (bacterin) is a simple, cost-effective, and to be safe solution, we evaluated, for the first time, a pentavalent formulation of recombinant bacterins containing the alpha, beta, and epsilon toxins of Clostridiumperfringens and C and D neurotoxins of Clostridiumbotulinum in sheep. METHODS: Subcutaneously, 18 Texel sheep received two doses (200 µg of each antigen) of recombinant bacterin (n = 7) or purified recombinant antigens (n = 6) on days 0 and 28, while the control group (n = 5) did not receive an immunization. Sera samples from days 0 (before the 1st dose), 28 (before the 2nd dose), and 56, 84, and 112 were used for measuring IgG (indirect ELISA) and neutralizing antibodies (mouse serum neutralization). RESULTS: Both formulations induced significant levels of IgG against all five toxins (p < 0.05) up to day 112, with peaks at days 28 and 56 post-immunization. The expected booster effect occurred only for the botulinum toxins. The neutralizing antibody titers were satisfactory against ETX (≥2 IU/ml for both formulations) and BoNT-D [5 IU/ml (bacterin) and 10 IU/ml (purified)]. CONCLUSION: While adjustments are required, the recombinant bacterin platform holds great potential for polyvalent vaccines due to its straightforward, safe, and cost-effective production, establishing it as a user-friendly technology for the veterinary immunobiological industry.
Assuntos
Anticorpos Antibacterianos , Anticorpos Neutralizantes , Vacinas Bacterianas , Botulismo , Enterotoxemia , Animais , Botulismo/prevenção & controle , Botulismo/veterinária , Botulismo/imunologia , Ovinos , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Anticorpos Antibacterianos/sangue , Enterotoxemia/prevenção & controle , Enterotoxemia/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Doenças dos Ovinos/prevenção & controle , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Imunoglobulina G/sangue , Escherichia coli/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , FemininoRESUMO
Lateral flow immunoassays (LFIA) for antibody detection represent cost-effective and user-friendly tools for serology assessment. This study evaluated a new LFIA prototype developed with a recombinant chimeric antigen from the spike/S and nucleocapsid/N proteins to detect anti-SARS-CoV-2 IgG antibodies. The evaluation of LFIA sensitivity and specificity used 811 serum samples from 349 hospitalized, SARS-CoV-2 RT-qPCR positive COVID-19 patients, collected at different time points and 193 serum samples from healthy controls. The agreement between ELISA results with the S/N chimeric antigen and LFIA results was calculated. The LFIA prototype for SARS-CoV-2 using the chimeric S/N protein demonstrated 85 % sensitivity on the first week post symptoms onset, reaching 94 % in samples collected at the fourth week of disease. The agreement between LFIA and ELISA with the same antigen was 92.7 %, 0.827 kappa Cohen value (95 % CI [0.765-0.889]). Further improvements are needed to standardize the prototype for whole blood use. The inclusion of the novel chimeric S + N antigen in the COVID-19 IgG antibody LFIA demonstrated optimal agreement with results from a comparable ELISA, highlighting the prototype's potential for accurate large-scale serologic assessments in the field in a rapid and user-friendly format.
RESUMO
Pediococcus pentosaceus is a lactic acid bacterium that has probiotic potential proven by studies. However, its viability can be affected by adverse conditions such as storage, heat stress, and even gastrointestinal passage. Thus, the aim of the present study was to microencapsulate and characterize microcapsules obtained by spray drying and produced only with whey powder (W) or whey powder combined with pectin (WP) or xanthan (WX) in the protection of P. pentosaceus P107. In the storage test at temperatures of - 20 °C and 4 °C, the most viable microcapsule was WP (whey powder and pectin), although WX (whey powder and xanthan) presented better stability at 25 °C. In addition, WX did not show stability to ensure probiotic potential (< 6 Log CFU mL-1) for 110 days and the microcapsule W (whey powder) maintained probiotic viability at the three temperatures (- 20 °C, 4 °C, and 25 °C) for 180 days. In the exposition to simulated gastrointestinal juice, the WX microcapsule showed the best results in all tested conditions, presenting high cellular viability. For the thermal resistance test, WP microcapsule was shown to be efficient in the protection of P. pentosaceus P107 cells. The Fourier transform infrared spectroscopy (FTIR) results showed that there was no chemical interaction between microcapsules of whey powder combined with xanthan or pectin. The three microcapsules produced were able to protect the cell viability of the microorganism, as well as the drying parameters were adequate for the microcapsules produced in this study.
Assuntos
Probióticos , Soro do Leite , Pectinas , Cápsulas/química , Pós , Proteínas do Soro do LeiteRESUMO
The present study was carried out to evaluate the intravaginal vaccine potential against bovine alphaherpesvirus type 5 (BoHV-5). Sixty three cows were divided into seven groups (n: 9) and inoculated intravaginally (VA) or intramuscularly (IM) with inactivated BoHV-5, associated with the recombinant B subunit of the heat-labile enterotoxin of E. coli (rLTB), 2-hydroxyethylcellulose (Drug Delivery System A - DDS-A) or Poloxamer 407 (Drug Delivery System B - DDS-B) as follows: G1 (DDS-A + BoHV-5 + rLTB), G2 (DDS-A + BoHV-5), G3 (DDS-B + BoHV-5 + rLTB), G4 (DDS-B + BoHV-5), G5 (BoHV-5 + rLTB), G6 (Negative control) e G7 (Positive control). The local and systemic humoral responses were measured by indirect ELISA (IgA and IgG) and serum neutralization tests, and the cellular response was measured by a quantitative direct ELISA (IL-2 and IFN-Gamma). The results showed the group inoculated by the IM route, G5, demonstrated the highest levels of IgG in the vaginal mucosa among the experimental groups (p < 0.05). In the groups tested with polymers (G1 and G3) in the vaginal mucosa, even higher levels of IgG were seen in comparison to the positive control (G7; p < 0.01). Higher levels of IgA were also noted in relation to the other groups (p < 0.05) on days 30, 60 and 90 post-inoculations. The groups G1 and G3 also provided higher titers of neutralizing antibodies (Log2) in relation to other treatments (p < 0.01) 90 days after inoculation. In the nasal mucosa, there was an increase in the levels of IgA and IgG with the use of vaccines from groups G1 and G3, in relation to the positive control, G7 (p < 0.05) at 60 and 90 days after the first inoculation. Moreover, neutralizing antibodies titers were detected at 60 and 90 days by serum neutralization. The inclusion of the evaluated polymers resulted in a superior response (p < 0.05) of immunoglobulins and IL-2 and IFN-Gamma in relation to the treatment using only rLTB (G5). This data demonstrates the capabilities of a vaccine with an intravaginal application in cattle to stimulate a local and systemic immune response.
Assuntos
Escherichia coli , Vacinas Virais , Animais , Feminino , Bovinos , Vacinas de Produtos Inativados , Interleucina-2 , Anticorpos Neutralizantes , Imunoglobulina G , Imunoglobulina A , Polímeros , Anticorpos AntiviraisRESUMO
Survival of Lactococcus lactis subsp. lactis R7, microencapsulated with whey and inulin, was analyzed when added to blueberry juice, milk, and cream. For 28 days, cell viability was evaluated for storage (4 °C), simulated gastrointestinal tract (GIT), and thermal resistance. All matrices demonstrated high cell concentration when submitted to GIT (11.74 and 12 log CFU mL-1), except for the blueberry juice. The thermal resistance analysis proved the need for microencapsulation, regardless of the food matrix. The results indicate that L. lactis R7 microcapsules have potential for application in different matrices and development of new probiotic products by thermal processing.
Assuntos
Lactococcus lactis , ProbióticosRESUMO
The emergence of recombinant DNA technology has led to the exploration of the use of the technology to develop novel vaccines. With a fundamental role in vaccines design, several immunoinformatics tools have been created to identify isolated epitopes that stimulate a specific immune response, contributing to effective vaccines development. In the past, vaccine development projects relied entirely on animal experimentation, a relatively expensive and time-consuming process. Currently, use of immunoinformatics tools play a vital role in the antigen analysis and refinement, allowing the identification of possible protective epitopes capable of stimulating convenient humoral or cellular immune responses, in addition to facilitating time and cost reduction of vaccine production. The vaccination aimed at bacterial species of Clostridium spp. has been considered a promising example of use of these approaches in recent years. Based on the literature search, it is possible to understand the best immunoinformatics software used by researchers that facilitate recombinant vaccine antigens design and development. This chapter presents an overview of how these tools are supporting the antigen engineering, aiming at increasing the efficiency of inducing protective immune response in animals.
Assuntos
Biologia Computacional , Desenvolvimento de Vacinas , Animais , Antígenos , Clostridium , Epitopos/genética , Epitopos de Linfócito T , Vacinas Sintéticas/genéticaRESUMO
This chapter describes a practical, industry-friendly, and efficient vaccine protocol based on the use of Escherichia coli cell fractions (inclusion bodies or cell lysate supernatant) containing the recombinant antigen. This approach was characterized and evaluated in laboratory and farm animals by the seroneutralization assay in mice, thereby showing to be an excellent alternative to induce a protective immune response against clostridial diseases.
Assuntos
Infecções por Escherichia coli , Vacinas contra Escherichia coli , Animais , Vacinas Bacterianas , Escherichia coli/genética , Escherichia coli/imunologia , Infecções por Escherichia coli/prevenção & controle , Infecções por Escherichia coli/veterinária , Corpos de Inclusão , Camundongos , Vacinas SintéticasRESUMO
Farm animals are frequently affected by a group of diseases with a rapid clinical course, caused by Clostridium spp. and immunization is essential to provide protection. However, the current manufacturing platform for these vaccines has disadvantages and the main alternative is the use of an expression system that uses Escherichia coli to obtain recombinant vaccine antigens. In this chapter we describe procedures for cloning, expression and characterization of recombinant toxins from Clostridium spp. produced in E. coli for veterinary vaccine applications.
Assuntos
Clostridium , Animais , Anticorpos Antibacterianos , Toxinas Bacterianas/genética , Vacinas Bacterianas , Escherichia coli/genética , Infecções por Escherichia coli , Vacinas SintéticasRESUMO
Electrochemical sensors and biosensors are useful techniques for fast, inexpensive, sensitive, and easy detection of innumerous specimen. In face of COVID-19 pandemic, it became evident the necessity of a rapid and accurate diagnostic test, so the impedimetric immunosensor approach can be a good alternative to replace the conventional tests due to the specific antibody-antigen binding interaction and the fast response in comparison to traditional methods. In this work, a modified electrode with electrosynthesized PEDOT and gold nanoparticles followed by the immobilization of truncated nucleoprotein (N aa160-406aa) was used for a fast and reliable detection of antibodies against COVID-19 in human serum sample. The method consists in analyzing the charge-transfer resistance (RCT) variation before and after the modified electrode comes into contact with the positive and negative serum sample for COVID-19, using [Fe(CN)6]3-/4- as a probe. The results show a linear and selective response for serum samples diluted in a range of 2.5 × 103 to 20 × 103. Also, the electrode material was fully characterized by Raman spectroscopy, transmission electron microscopy and scanning electron microscopy coupled with EDS, indicating that the gold nanoparticles were well distributed around the polymer matrix and the presence of the biological sample was confirmed by EDS analysis. EIS measurements allowed to differentiate the negative and positive samples by the difference in the RCT magnitude, proving that the material developed here has potential properties to be applied in impedimetric immunosensors for the detection of SARS-CoV-2 antibodies in about 30 min.
RESUMO
A case of subcutaneous phaeohyphomycosis in a dog with an ulcerative lesion on the right limb during a post-operative period of castration was described for the first time. The macroscopic and microscopic characteristics of the fungal colonies growth on the Sabourauddextrose agar were detailed. The fungus was identified as Aureobasidium pullulans on the basis of the phenotypic analysis, which was confirmed by sequencing of the internal transcribed spacers (ITS) region of rDNA. The patient might have acquired the infection through traumatic inoculation by environmental contact, along with the immunological condition during the stressful period of postoperative. The spontaneous remission of the lesion was observed in five weeks without antifungal treatment. This work highlights the importance of considering the pathogenic potential of this environmental fungus and the need of including it in the differential diagnosis of cutaneous lesions in dogs.