Hantavirus: Preliminary assessment of lateral flow immune assay prototypes to detect IgM and IgG antibodies.

Three lateral flow immunoassay prototypes developed to detect IgM, IgG and IgM/IgG antibodies against Hantavirus were evaluated. A total of 163 samples were tested: 10 from Hantavirus patients, 103 from related diseases, and 50 from healthy controls. The prototypes exhibited 100 % sensitivity, 97.5 % to 99.3 % specificity, indicating promising improved diagnosis.

Immunogenicity of a pentavalent recombinant Escherichiacoli bacterin against enterotoxemia and botulism in sheep.

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.

Clostridium perfringens antigens and challenges for development of vaccines against necrotic enteritis in poultry.

INTRODUCTION: Chickens with Necrotic Enteritis (NE), caused by Clostridium perfringens, exhibit acute and chronic symptoms that are difficult to diagnose, leading to significant economic losses. Vaccination is the best method for controlling and preventing NE. However, only two vaccines based on the CPA and NetB toxins have been commercialized, offering partial protection, highlighting the urgent need for more effective vaccines. OBJECTIVE: This review aimed to identify promising antigens for NE vaccine formulation and discuss factors affecting their effectiveness. METHODS: A systematic review using five scientific databases identified 30 eligible studies through the Rayyan tool, which were included for quality review. RESULTS: We identified 25 promising antigens, including CPA, NetB, FBA, ZMP, CnaA, FimA, and FimB, categorized by their role in disease pathogenesis. This review discusses the biochemical, physiological, and genetic traits of recombinant antigens used in vaccine prototypes, their expression systems, and immunization potential in chickens challenged with virulent C. perfringens strains. Market supply challenges, immunogenic potential, vaccine platforms, adjuvants, and factors related to vaccination schedules-such as administration routes, dosing intervals, and age at immunization-are also addressed. Additionally, the study notes that vaccine formulations tested under mild challenges may not offer adequate field-level protection due to issues replicating aggressive conditions, strain virulence loss, and varied methodologies. CONCLUSIONS: An ideal NE vaccine should incorporate multiple antigens, molecular adjuvants, and delivery systems via in ovo and oral routes. The review underscores the challenges in developing and validating NE vaccines and the urgent need for a standardized protocol to replicate aggressive challenges accurately.

Development and evaluation of a Lateral flow immunoassay (LFIA) prototype for the detection of IgG anti-SARS-CoV-2 antibodies.

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.

Development of a microencapsulated probiotic delivery system with whey, xanthan, and pectin.

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.

Immunogenicity of an inactivated vaccine for intravaginal application against bovine alphaherpesvirus type 5 (BoHV-5).

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.

Survival of Microencapsulated Lactococcus lactis Subsp. lactis R7 Applied in Different Food Matrices.

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.

Recombinant Vaccine Design Against Clostridium spp. Toxins Using Immunoinformatics Tools.

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.

Recombinant Escherichia coli Cell Lysates as a Low-Cost Alternative for Vaccines Against Veterinary Clostridial Diseases.

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.

Clostridium spp. Toxins: A Practical Guide for Expression and Characterization in Escherichia coli.

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.

PEDOT-AuNPs-based impedimetric immunosensor for the detection of SARS-CoV-2 antibodies.

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.

Subcutaneous phaeohyphomycosis due to Aureobasidium pullulans infection in a dog

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.

Human sporotrichosis outbreak caused by Sporothrix brasiliensis in a veterinary hospital in Southern Brazil.

Sporotrichosis is a mycotic disease caused by Sporothrix spp., whose zoonotic transmission by sick cats is the main infection route in Brazil. The aim of the current study is to report a human sporotrichosis outbreak caused by zoonotic transmission from a feline, with emphasis on the importance of making differential diagnosis and of using personal protective equipment. A hospital team member presented injury in the arm after having handled a cat that had been hospitalized for being hit by a car. The animal presented skin lacerations, myiasis, and full tibial fracture - there were no other signs of skin lesions. Clinical samples were collected from both the human and the suspected cat, for mycological culture; results have shown Sporothrix sp. growth. A search was conducted to identify other hospital team members who also had contact with the animal. Other six individuals also had suspected lesions in their arms, hands and ocular area; they were all subjected to sample collection. Mycological results have also confirmed Sporothrix spp.; sequencing analysis has shown that all seven humans were infected with Sporothrix brasiliensis. Since Southern Brazil is endemic of this disease, it is worth emphasizing the importance of taking into consideration zoonotic risks at the time to provide emergency care to stray animals, mainly felines, as well as of using Personal Protective Equipment while handling them - regardless of whether they present, or not, typical clinical symptoms or history of the disease, given the potential zoonotic risk posed by Sporothrix brasiliensis.

Immunomodulatory effect of short-term supplementation with Bacillus toyonensis BCT-7112T and Saccharomyces boulardii CNCM I-745 in sheep vaccinated with Clostridium chauvoei.

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.

Evaluation of the expression and immunogenicity of four versions of recombinant Clostridium perfringens beta toxin designed by bioinformatics tools.

Beta toxins (CPB) produced by Clostridium perfringens type B and C cause various diseases in animals, and the use of toxoids is an important prophylactic measure against such diseases. Promising recombinant toxoids have been developed recently. However, both soluble and insoluble proteins expressed in Escherichia coli can interfere with the production and immunogenicity of these antigens. In this context, bioinformatics tools have been used to design new versions of the beta toxin, and levels of expression and solubility were evaluated in different strains of E. coli. The immunogenicity in sheep was assessed using the molecule with the greatest potential that was selected on analyzing these results. In silico analyzes, greater mRNA stability (-169.70 kcal/mol), solubility (-0.755), and better tertiary structure (-0.12) were shown by rCPB-C. None of the strains of E. coli expressed rFH8-CPB, but a high level of expression and solubility was shown by rCPB-C. Higher levels of total and neutralizing anti-CPB antibodies were observed in sheep inoculated with bacterins containing rCPB-C. Thus, this study suggests that due to higher productivity of rCPB-C in E. coli and immunogenicity, it is considered as the most promising molecule for the production of a recombinant vaccine against diseases caused by the beta toxin produced by C. perfringens type B and C.

Feline sporotrichosis: a case series of itraconazole-resistant Sporothrix brasiliensis infection.

The treatment of feline sporotrichosis is a challenge for veterinary clinicians since refractory cases may occur, due either to patient and/or to pharmacological management errors or due to the development of antifungal resistance. Thus, we aimed to describe the therapeutic history of feline cases infected by itraconazole-resistant Sporothrix brasiliensis in an endemic region of Southern Brazil. Medical records of cats attended at the Veterinary Clinic Hospital (Pelotas/RS, Brazil) between 2016 and 2017 were reviewed. Twelve cases of infection by S. brasiliensis with that showed high minimum inhibitory concentration (MIC) values (≥ 4 µg/mL) to itraconazole by M38-A2 of CLSI were selected. At the hospital consultation, disseminated (cats 1-l0, 12) and localized (cat 11) skin lesions remained in the cats, even after treatment with fluconazole, ketoconazole (02/12), and itraconazole (ITZ, 09/12) performed before this study. High doses (25-100 mg/kg/day) of ITZ for up to 4 months (03/12, cats 2, 6, 12) or over 12 months (05/12, cats 1, 5, 7, 8, 11) did not provide a clinical cure, except for the association of ITZ plus potassium iodide (01/12, cat 12) for 3 months, which proved useful in infections with itraconazole-resistant S. brasiliensis. However, the combined issues of abandonment of therapy by owners for financial reasons, difficulties surrounding therapy administration (03/12, cats 6, 11, 12), and the inappropriate choice of medication (01/12, cat 6), together reflect the reality of this endemic region, which greatly compromises clinical healing. This study highlighted the occurrence of refractory cases by itraconazole-resistant S. brasiliensis in cats from Southern Brazil, as well as the abandonment of treatment and therapeutic errors. We warn of the need for antifungal susceptibility tests to adapt therapeutic protocols in feline sporotrichosis.

Novel phage display-derived recombinant antibodies recognizing both MPT64 native and mutant (63-bp deletion) are promising tools for tuberculosis diagnosis

Tuberculosis (TB) is one of the top 10 causes of death in humans worldwide. The most important causative agents of TB are bacteria from the Mycobacterium tuberculosis complex (MTC), although nontuberculous mycobacteria (NTM) can also cause similar infections. The ability to identify and differentiate MTC isolates from NTM is important for the selection of the correct antimicrobial therapy. Immunochromatographic assays with antibodies anti-MPT64 allow differentiation between MTC and NTM since the MPT64 protein is specific from MTC. However, studies reported false-negative results mainly due to mpt64 63-bp deletion. Considering this drawback, we selected seven human antibody fragments against MPT64 by phage display and produced them as scFv-Fc. Three antibodies reacted with rMPT64 mutant (63-bp deletion) protein and native MPT64 from M. tuberculosis H37Rv in ELISA and Western blot. These antibodies are new biological tools with the potential for the development of TB diagnosis helping to overcome limitations of the MPT64-based immunochromatographic tests currently available.

Molecular Characterization of Carbapenem-Resistant Acinetobacter baumannii Associated with Nosocomial Infection in the Pelotas, RS, Brazil.

Acinetobacter calcoaceticus-Acinetobacter baumannii complex (ACB) comprises some opportunistic pathogens associated with infectious outbreaks in hospital settings. A. baumannii is the most relevant species owing to its capacity to develop resistance to the different classes of antimicrobials. The aim of this study was to identify the species, establish the genetic patterns, resistance and biofilm profiles in ACB isolates associated with nosocomial infection in a hospital of Pelotas, Rio Grande do Sul, Brazil. Twenty-two clinical isolates were characterized at the species level through multiplex polymerase chain reaction (PCR) for the gyrB and blaOXA51-like genes, and the genetic relationship was determined through pulsed-field gel electrophoresis (PFGE). Their antibiotic resistance profiles and carbapenemases synthesis were evaluated following CLSI guidelines. PCR was carried out to evaluate the presence of carbapenemases genes and the isolates were classified for their biofilm-forming ability. All isolates obtained in the study were identified as A. baumannii and 72.7% of the isolates were classified as strong biofilm formers. In the class carbapenems, 95.4% and 77.3% of the isolates were resistant to meropenem and imipenem, respectively. The blaVIM gene was identified in 90.9% of isolates and carbapenemases synthesis were confirmed in 95.4% of the isolates. Fourteen genetic patterns were confirmed through PFGE analyses. The isolates collected within a time gap of 2 years demonstrated a genetic relationship, and the same clone was identified in different departments in the hospital. To the best of our knowledge, this is the first report of identification and characterization of A. baumannii nosocomial isolates in Pelotas, RS, Brazil.

Saccharomyces boulardii reduces the mean intensity of infection in mice caused by the consumption of liver contaminated by Toxocara canis.

Probiotics have shown promising results as a potential method to control toxocariasis in mice inoculated with embryonated eggs of Toxocara canis. This study aimed to evaluate the protective effect of Saccharomyces boulardii in mice fed in natura chicken livers infected with T. canis. Twenty 15-day-old male Sussex chickens were inoculated with 300 T. canis embryonated eggs via intragastric catheter (GI). After 72 h of infection, each liver was collected and individually offered to a group of 20 mice. Mice that received supplemented ration with S. boulardii (1.107 colony forming units) and consumed in natura chicken liver showed reduction in infection intensity of 67.1%. This study demonstrated that administration of S. boulardii has potential as a probiotic to assist in controlling visceral toxocariasis caused by the consumption of viscera from paratenic hosts containing infective parasite larvae.

Biopolymer Xanthan: A New Adjuvant for DNA Vaccines

Abstract DNA vaccines have been evaluated as an option to prevent several diseases. In this study, the capacity of the xanthan biopolymer to improve the DNA vaccines immune response, administered intramuscularly, was evaluated. The experimental vaccines consisted of genes encoding fragments of the proteins LigA and LigB of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130. The humoral immune response was evaluated by indirect ELISA. Cytokine expression levels were determined by RT-qPCR. Compared to the control group, the IgG antibody levels of animals immunized with pTARGET/ligAni and pTARGET/ligBrep plasmids associated with xanthan biopolymer were significantly higher than the control group. Additionally, there was a significant increase in IL-17 expression in animals vaccinated with pTARGET/ligBrep and xanthan.