Evaluation of long-term immune response in cattle to botulism using a recombinant E. coli bacterin formulated with Montanide™ ISA 50 and aluminum hydroxide adjuvants.

Botulism is a severe disease caused by potent botulinum neurotoxins (BoNTs) produced by Clostridium botulinum. This disease is associated with high-lethality outbreaks in cattle, which have been linked to the ingestion of preformed BoNT serotypes C and D, emphasizing the need for effective vaccines. The potency of current commercial toxoids (formaldehyde-inactivated BoNTs) is assured through tests in guinea pigs according to government regulatory guidelines, but their short-term immunity raises concerns. Recombinant vaccines containing the receptor-binding domain have demonstrated potential for eliciting robust protective immunity. Previous studies have demonstrated the safety and effectiveness of recombinant E. coli bacterin, eliciting high titers of neutralizing antibodies against C. botulinum and C. perfringens in target animal species. In this study, neutralizing antibody titers in cattle and the long-term immune response against BoNT/C and D were used to assess the efficacy of the oil-based adjuvant compared with that of the aluminum hydroxide adjuvant in cattle. The vaccine formulation containing Montanide™ ISA 50 yielded significantly higher titers of neutralizing antibody against BoNT/C and D (8.64 IU/mL and 9.6 IU/mL, respectively) and induced an immune response that lasted longer than the response induced by aluminum, extending between 30 and 60 days. This approach represents a straightforward, cost-effective strategy for recombinant E. coli bacterin, enhancing both the magnitude and duration of the immune response to botulism.

Immunization of sheep with a recombinant vaccine containing immunogenic nontoxic domains of Clostridium perfringens alpha and beta toxins.

Clostridium perfringens (types A and C) can cause several diseases by secreting alpha (CPA) and beta (CPB) exotoxins in the gastrointestinal tract. Although vaccination is the main measure of immunization against C. perfringens, available vaccines have limitations in terms of productivity and safety. Thus, recombinant vaccines are an important, more effective, practical, and safer strategy in the immunization of animals. In this study, we evaluated the immunization of sheep with recombinant Escherichia coli bacterins expressing CPA and CPB complete proteins (co-administered), the immunogenic nontoxic domains rCPA-C247-370 and rCPB-C143-311 co-administered or fused as a bivalent chimera (rCPBcAc). For this, in silico analysis was performed to design rCPBcAc, considering the stability of the mRNA (-278.80 kcal/mol), the degree of antigenicity (0.7557), the epitopes of the B cell ligand, and different physicochemical characteristics. All proteins were expressed in vitro. In vivo, animals vaccinated with the co-administered antigens rCPA + rCPB and rCPA-C+ rCPB-C (200 µg each) had mean CPA and CPB neutralizing antitoxin titers of 4, 10, 4.8, and 14.4 IU/mL, respectively, while those vaccinated with 200 µg of rCPBcAc chimera (approximately 100 µg of each antigen) had titers of <4 and 12 IU/mL of CPA and CPB antitoxins, respectively, 56 days after the administration of the first dose. In addition, the chimera was considered to be immunogenic for inducing antitoxin titers using the half dose. In this study, we presented a new recombinant antigen potentially applicable for vaccines against the CPA and CPB toxins for preventing diseases caused by Clostridium perfringens.

Occurrence of ESBL-producing avian pathogenic Escherichia coli (APEC) isolates in spiced chicken meat in Goias, Brazil.

Some extraintestinal pathogenic Escherichia coli isolates (ExPEC), obtained from humans and chickens avian pathogenic E. coli (APEC), share similar virulence genes. Thus, products of avian origin can be a source of human infection. Moreover, these APEC isolates are resistant to antimicrobials and can spread in the environment through the chicken feces. Although the development of multidrug-resistant (MDR) microorganisms in poultry is on the rise, healthcare entities have raised concerns since MDRs can horizontally transfer resistance genes to other microorganisms and complicate the management of human infections by MDR APEC. The results of our study showed that of 80 investigated spiced chicken meat samples, 55% were contaminated with E. coli, of which 34% (15/44) contaminate with APEC. No diarrheagenic E. coli (DEC) pathotypes were found. Twenty-six isolates were MDR E. coli. Among the APEC isolates, 87% (13/15) produced extended-spectrum beta-lactamase (ESBL). The emergence of MDR/ESBL-producing APEC with zoonotic potential for humans is extremely worrying. Therefore, further studies are required to identify the prevalence of MDR/ESBL-producing APEC in the entire chicken production chain from creation, slaughter, processing, and butchery.

Increased Neutrophil Percentage and Neutrophil-T Cell Ratio Precedes Clinical Onset of Experimental Cerebral Malaria.

Newly emerging data suggest that several neutrophil defense mechanisms may play a role in both aggravating and protecting against malaria. These exciting findings suggest that the balance of these cells in the host body may have an impact on the pathogenesis of malaria. To fully understand the role of neutrophils in severe forms of malaria, such as cerebral malaria (CM), it is critical to gain a comprehensive understanding of their behavior and functions. This study investigated the dynamics of neutrophil and T cell responses in C57BL/6 and BALB/c mice infected with Plasmodium berghei ANKA, murine models of experimental cerebral malaria (ECM) and non-cerebral experimental malaria, respectively. The results demonstrated an increase in neutrophil percentage and neutrophil-T cell ratios in the spleen and blood before the development of clinical signs of ECM, which is a phenomenon not observed in the non-susceptible model of cerebral malaria. Furthermore, despite the development of distinct forms of malaria in the two strains of infected animals, parasitemia levels showed equivalent increases throughout the infection period evaluated. These findings suggest that the neutrophil percentage and neutrophil-T cell ratios may be valuable predictive tools for assessing the dynamics and composition of immune responses involved in the determinism of ECM development, thus contributing to the advancing of our understanding of its pathogenesis.

Sporotrichosis in dogs: epidemiological and clinical-therapeutic profile and the emergence of itraconazole-resistant isolates.

Sporotrichosis is a neglected and emerging mycosis caused by the traumatic implantation of Sporothrix propagules into the (sub)cutaneous tissues of humans and animals. We evaluated canine sporotrichosis's clinical-therapeutic, epidemiological profile, and in vitro susceptibility of isolates to itraconazole. The variables were evaluated by a chi-square test. A total of 69 dogs were infected with Sporothrix spp., and the molecular identification revealed an overwhelming occurrence of S. brasiliensis as the etiological agent. The epidemiological profile was male (56.5%), adults (4.9 ± 1.92 years old; 69.6%), and mongrels (53.6%). The clinical signs were 76.8%, ulcers, draining tracts, and nodules were predominant, mainly in the nasal region (82.2%). Dogs were diagnosed late with an evolution time of up to 3 months (34.8%). According to the prior therapeutic information, 52.2% received empirical therapy, 79.2% antibiotics, and had a 0.29 significantly greater chance of presenting lesion evolution time ˃ 3 months (P < .05; Odds Ratio [OR] 1/0.29). Additionally, 25 S. brasiliensis isolates recovered between 2006-2012 (n = 15; Minimal inhibitory concentration (MIC): 0.06-2 µg/ml) and 2013-2018 (n = 10; MIC: 2→16 µg/ml) were tested against itraconazole (ITZ). These findings highlighted the resistance to ITZ in clinical cases due to S. brasiliensis occurring after 2013, showing the temporal evolution of ITZ-resistance. We warn of the importance of accurate and early diagnosis in Sporothrix-affected areas, and we report the emergence of ITZ-resistant isolates in Southern Brazil.

Sporotrichosis is a fungal zoonosis. We investigated the clinical-therapeutic, epidemiological profile, and in vitro susceptibility of isolates to itraconazole (ITZ) in canine cases in Southern Brazil. Our study highlighted the emergence of ITZ-resistant Sporothrix brasiliensis and the main challenges for clinical control of this neglected disease.

Discovery of novel benzothiophene derivatives as potent and narrow spectrum inhibitors of DYRK1A and DYRK1B.

The discovery of potent and selective inhibitors for understudied kinases can provide relevant pharmacological tools to illuminate their biological functions. DYRK1A and DYRK1B are protein kinases linked to chronic human diseases. Current DYRK1A/DYRK1B inhibitors also antagonize the function of related protein kinases, such as CDC2-like kinases (CLK1, CLK2, CLK4) and DYRK2. Here, we reveal narrow spectrum dual inhibitors of DYRK1A and DYRK1B based on a benzothiophene scaffold. Compound optimization exploited structural differences in the ATP-binding sites of the DYRK1 kinases and resulted in the discovery of 3n, a potent and cell-permeable DYRK1A/DYRK1B inhibitor. This compound has a different scaffold and a narrower off-target profile compared to current DYRK1A/DYRK1B inhibitors. We expect the benzothiophene derivatives described here to aid establishing DYRK1A/DYRK1B cellular functions and their role in human pathologies.

Peroxisome proliferator-activated receptor delta-PPARδ agonist (L-165041) enhances bovine embryo survival and post vitrification viability.

The effect of L-165041 (PPARδ-agonist) on decreasing apoptosis and intracellular lipid content was assessed in fresh and vitrified-warmed in vitro -produced bovine embryos. It was hypothesised that the addition of L-165041 to the culture medium enhances development and cryopreservation. Oocytes were allocated to one of two treatments: control-standard culture medium, or L-165041 added to the medium on day1 with no media change. Ultrastructure, cleavage, and blastocyst rates were evaluated in fresh, and in post-vitrification cultured embryos by optical and electronic microscopy. A subset of fresh embryos were fixed for TUNEL assay and for Sudan-Black-B histochemical staining. Vitrified-warmed embryos were assessed using MALDI-MS technique. Cleavage and blastocyst rates (control 49.4±5.2, L-165041 51.8±4.3) were not influenced by L-165041. The proportion of inner cell mass cells (ICM) was higher in fresh embryos, and the rate of total and ICM apoptosis was lower in L-165041. In warmed-embryos, total and ICM apoptosis was lower in L-165041. The overall hatching rate was higher in L-165041 (66.62±2.83% vs 53.19±2.90%). There was less lipid accumulation in fresh L-165041-embryos. In conclusion, the use of L-165041 is recommended to improve the viability of in vitro -derived bovine embryos.

Protective effect of kavain in meristematic cells of Allium cepa L.

Kavain is one of the main kavalactones of Piper methysticum (Piperaceae) with anxiolytic, analgesic, and antioxidant activities. Therefore, the aim of the study was to evaluate the cytotoxic, mutagenic, and antimutagenic potential of kavain in Allium cepa cells. Roots of A. cepa were transferred to the negative (2% acetone) and positive (10 µg/mL of Methylmethanesulfonate, MMS) controls and to the concentrations of kavain (32, 64 and 128 µg/mL) for 48 h. A total of 5,000 meristematic cells were analyzed under an optical microscope to determine the mitotic index, mean number of chromosomal alterations and percentage of damage reduction. Data were analyzed by Kruskal-Wallis test (p <0.05). All concentrations of kavain were not cytotoxic and did not show significant chromosomal changes when compared to 2% acetone. Kavain showed a cytoprotective effect in the pre (128 µg/mL) and in the post-treatment (32 and 64 µg/mL) and reduced damage against the mutagenic action of MMS in all concentrations of the pre and simultaneous and at the highest of post (128 µg/mL). Kavain promoted a significant reduction in micronuclei, nuclear buds and chromosomal losses in relation to MMS. The observed data indicate the importance of kavain for the inhibition of damage and chemoprevention.

Evaluation of WALA ridge in different facial patterns: A cone-beam computed tomography study.

INTRODUCTION: This retrospective study evaluated the buccal bone thickness in mandibular canine, premolar, and molar areas, using as a reference the WALA ridge in patients with various facial patterns. METHODS: The sample comprised 51 cone-beam computerized tomography scans of subjects divided into 3 groups according to the facial pattern, determined by the Ricketts' VERT index, brachyfacial (group 1), mesofacial (group 2), and dolichofacial (group 3). A quantitative analysis of the buccal bone thickness was made in cone-beam computerized tomography scans in the region of the mandibular dental arch corresponding to the WALA ridge. The intergroup comparison of buccal bone thickness was performed with a 1-way analysis of variance and Tukey tests. RESULTS: Buccal bone thickness was similar among the groups, and it increased progressively from the mandibular first to the second molars. In the canine and premolar regions, the thickness was less and often not visible on tomography. There was no difference in the buccal bone thickness in the mandibular dental arch region corresponding to the WALA ridge reference among facial patterns, except for the mesial roots of the second molars, which were thicker in brachyfacial subjects than in dolichofacial subjects. CONCLUSIONS: The use of the WALA ridge as a clinical reference for individualized orthodontic arch diagramming should be made with caution and independent of the facial pattern.

Fertility analysis of bovine semen by in vitro fertilization.

The aim of the present study was to evaluate the efficiency of using in vitro fertilization to validate semen fertility for artificial insemination. Cryopreserved semen from ten bulls (five Nelore and five Brangus bulls) was evaluated using in vitro production of embryos (IVPE) and via fixed-time artificial insemination (FTAI). There was variation (p < 0.05) in the IVPE (20.9 to 53.7% of blastocyst production) and in the FTAI (42.0 to 56.0% of pregnant cows) results among the semen evaluated. According to the results, there was a positive correlation (rs = 0.8378; p = 0.0001) between the rate of blastocyst production (using IVPE) and the rate of pregnancy (using FTAI) using Nelore bull semen. Variation (p < 0.05) was also found using semen from Brangus bulls, in the rates of blastocyst production (36.5 to 47.0%) and pregnancy (45.6 to 52.2%) via FTAI. There was also a positive correlation (rs = 0.8786; p = 0.0001) between the rates of blastocyst production (IVPE) and pregnancy (FTAI) when using Brangus bull semen. According to the results, IVPE may be used in addition to conventional semen analysis to evaluate and validate the semen fertility of bulls for artificial insemination programs.

Regulation of muscle and mitochondrial health by the mitochondrial fission protein Drp1 in aged mice.

KEY POINTS: The maintenance of mitochondrial integrity is critical for skeletal muscle health. Mitochondrial dynamics play key roles in mitochondrial quality control; however, the exact role that mitochondrial fission plays in the muscle ageing process remains unclear. Here we report that both Drp1 knockdown and Drp1 overexpression late in life in mice is detrimental to skeletal muscle function and mitochondrial health. Drp1 knockdown in 18-month-old mice resulted in severe skeletal muscle atrophy, mitochondrial dysfunction, muscle degeneration/regeneration, oxidative stress and impaired autophagy. Overexpressing Drp1 in 18-month-old mice resulted in mild skeletal muscle atrophy and decreased mitochondrial quality. Our data indicate that silencing or overexpressing Drp1 late in life is detrimental to skeletal muscle integrity. We conclude that modulating Drp1 expression is unlikely to be a viable approach to counter the muscle ageing process. ABSTRACT: Sarcopenia, the ageing-related loss of skeletal muscle mass and function, is a debilitating process negatively impacting the quality of life of afflicted individuals. Although the mechanisms underlying sarcopenia are still only partly understood, impairments in mitochondrial dynamics, and specifically mitochondrial fission, have been proposed as an underlying mechanism. Importantly, conflicting data exist in the field and both excessive and insufficient mitochondrial fission were proposed to contribute to sarcopenia. In Drosophila melanogaster, enhancing mitochondrial fission in midlife through overexpression of dynamin-1-like protein (Drp1) extended lifespan and attenuated several key hallmarks of muscle ageing. Whether a similar outcome of Drp1 overexpression is observed in mammalian muscles remains unknown. In this study, we investigated the impact of knocking down and overexpressing Drp1 protein for 4 months in skeletal muscles of late middle-aged (18 months) mice using intra-muscular injections of adeno-associated viruses expressing shRNA targeting Drp1 or full Drp1 cDNA. We report that knocking down Drp1 expression late in life triggers severe muscle atrophy, mitochondrial dysfunctions, degeneration/regeneration, oxidative stress and impaired autophagy. Drp1 overexpression late in life triggered mild muscle atrophy and decreased mitochondrial quality. Taken altogether, our results indicate that both overexpression and silencing of Drp1 in late middle-aged mice negatively impact skeletal muscle mass and mitochondrial health. These data suggest that Drp1 content must remain within a narrow physiological range to preserve muscle and mitochondrial integrity during ageing. Altering Drp1 expression is therefore unlikely to be a viable target to counter sarcopenia.

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.

Distribution of genetically characterized yeasts and its antifungals susceptibility in the hospital environment.

Candida spp. is one of the main pathogens associated with nosocomial infection in Brazil and worldwide. The aim of this study was to evaluate the distribution of Candida yeasts in the ICU and their susceptibility to the antifungal agents terbinafine and fluconazole. The samples were collected by swabbing nine surfaces in the ICU of a hospital located in Pelotas, RS. These isolates were genetically characterized by sequencing the internal transcript spacer (ITS) using the primers ITS1 and ITS4. The test against antifungals was performed by Microdilution in Broth (CLSI-M27-A4). 64 yeasts identified as Candida parapsilosis (45.31%; n = 29), Meyerozyma (Pichia) guilliermondii (28.12%; n = 18), Claviceps lusitaneae (25%; n = 16) and Candida tropicalis (1, 56%; n = 1) mostly at the counter used for handling medicines and food distribution (68.75%; n = 44). Susceptibility to antifungals varied between species. These results describe potentially pathogenic Candida species as contaminants in the ICU environment. The study environment is a potential source of exogenous infection for hospitalized patients.

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.

Non-invasive quantification of vitamin C, citric acid, and sugar in 'Valência' oranges using infrared spectroscopies.

Near (NIR) and mid (MIR) infrared spectroscopies have been studied as potential methods for non-destructive analyses of the fresh fruits quality. In this study, vitamin C, citric acid, total and reducing sugar content in 'Valência' oranges were evaluated using NIR and MIR spectroscopy with multivariate analysis. The spectral data were used to build up prediction models based on PLS (Partial Least Squares) regression. For vitamin C and citric acid, both NIR (r = 0.72 and 0.77, respectively) and MIR (0.81 and 0.91, respectively) resulted in feasible models. For sugars determination the two techniques presented a strong correlation between the reference values and analytical signals, with low RMSEP and r > 0.70 (NIR: sucrose RMSEP = 12.2 and r = 0.75; glucose RMSEP = 6.77 and r = 0.82; fructose RMSEP = 5.07 and r = 0.81; total sugar RMSEP = 12.1 and r = 0.80; reducing sugar RMSEP = 20.32 and r = 0.82; MIR: sucrose RMSEP = 9.47 and r = 0.80; glucose RMSEP = 6.70 and r = 0.82; fructose RMSEP = 5.20 and r = 0.81; total sugar RMSEP = 11.72 and r = 0.81; reducing sugar RMSEP = 20.42 and r = 0.81). The models developed with MIR presented lower prediction error rates than those made with NIR. Therefore, infrared techniques show applicability to determine of orange quality parameters in a non-destructive way.

Origanum vulgare essential oil: antibacterial activities and synergistic effect with polymyxin B against multidrug-resistant Acinetobacter baumannii.

Antimicrobial resistance is increasing around the world and the search for effective treatment options, such as new antibiotics and combination therapy is urgently needed. The present study evaluates oregano essential oil (OEO) antibacterial activities against reference and multidrug-resistant clinical isolates of Acinetobacter baumannii (Ab-MDR). Additionally, the combination of the OEO and polymyxin B was evaluated against Ab-MDR. Ten clinical isolates were characterized at the species level through multiplex polymerase chain reaction (PCR) for the gyrB and blaOXA-51-like genes. The isolates were resistant to at least four different classes of antimicrobial agents, namely, aminoglycosides, cephems, carbapenems, and fluoroquinolones. All isolates were metallo-ß-lactamase (MßL) and carbapenemase producers. The major component of OEO was found to be carvacrol (71.0%) followed by ß-caryophyllene (4.0%), γ-terpinene (4.5%), p-cymene (3,5%), and thymol (3.0%). OEO showed antibacterial effect against all Ab-MDR tested, with minimum inhibitory concentrations (MIC) ranging from 1.75 to 3.50 mg mL-1. Flow cytometry demonstrated that the OEO causes destabilization and rupture of the bacterial cell membrane resulting in apoptosis of A. baumannii cells (p < 0.05). Synergic interaction between OEO and polymyxin B (FICI: 0.18 to 0.37) was observed, using a checkerboard assay. When combined, OEO presented until 16-fold reduction of the polymyxin B MIC. The results presented here indicate that the OEO used alone or in combination with polymyxin B in the treatment of Ab-MDR infections is promising. To the best of our knowledge, this is the first report of OEO and polymyxin B association against Ab-MDR clinical isolates.

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.

Formaldehyde effects on kanamycin resistance gene of inactivated recombinant Escherichia coli vaccines.

OBJECTIVES: Earlier studies have demonstrated the use of inactivated recombinant E. coli (bacterins), to protect against Clostridium spp. in vaccinated animals. These bacterins have a simpler, safer, and faster production process. However, these bacterins carry expression plasmids, containing antibiotic resistance gene, which could be assimilate accidentally by environmental microorganisms. Considering this, we aimed to impair this plasmids using formaldehyde at different concentrations. RESULTS: This compound inactivated the highest density of cells in 24 h. KanR cassette amplification was found to be impaired with 0.8% for 24 h or 0.4% for 72 h. Upon electroporation, E. coli DH5α ultracompetent cells were unable to acquire the plasmids extracted from the bacterins after inactivation procedure. Formaldehyde-treated bacterins were incubated with other viable strains of E. coli, leading to no detectable gene transfer. CONCLUSIONS: We found that this compound is effective as an inactivation agent. Here we demonstrate the biosafety involving antibiotic resistance gene of recombinant E. coli vaccines allowing to industrial production and animal application.

Recombinant vaccine against botulism in buffaloes: Evaluation of the humoral immune response over 12 months.

Botulism is a neuroparalytic intoxication, usually fatal, caused by the botulinum toxins (BoNTs). Vaccination is the best-known strategy to prevent this disease in ruminants. Serotypes C and D and their variants CD and DC are the main types responsible for botulism in bovine and buffaloes in Brazil and cattle in Japan and Europe. Brazil has a herd of approximately 1.39 million buffaloes and is the largest producer in the Western world. This study aimed to assess the humoral immune response of buffaloes during the 12-month period after vaccination against BoNT serotypes C and D with a recombinant vaccine in three different concentrations (100, 200, and 400 µg) of non-purified recombinant proteins (Vrec) and also with a bivalent commercial toxoid (Vcom). Vrec400 was the best vaccine among those tested because it induced higher levels of antibodies and maintained higher levels of antibodies for the longest time, while Vrec200 could be considered the most cost-effective vaccine for large-scale production. None of the vaccines were able to promote continuous immunological protection within the timeframe proposed by the current Brazilian vaccination protocol. Further studies should focus on vaccine adjustments to ensure continued humoral protection against botulism.

In vitro fertilization: productivity of donors of different bovine breeds.

The objective of the present study was to evaluate efficiency of in vitro fertilization (IVF) in Nelore, Brangus, and Girolando oocyte donors. Ovum pickup (OPU) from the donors was conducted every 15 days to assess oocyte recovery, IVF, and post-transfer pregnancy percentage. For Nelore, the mean numbers of total and viable oocytes recovered (23.5 ± 1.1 and 14.0 ± 1.0, respectively) were higher (p < 0.05) than those for Brangus (12.7 ± 1.9 and 6.6 ± 1.0, respectively) and Girolando (12.5 ± 1.4 and 6.8 ± 0.7, respectively); Brangus and Girolando did not differ from each other (p > 0.05). The percentage of blastocyst production differed (p < 0.05) between Nelore (48.4 ± 2.4%), Brangus (40.3 ± 3.6%), and Girolando (38.9 ± 2.6%), but those in Brangus and Girolando did not differ (p > 0.05). The percentage of blastocysts (transferred) that resulted in pregnancy did not differ (p > 0.05) between Nelore (45.5 ± 3.8%), Brangus (41.7 ± 4.1%), and Girolando (40.7 ± 3.7%). Of the breeds studied, Nelore donors are more efficient for IVF, but conditions of this study.