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.

Regular Voluntary Running is Associated with Increased Tumor Vascularization and Immune Cell Infiltration and Decreased Tumor Growth in Mice.

Tumors present dysfunctional vasculature that limits blood perfusion and hinders immune cells delivery. We aimed to investigate if regular voluntary running promotes tumor vascular remodelling, improves intratumoral immune cells infiltration and inhibits tumor growth. Tumors were induced in C57BL/6 male mice (n=28) by subcutaneous inoculation in the dorsal region with a suspension of RM1 cells (1.5×105 cells/500 µL PBS) and randomly allocated into two groups: sedentary (n=14) and voluntarily exercised on a wheel (n=14). Seven mice from each group were sacrificed 14 and 28 days after cells' inoculation to evaluate tumor weight, microvessel density, vessels' lumen regularity and the intratumoral quantity of NKG2D receptors, CD4+and CD8+T cells, by immunohistochemistry. The statistical inference was done through a two-way ANOVA. Exercised mice developed smaller tumors at 14 (0.17±0.1 g vs. 0.48±0.2 g, p<0.05) and 28 (0.92±0.7 g vs. 2.09±1.3 g, p<0.05) days, with higher microvessel density (21.20±3.2 vs. 15.86±4.0 vessels/field, p<0.05), more regular vessels' lumen (1.06±0.2 vs. 1.43±0.2, p<0.05), and higher CD8+T cells (464.95±48.0 vs. 364.70±49.4 cells/mm2, p<0.01), after 28 days. NKG2D expression was higher in exercised mice at 14 (263.27±25.8 cells/mm2, p<0.05) and 28 (295.06±56.2 cells/mm2, p<0.001) days. Regular voluntary running modulates tumor vasculature, increases immune cells infiltration and attenuates tumor growth, in mice.

Combined antibodies against internalins A and B proteins have potential application in immunoassay for detection of Listeria monocytogenes.

Listeria monocytogenes is a food-borne bacterium that causes listeriosis upon the ingestion of contaminated food. Traditional methods to detect L. monocytogenes require pre-enrichment broths to increase its concentration. To improve the screening of contaminated food and prevent listeriosis outbreaks, rapid, specific and sensitive assays are needed to detect L. monocytogenes. This study developed a prototype lateral flow immunochromatographic assay (LFIA) employing antibodies against L. monocytogenes Internalin A (InlA) and Internalin B (InlB) proteins, that are involved in non-phagocytic cell invasion. The following antibodies were used to capture L. monocytogenes antigenic targets: mouse anti-Internalin A monoclonal antibody (MAb-2D12) conjugated to colloidal gold nanoparticles and a mouse anti-Internalin B polyclonal antibody. This test was able to detect pure L. monocytogenes from culture with a limit of detection (LOD) ranging from 5.9 × 103 to 1.5 × 104 CFU/mL. In milk artificially contaminated with L. monocytogenes, the LOD was 1 × 105 CFU/mL. This prototype test discriminated L. monocytogenes from other bacterial species (Listeria innocua, Enterobacter cloacae, Bacillus cereus). Results indicate that this LFIA developed using antibodies against L. monocytogenes InlA and InlB proteins is a sensitive and specific tool that can be potentially useful to rapidly detect L. monocytogenes in contaminated food. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05597-9.

Komagataella pastoris KM71H modulates neuroimmune and oxidative stress parameters in animal models of depression: A proposal for a new probiotic with antidepressant-like effect.

Many studies have suggested that imbalance of the gut microbial composition leads to an increase in pro-inflammatory cytokines and promotes oxidative stress, and this are directly associated with neuropsychiatric disorders, including major depressive disorder (MDD). Clinical data indicated that the probiotics have positive impacts on the central nervous system and thus may have a key role to treatment of MDD. This study examined the benefits of administration of Komagataella pastoris KM71H (8 log UFC·g-1/animal, intragastric route) in attenuating behavioral, neurochemical, and neuroendocrine changes in animal models of depressive-like behavior induced by repeated restraint stress and lipopolysaccharide (0.83 mg/kg). We demonstrated that pretreatment of mice with this yeast prevented depression-like behavior induced by stress and an inflammatory challenge in mice. We believe that this effect is due to modulation of the permeability of the blood-brain barrier, restoration in the mRNA levels of the Nuclear factor kappa B, Interleukin 1ß, Interferon γ, and Indoleamine 2 3-dioxygenase, and prevention of oxidative stress in the prefrontal cortices, hippocampi, and intestine of mice and of the decrease the plasma corticosterone levels. Thus, we conclude that K. pastoris KM71H has properties for a new proposal of probiotic with antidepressant-like effect, arising as a promising therapeutic strategy for MDD.

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.

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.

Inactivated recombinant Escherichia coli as a candidate vaccine against Clostridium perfringens alpha toxin in sheep.

Clostridium perfringens type A is the causative agent of gas gangrene and gastroenteric ("yellow lamb disease") disease in ruminants, with C. perfringens alpha toxin (CPA) being the main virulence factor in the pathogenesis of these illnesses. In the present study, we have developed recombinant Escherichia coli bacteria expressing rCPA and used it to vaccinate rabbits and sheep. Doses of up to 200 µg of rCPA used for inoculation, induced 13.82 IU.mL-1 of neutralizing antitoxin in rabbits, which is three times higher than that recommended by the USDA (4 IU.mL-1). In sheep, recombinant bacteria induced antitoxin titers of 4 IU.mL-1, 56 days after the first dose. rCPA which was expressed, mainly, in inclusion bodies, was not found to influence the immunogenicity of the vaccine. The recombinant Escherichia coli bacterin, produced simply and safely, is capable of affording protection against diseases caused by C. perfringens CPA. The current findings represent a novel production method for CPA vaccines potentially applicable to veterinary medicine.

Reactivity of recombinant Toxocara canis TES-30/120 in experimentally infected mice.

AIM: While the use of recombinant antigens is being widely investigated in the diagnosis of human toxocariasis, relatively little attention has been given to animal diagnostic models. For this reason, this study aimed to investigate the diagnosis potential of Toxocara canis TES-30 and TES-120 recombinant antigens in mice, the animal model for toxocariasis studies. METHODS AND RESULTS: Serum samples obtained from mice infected with T. canis or Toxocara cati were tested by indirect ELISA using T. canis TES-30 and TES-120 recombinant antigens produced in Escherichia coli. 90% of the samples reacted with rTES-30, whereas there was almost no reactivity with rTES-120. CONCLUSION: Despite rTES-120 being a good antigen for diagnosis in humans, it could not reproduce its reactivity in this animal model. As rTES-30 has good reactivity in mice, it is a valuable tool for diagnosis.

Bioremediation and biomass production from the cultivation of probiotic Saccharomyces boulardii in parboiled rice effluent.

The parboilization of rice generates 2 L of effluent per kilogram of processed grain. Several methodologies have previously been tested with the aim of reducing the environmental impact of this effluent. The objective of this study was to evaluate the bioremediation of parboiled rice effluent supplemented with sucrose or residual glycerol from the biodiesel during the cultivation of the Saccharomyces boulardii probiotic. In the first stage of the experiment, cultures were grown in orbital shaker, and five media compositions were evaluated: 1) parboiled rice effluent; 2) effluent supplemented with 1% sucrose; 3) effluent supplemented with 3% sucrose; 4) effluent supplemented with 15 g.L-1 of biodiesel glycerol and 5) standard yeast culture medium (YM). The addition of 1% of sucrose generated the most promising results in terms of cell viability, removal of nitrogen, phosphorus and chemical oxygen demand (COD). From these results, four independent cultures were grown in a bioreactor using effluent +1% of sucrose as the medium. This assays generated a mean of 3.8 g.L-1 of biomass, 1.8 × 1011 CFU.L-1, and removal of 74% of COD and 78% of phosphorus. Therefore, the cultivation of Saccharomyces boulardii in parboiled rice effluent supplemented with 1% sucrose may represent a viable method by which the environmental impact of this effluent can be reduced while simultaneously producing probiotic culture for use in animal production.

Ultrasound-guided Liver Biopsies in Children: A Single-center Experience and Risk Factors for Minor Bleeding.

OBJECTIVES: The aim of the study was to analyze the institution's experience in ultrasound-guided liver biopsies performed on children and identify risk factors for complications, following a previous study performed in our institution. METHODS: Retrospective analysis of a consecutive series of ultrasound-guided pediatric liver biopsies, between 2011 and 2016. Demographic and anthropometric data, biopsy indications, international normalized ratio (INR) and platelet count, biopsy technique, complications, and pathologic outcomes were recorded. RESULTS: A total of 228 procedures were performed on 203 children with a median age of 9.25 years (range: 0.08-18.42): 107 girls (47%) and 121 boys (53%). One hundred twenty-seven biopsies were performed on transplanted livers (55.7%) and 101 on native livers (44.3%). There were 27 cases with immediate complications (11.84%), all due to minor bleeding. There were no major complications. Increasing needle passes were shown to be a reliable predictor for bleeding (P = 0.0023), whereas transplanted livers predicted protection against bleeding (P = 0.0007). Age younger than 3 years, bodyweight <16 kg, platelet count <70 g/L and INR >1.25 revealed association with increased bleeding incidence, but no predictive value. CONCLUSION: Ultrasound-guided liver biopsies in pediatric age are a safe procedure with a high diagnostic yield. Increasing the number of needle passes predicts a higher incidence of minor bleeding. Other factors to account for minor bleeding risk may include age younger than 3 years, bodyweight <16 kg, platelet count <70 G/L, and INR >1.25. Transplanted livers present a lower bleeding risk.

Parenteral adjuvant potential of recombinant B subunit of Escherichia coli heat-labile enterotoxin.

BACKGROUND: The B subunit of Escherichia coli heat-labile enterotoxin (LTB) is a potent mucosal immune adjuvant. However, there is little information about LTB's potential as a parenteral adjuvant. OBJECTIVES: We aimed at evaluating and better understanding rLTB's potential as a parenteral adjuvant using the fused R1 repeat of Mycoplasma hyopneumoniae P97 adhesin as an antigen to characterise the humoral immune response induced by this construct and comparing it to that generated when aluminium hydroxide is used as adjuvant instead. METHODS: BALB/c mice were immunised intraperitoneally with either rLTBR1 or recombinant R1 adsorbed onto aluminium hydroxide. The levels of systemic anti-rR1 antibodies (total Ig, IgG1, IgG2a, and IgA) were assessed by enzyme-linked immunosorbent assay (ELISA). The ratio of IgG1 and IgG2a was used to characterise a Th1, Th2, or mixed Th1/Th2 immune response. FINDINGS: Western blot confirmed rR1, either alone or fused to LTB, remained antigenic; anti-cholera toxin ELISA confirmed that LTB retained its activity when expressed in a heterologous system. Mice immunised with the rLTBR1 fusion protein produced approximately twice as much anti-rR1 immunoglobulins as mice vaccinated with rR1 adsorbed onto aluminium hydroxide. Animals vaccinated with either rLTBR1 or rR1 adsorbed onto aluminium hydroxide presented a mixed Th1/Th2 immune response. We speculate this might be a result of rR1 immune modulation rather than adjuvant modulation. Mice immunised with rLTBR1 produced approximately 1.5-fold more serum IgA than animals immunised with rR1 and aluminium hydroxide. MAIN CONCLUSIONS: The results suggest that rLTB is a more powerful parenteral adjuvant than aluminium hydroxide when administered intraperitoneally as it induced higher antibody titres. Therefore, we recommend that rLTB be considered an alternative adjuvant, even if different administration routes are employed.

Protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D.

Botulinum neurotoxin (BoNT) serotypes C and D are responsible for cattle botulism, a fatal paralytic disease that results in great economic losses in livestock production. Vaccination is the main approach to prevent cattle botulism. However, production of commercially available vaccines (toxoids) involves high risk and presents variation of BoNT production between batches. Such limitations can be attenuated by the development of novel nontoxic recombinant vaccines through a simple and reproducible process. The aim of this study was to evaluate the protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Bivalent vaccines containing 200 µg rHCC and rHCD each were formulated in three different ways: (1) purified antigens; (2) recombinant Escherichia coli bacterins; (3) recombinant E. coli cell lysates (supernatant and inclusion bodies). Guinea pigs immunized subcutaneously with recombinant formulations developed a protective immune response against the respective BoNTs as determined by a mouse neutralization bioassay with pooled sera. Purified recombinant antigens were capable of inducing 13 IU/mL antitoxin C and 21 IU/mL antitoxin D. Similarly, both the recombinant bacterins and the cell lysate formulations were capable of inducing 12 IU/mL antitoxin C and 20 IU/mL antitoxin D. These values are two times as high as compared to values induced by the commercial toxoid used as control, and two to ten times as high as the minimum amount required by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), respectively. Therefore, we used a practical, industry-friendly, and efficient vaccine production process that resulted in formulations capable of inducing protective immune response (neutralizing antitoxins) against botulism serotypes C and D.

Pichia pastoris X-33 has probiotic properties with remarkable antibacterial activity against Salmonella Typhimurium.

Probiotics are live microorganisms which are beneficial for the host when ingested at high enough concentrations. The methylotrophic yeast Pichia pastoris is widely used as heterologous protein production platform. However, its use as probiotic is poorly studied. The objective of this study was to evaluate some probiotic properties of the P. pastoris strain X-33 wild type. The resistance to in vitro and in vivo gastrointestinal conditions, stability in feed, safety, and antibacterial activity against Salmonella Typhimurium were evaluated. The yeast remained viable and persisted at appropriate concentration in the diet for at least 2 months, survived the stresses of the gastrointestinal tract in vitro and in vivo, caused no behavioral changes or lesions when administered to mice, inhibited the growth of S. Typhimurium in culture media, and reduced adhesion of the bacteria to the intestinal cells HCT-116. In the challenge experiment with a LD50 of virulent S. Typhimurium strain, mice supplemented with the yeast had a higher survival rate (50 % when administered by gavage and 80 % via the diet, compared with 20 and 50 %, respectively, in the control group). In addition, the S. Typhimurium concentration in the intestine of the surviving mice was lower; the score of intestinal lesions, lower; and the pathogen, not detected in the liver, spleen, and feces when compared to the control group (p < 0.05). It was concluded that the yeast Pichia pastoris X-33 has probiotic properties with remarkable antibacterial activity against S. Typhimurium.

Potentially functional lactose-free ice cream with Lacticaseibacillus casei CSL3, ginger, and honey.

AIMS: To develop and characterize a functional lactose-free ice cream with added ginger and honey, evaluate the survival of Lacticaseibacillus casei CSL3 under frozen storage and the simulated gastrointestinal tract (GIT), as well as antioxidant activity and product acceptability. METHODS AND RESULTS: The survival of Lacticaseibacillus casei CSL3 was evaluated for 180 days, under frozen storage, and GIT at 60 days. At 15 days of storage, proximal composition, antioxidant activity, color, pH, acidity, fusion, density, overrun, and sensory analysis were performed. Ice cream was an effective food matrix for maintaining the viability of CSL3, with concentrations > 7 log CFU g- 1 during storage and GIT. In addition, the analysis showed overrun and prebiotic characteristics through high values of antioxidant activity and phenolic compounds, good acceptability, and purchase intention. CONCLUSIONS: The product has satisfactory market potential (acceptance rate of 95.19% and purchase intention rate > 96%), and it could become another means of inserting probiotics in food.

Highly specific fiber optic immunosensor coupled with immunomagnetic separation for detection of low levels of Listeria monocytogenes and L. ivanovii.

BACKGROUND: Immunomagnetic separation (IMS) and immunoassays are widely used for pathogen detection. However, novel technology platforms with highly selective antibodies are essential to improve detection sensitivity, specificity and performance. In this study, monoclonal antibodies (MAbs) against Internalin A (InlA) and p30 were generated and used on paramagnetic beads of varying diameters for concentration, as well as on fiber-optic sensor for detection. RESULTS: Anti-InlA MAb-2D12 (IgG2a subclass) was specific for Listeria monocytogenes and L. ivanovii, and p30-specific MAb-3F8 (IgM) was specific for the genus Listeria. At all bacterial concentrations (10³-108 CFU/mL) tested in the IMS assay; the 1-µm diameter MyOne beads had significantly higher capture efficiency (P < 0.05) than the 2.8-µm diameter M-280 beads with both antibodies. The highest capture efficiency for MyOne-2D12 (49.2% for 105 CFU/mL) was significantly higher (P < 0.05) than that of MyOne-3F8 (16.6 %) and Dynabeads anti-Listeria antibody (9 %). Furthermore, capture efficiency for MyOne-2D12 was highly specific for L. monocytogenes and L. ivanovii. Subsequently, we captured L. monocytogenes by MyOne-2D12 and MyOne-3F8 from hotdogs inoculated with mono- or co-cultures of L. monocytogenes and L. innocua (10-40 CFU/g), enriched for 18 h and detected by fiber-optic sensor and confirmed by plating, light-scattering, and qPCR assays. The detection limit for L. monocytogenes and L. ivanovii by the fiber-optic immunosensor was 3 × 10² CFU/mL using MAb-2D12 as capture and reporter antibody. Selective media plating, light-scattering, and qPCR assays confirmed the IMS and fiber-optic results. CONCLUSIONS: IMS coupled with a fiber-optic sensor using anti-InlA MAb is highly specific for L. monocytogenes and L. ivanovii and enabled detection of these pathogens at low levels from buffer or food.

Production and characterization of Clostridium perfringens recombinant ß toxoid.

In this work, we produced and evaluated a vaccine based on a ß toxoid of Clostridium perfringens type C produced in Escherichia coli (rBT). The non-toxic rBT was innocuous for mice and induced 14 IU mL(-1) of ß antitoxin in rabbits, complying with the European Pharmacopeia and CFR9 - USDA guidelines.

Phage Display-Derived Monoclonal Antibodies Against Internalins A and B Allow Specific Detection of Listeria monocytogenes.

Listeria monocytogenes is the causative agent of listeriosis, a highly lethal disease initiated after the ingestion of Listeria-contaminated food. This species comprises different serovars, from which 4b, 1/2a, and 1/2b cause most of the infections. Among the different proteins involved in pathogenesis, the internalins A (InlA) and B (InlB) are the best characterized, since they play a major role in the enterocyte entry of Listeria cells during early infection. Due to their covalent attachment to the cell wall and location on the bacterial surface, along with their exclusive presence in the pathogenic L. monocytogenes, these proteins are also used as detection targets for this species. Even though huge advancements were achieved in the enrichment steps for subsequent Listeria detection, few studies have focused on the improvement of the antibodies for immunodetection. In the present study, recombinant InlA and InlB produced in Escherichia coli were used as targets to generate antibodies via phage display using the human naïve antibody libraries HAL9 and HAL10. A set of five recombinant antibodies (four against InlA, and one against InlB) were produced in scFv-Fc format and tested in indirect ELISA against a panel of 19 Listeria strains (17 species; including the three main serovars of L. monocytogenes) and 16 non-Listeria species. All five antibodies were able to recognize L. monocytogenes with 100% sensitivity (CI 29.24-100.0) and specificity (CI 88.78-100.0) in all three analyzed antibody concentrations. These findings show that phage display-derived antibodies can improve the biological tools to develop better immunodiagnostics for L. monocytogenes.

Regular Voluntary Running Inhibits Androgen-Independent Prostate Cancer Growth in Mice.

INTRODUCTION: Benefits of regular physical exercise were demonstrated as preventive and coadjuvant nonpharmacological anticancer therapy. However, the role of exercise in modulating prostate cancer behavior has yet to be established. METHODS: Prostate tumors were induced in C57BL/6 male mice (n = 28) by subcutaneous inoculation of a suspension of murine androgen-independent RM1 cells (1.5 × 105 cells/500 µL phosphate-buffered saline) in the dorsal region. Mice were randomly allocated into 2 study groups: sedentary tumor-induced (n = 14) and exercised tumor-induced (n = 14). Exercise consisted of voluntary running in wheeled cages. Mice (n = 7 per group) were sacrificed either 14 or 28 days after cell inoculation to evaluate tumor weight and percentage of area occupied by immunohistochemistry stained cells for Ki-67 and TdT-mediated dUTP-biotin nick end labeling, used as surrogate markers of cell proliferation and apoptosis, respectively. RESULTS: Compared with sedentary tumor-induced mice, the tumors developed by exercised tumor-induced mice were significantly smaller at 14 days (0.17 [0.12] g vs 0.48 [0.24] g, P < .05) and at 28 days (0.92 [0.73] g vs 2.09 [1.31] g, P < .05), with smaller Ki-67 and greater TdT-mediated dUTP-biotin nick end-labeling stained areas (P < .05). CONCLUSION: These results suggest that regular voluntary running inhibits prostate cancer cell growth by reducing cell proliferation and enhancing apoptosis.

Pyruvate dehydrogenase complex-enzyme 2, a new target for Listeria spp. detection identified using combined phage display technologies.

The genus Listeria comprises ubiquitous bacteria, commonly present in foods and food production facilities. In this study, three different phage display technologies were employed to discover targets, and to generate and characterize novel antibodies against Listeria: antibody display for biomarker discovery and antibody generation; ORFeome display for target identification; and single-gene display for epitope characterization. With this approach, pyruvate dehydrogenase complex-enzyme 2 (PDC-E2) was defined as a new detection target for Listeria, as confirmed by immunomagnetic separation-mass spectrometry (IMS-MS). Immunoblot and fluorescence microscopy showed that this protein is accessible on the bacterial cell surface of living cells. Recombinant PDC-E2 was produced in E. coli and used to generate 16 additional antibodies. The resulting set of 20 monoclonal scFv-Fc was tested in indirect ELISA against 17 Listeria and 16 non-Listeria species. Two of them provided 100% sensitivity (CI 82.35-100.0%) and specificity (CI 78.20-100.0%), confirming PDC-E2 as a suitable target for the detection of Listeria. The binding region of 18 of these antibodies was analyzed, revealing that ≈ 90% (16/18) bind to the lipoyl domains (LD) of the target. The novel target PDC-E2 and highly specific antibodies against it offer new opportunities to improve the detection of Listeria.

Protective efficacy of recombinant bacterin vaccine against botulism in cattle.

Botulism is a paralytic disease caused by the intoxication of neurotoxins produced by Clostridium botulinum. Among the seven immunologically distinct serotypes of neurotoxins (BoNTs A - G), serotypes C and D, or a chimeric fusion termed C/D or D/C, are responsible for animal botulism. The most effective way to prevent botulism in cattle is through vaccination; however, the commercially available vaccines produced by detoxification of native neurotoxins are time-consuming and hazardous. To overcome these drawbacks, a non-toxic recombinant vaccine was developed as an alternative. In this study, the recombinant protein vaccine was produced using an Escherichia coli cell-based system. The formaldehyde-inactivated E. coli is able to induce 7.45 ± 1.77 and 6.6 ± 1.28 IU/mL neutralizing mean titers against BoNTs C and D in cattle, respectively, determined by mouse neutralization bioassay, and was deemed protective by the Brazilian legislation. Moreover, when the levels of anti-BoNT/C and D were compared with those achieved by the recombinant purified vaccines, no significant statistical difference was observed. Cattle vaccinated with the commercial vaccine developed 1.33 and 3.33 IU/mL neutralizing mean titers against BoNT serotypes C and D, respectively. To the best of our knowledge, this study is the first report on recombinant E. coli bacterin vaccine against botulism. The vaccine was safe and effective in generating protective antibodies and, thus, represents an industry-friendly alternative for the prevention of cattle botulism.