Production and purification of Clostridium perfringens type C beta-toxin and IgG and IgY antitoxins.

OBJECTIVES: This study aimed to produce and purify Clostridium perfringens type C beta-toxin, sheep anti-beta toxin immunoglobulin G (IgG) and chicken immunoglobulin Y (IgY). METHODS: Two methods were used for beta-toxin purification: single-step metal affinity chromatography (MAC) using zinc as a chelator and ion exchange chromatography (IEX). The purified and inactivated beta-toxoids were then administered to sheep and chickens in order to produce IgG and IgY. RESULTS: All assays using the IEX failed. In contrast, MAC purified more than 21 mg of toxin per run in a single-step protocol. The purified and inactivated beta-toxoids were then administered to sheep and chickens, and IgG and IgY were purified with a high yield, medium antibody titer of 50 IU/mL, and high avidity (73.2 %). CONCLUSIONS: C. perfringens type C beta-toxin and sheep or chicken anti-beta toxin IgG and IgY antibodies were successfully produced and purified using a simple protocol. This protocol can be used for the production of components used in the diagnosis and research of necrotic enteritis caused by C. perfringens type C, as well as for the evaluation of existing vaccines and the development of new preventive methods against this disease.

The membrane-binding bacterial toxin long direct repeat D inhibits protein translation.

Bacterial plasmids and chromosomes widely contain toxin-antitoxin (TA) loci, which are implicated in stress response, growth regulation and even tolerance to antibiotics and environmental stress. Type I TA systems consist of a stable toxin-expressing mRNA, which is counteracted by an unstable RNA antitoxin. The Long Direct Repeat (LDR-) D locus, a type I TA system of Escherichia Coli (E. coli) K12, encodes a 35 amino acid toxic peptide, LdrD. Despite being characterized as a bacterial toxin, causing rapid killing and nucleoid condensation, little was known about its function and its mechanism of toxicity. Here, we show that LdrD specifically interacts with ribosomes which potentially blocks translation. Indeed, in vitro translation of LdrD-coding mRNA greatly reduces translation efficiency. The structure of LdrD in a hydrophobic environment, similar to the one found in the interior of ribosomes was determined by NMR spectroscopy in 100% trifluoroethanol solution. A single compact α-helix was found which would fit nicely into the ribosomal exit tunnel. Therefore, we conclude that rather than destroying bacterial membranes, LdrD exerts its toxic activity by inhibiting protein synthesis through binding to the ribosomes.

Evaluation of toxin-antitoxin genes, antibiotic resistance, and virulence genes in Pseudomonas aeruginosa isolates.

OBJECTIVE: Toxin-antitoxin genes RelBE and HigBA are known to be involved in the formation of biofilm, which is an important virulence factor for Pseudomonas aeruginosa. The purpose of this study was to determine the presence of toxin-antitoxin genes and exoenzyme S and exotoxin A virulence genes in P. aeruginosa isolates and whether there is a relationship between toxin-antitoxin genes and virulence genes as well as antibiotic resistance. METHODS: Identification of the isolates and antibiotic susceptibilities was determined by a VITEK 2 (bioMérieux, France) automated system. The presence of toxin-antitoxin genes, virulence genes, and transcription levels were detected by real-time polymerase chain reaction. RESULTS: RelBE and HigBA genes were detected in 94.3% (82/87) of P. aeruginosa isolates, and exoenzyme S and exotoxin A genes were detected in all of the isolates (n=87). All of the isolates that harbor the toxin-antitoxin and virulence genes were transcribed. There was a significant increase in the RelBE gene transcription level in imipenem- and meropenem-sensitive isolates and in the HigBA gene transcription level in amikacin-sensitive isolates (p<0.05). There was a significant correlation between RelBE and exoenzyme S (p=0.001). CONCLUSION: The findings suggest that antibiotic resistance may be linked to toxin-antitoxin genes. Furthermore, the relationship between RelBE and exoenzyme S indicates that toxin-antitoxin genes in P. aeruginosa isolates are not only related to antibiotic resistance but also play an influential role in bacterial virulence. Larger collections of comprehensive studies on this subject are required. These studies should contribute significantly to the solution of the antibiotic resistance problem.

Role of PumB antitoxin as a transcriptional regulator of the PumAB type-II toxin-antitoxin system and its endoribonuclease activity on the PumA (toxin) transcript.

The PumAB type-II toxin-antitoxin (TA) system is encoded by pumAB genes that are organized into an operon. This system is encoded by the pUM505 plasmid, isolated from a Pseudomonas aeruginosa clinical strain. The pumA gene encodes a putative RelE toxin protein (toxic component), whereas the pumB gene encodes a putative HTH antitoxin protein. The expression of the PumAB system in Escherichia coli confers plasmid stability. In addition, PumA toxin overexpression in P. aeruginosa possesses the capability to increase bacterial virulence, an effect that is neutralized by the PumB antitoxin. The aim of this study was to establish the mechanism of regulation of the PumAB toxin-antitoxin system from pUM505. By an in silico analysis of the putative regulatory elements, we identified two putative internal promoters, PpumB and PpumB-AlgU (in addition to the already reported PpumAB), located upstream of pumB. By RT-qPCR assays, we determined that the pumAB genes are transcribed differentially, in that the mRNA of pumB is more abundant than the pumA transcript. We also observed that pumB could be expressed individually and that its mRNA levels decreased under oxidative stress, during individual expression as well as co-expression of pumAB. However, under stressful conditions, the pumA mRNA levels were not affected. This suggests the negative regulation of pumB by stressful conditions. The PumB purified protein was found to bind to a DNA region located between the PpumAB and the pumA coding region, and PumA participates in PumB binding, suggesting that a PumA-PumB complex co-regulates the transcription of the pumAB operon. Interestingly, the pumA mRNA levels decreased after incubation in vitro with PumB protein. This effect was repressed by ribonuclease inhibitors, suggesting that PumB could function as an RNAse toward the mRNA of the toxin. Taken together, we conclude that the PumAB TA system possesses multiple mechanisms to regulate its expression, as well as that the PumB antitoxin generates a decrease in the mRNA toxin levels, suggesting an RNase function. Our analysis provides new insights into the understanding of the control of TA systems from mobile plasmid-encoded genes from a human pathogen.

Botulismo alimentar: relato de caso de uma doença rara, grave e de relevância para a saúde pública / Foodborne botulism: case report of a rare, serious disease of public health relevance

Este resumo apresenta o relato de um caso de Botulismo ocorrido em maio de 2021 no Hospital do Servidor Público Municipal (HSPM) em São Paulo. O objetivo principal é sensibilizar os profissionais de saúde para considerar o Botulismo como uma possível causa de síndrome neuroparalítica aguda, apesar de sua raridade. Destaca-se a importância do diagnóstico precoce e do tratamento com antitoxina, juntamente com cuidados intensivos, para reduzir a mortalidade. O estudo é observacional e descritivo, relatando o caso de uma paciente hospitalizada com Botulismo de origem alimentar entre maio e agosto de 2021. Diante de uma síndrome neuroparalítica aguda, a suspeita de Botulismo e uma revisão da epidemiologia da doença são cruciais. Destaca-se a importância da antitoxina e dos cuidados intensivos no tratamento para reduzir a mortalidade. Complicações pós-infecção, como sequelas motoras, são comuns em pacientes de Botulismo, tornando essencial uma abordagem multidisciplinar de reabilitação física para uma recuperação eficaz. A Vigilância Epidemiológica e Sanitária desempenham um papel vital na prevenção e controle do Botulismo, incluindo a coleta e transporte oportunos de amostras, busca ativa de casos suspeitos e orientação à população sobre medidas preventivas. A qualidade dos dados notificados é fundamental para a eficácia dessas ações. Em vista da alta letalidade do Botulismo, destaca-se a importância de alertar os profissionais de saúde para identificar casos suspeitos, bem como treiná-los na integração com as unidades de Vigilância Sanitária e Epidemiológica. Isso permite uma identificação precoce e tratamento oportuno de casos suspeitos, contribuindo para a saúde pública. Palavras-chave: Botulismo. Neurotoxina botulínica. Intoxicação alimentar.

Ex vivo and in vitro poultry intestinal models to evaluate antimycotoxins additives / Uso de modelos ex vivo e in vitro de intestino de aves para avaliar aditivos antimicotoxinas

In vitro tests are performed to evaluate the efficacy of antimycotoxins additives (AMAs); nevertheless, such assays show a low correlation with in vivo trials, which are also required to determine AMAs' efficacy. In search of an alternative method, the current study investigated the use of an ex vivo technique. Six AMAs (AMA1 to AMA6) had their ability to reduce intestinal absorption of aflatoxin B1 (AFB1) evaluated. Jejunal explants were obtained from broilers and subjected to two treatments per AMA in Ussing chambers: T1 (control) - 2.8 mg/L AFB1, and T2 - 2.8 mg/L AFB1 + 0.5% AMA. AMAs were also tested in vitro to assess adsorption of AFB1 in artificial intestinal fluid. In the ex vivo studies, AMA1 to AMA6 decreased intestinal absorption of AFB1 by 67.11%, 73.82%, 80.70%, 85.86%, 86.28% and 82.32%, respectively. As for the in vitro results, AMA1 to AMA6 presented an adsorption of 99.72%, 99.37%, 99.67%, 99.53%, 99.04% and 99.15%, respectively. The evaluated ex vivo model proved useful in the assessment of AMAs. No correlation was reported between ex vivo and in vitro findings. Further studies are needed to elucidate the correlation between ex vivo and in vivo results seeking to reduce animal testing.

Testes in vitro são realizados para avaliar a eficácia de aditivos antimicotoxinas (AAMs); entretanto, tais experimentos apresentam uma baixa correlação com ensaios in vivo, que também são exigidos para determinar a eficácia de AAMs. Em busca de um método alternativo, o presente estudo investigou o uso de uma técnica ex vivo. A capacidade de seis AAMs (AAM1 a AAM6) de reduzir a absorção intestinal de aflatoxina B1 (AFB1) foi avaliada. Explantes jejunais foram coletados de frangos de corte e submetidos a dois tratamentos por AAM em câmaras de Ussing: T1 (controle) - 2,8 mg/L AFB1, e T2 - 2.8 mg/L AFB1 + 0,5% AAM. Os AAMs também foram testados in vitro para verificar a adsorção de AFB1 em fluido intestinal artificial. Nos ensaios ex vivo, AAM1 ao AAM6 diminuíram a absorção intestinal de AFB1 em 67,11%, 73,82%, 80,70%, 85,86%, 86,28% e 82,32%, respectivamente. Quanto aos achados in vitro, AAM1 ao AAM6 apresentaram adsorção de 99,72%, 99,37%, 99,67%, 99,53%, 99,04% e 99,15%, respectivamente. O modelo ex vivo avaliado mostrou-se eficiente na avaliação de AAMs. Não houve correlação entre os resultados ex vivo e in vitro. Estudos adicionais são necessários para definir a correlação entre achados ex vivo e in vivo na tentativa de reduzir os testes em animais.

Zebrafish embryo sensitivity test as in vivo platform to anti-Shiga toxin compound screening.

Shiga toxin-producing Escherichia coli (STEC) pathotype secretes two types of AB5 cytotoxins (Stx1 and Stx2), responsible for complications such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in infected patients, which could lead to sequels and death. Currently, there is no effective treatment against the cytotoxic effect of these toxins. However, in order to approve any therapy molecule, an animal experiment is required in order to evaluate the efficacy and safety of therapeutic approaches. The use of alternative small host models is growing among human infectious disease studies, particularly the vertebrate zebrafish model, since relevant results have been described for pathogen-host interaction. In this sense, the present work aimed to analyze the toxic effect of Shiga toxins in zebrafish embryo model in order to standardize this method in the future to be used as a fast, simple, and efficient methodology for the screening of therapeutic molecules. Herein, we demonstrated that the embryos were sensitive in a dose-dependent manner to both Stx toxins, with LD50 of 22 µg/mL for Stx1 and 33 µg/mL for Stx2, and the use of anti-Stx polyclonal antibody abolished the toxic effect. Therefore, this methodology can be a rapid alternative method for selecting promising compounds against Stx toxins, such as recombinant antibodies.

Large-Scale Comparison of Toxin and Antitoxins in Listeria monocytogenes.

Toxin-antitoxin systems (TASs) are widely distributed in prokaryotes and encode pairs of genes involved in many bacterial biological processes and mechanisms, including pathogenesis. The TASs have not been extensively studied in Listeria monocytogenes (Lm), a pathogenic bacterium of the Firmicutes phylum causing infections in animals and humans. Using our recently published TASmania database, we focused on the known and new putative TASs in 352 Listeria monocytogenes genomes and identified the putative core gene TASs (cgTASs) with the Pasteur BIGSdb-Lm database and, by complementarity, the putative accessory gene TAS (acTASs). We combined the cgTASs with those of an additional 227 L. monocytogenes isolates from our previous studies containing metadata information. We discovered that the differences in 14 cgTAS alleles are sufficient to separate the four main lineages of Listeria monocytogenes. Analyzing these differences in more details, we uncovered potentially co-evolving residues in some pairs of proteins in cgTASs, probably essential for protein-protein interactions within the TAS complex.

The ecnA Antitoxin Is Important Not Only for Human Pathogens: Evidence of Its Role in the Plant Pathogen Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri causes citrus canker disease worldwide in most commercial varieties of citrus. Its transmission occurs mainly by wind-driven rain. Once X. citri reaches a leaf, it can epiphytically survive by forming a biofilm, which enhances the persistence of the bacteria under different environmental stresses and plays an important role in the early stages of host infection. Therefore, the study of genes involved in biofilm formation has been an important step toward understanding the bacterial strategy for survival in and infection of host plants. In this work, we show that the ecnAB toxin-antitoxin (TA) system, which was previously identified only in human bacterial pathogens, is conserved in many Xanthomonas spp. We further show that in X. citri, ecnA is involved in important processes, such as biofilm formation, exopolysaccharide (EPS) production, and motility. In addition, we show that ecnA plays a role in X. citri survival and virulence in host plants. Thus, this mechanism represents an important bacterial strategy for survival under stress conditions.IMPORTANCE Very little is known about TA systems in phytopathogenic bacteria. ecnAB, in particular, has only been studied in bacterial human pathogens. Here, we showed that it is present in a wide range of Xanthomonas sp. phytopathogens; moreover, this is the first work to investigate the functional role of this TA system in Xanthomonas citri biology, suggesting an important new role in adaptation and survival with implications for bacterial pathogenicity.

Humoral responses in cattle to commercial vaccines containing Clostridium perfringens epsilon toxoid and C. botulinum types C and D toxoids last less than a-year.

The aim of this study was to evaluate the titers of neutralizing antibodies in cattle inoculated with multivalent commercial clostridial vaccines containing C. botulinum type C (BoNTC), C. botulinum type D (BoNTD), and C. perfringens epsilon (ETX) toxoids for a period of one year. Cattle (Bos taurus), aged 4-6 months and not previously immunized, were vaccinated under four different protocols at days 0 and 30 and followed over one year. Individual serum titration was performed by a serum neutralization test in mice or in MDCK cells. The number of animals with detectable neutralizing antibodies ranged from 40.6% to 78.1%, but only 12.5% of animals showed neutralizing antibodies against all tested antigens. Neutralizing antibodies were found only until 60 days for ETX, 120 days for BoNTC, and 180 days for BoNTD. The absence of detectable neutralizing antibodies against the three antigens before 360 days, suggests that cattle remained unprotected for a long period before the recommended booster vaccination.

Characterization of an OrtT-like toxin of Salmonella enterica serovar Houten.

The Escherichia coli GhoT/GhoS system is a type V toxin-antitoxin system in which the antitoxin GhoS cleaves the GhoT mRNA, controlling its translation. GhoT is a small hydrophobic protein that damages bacterial membranes. OrtT is a GhoT-like toxin, but it apparently lacks a corresponding antitoxin and serves a different physiologic role. Using a profile hidden Markov model approach, a Salmonella enterica serovar Houten genome was screened to obtain homologs of GhoT/OrtT. We only found one protein (referred to here as OrtT-Sal) that shared more sequence identity with OrtT than GhoT. The chromosomal region around the coding sequence of OrtT-Sal suggests that it is an orphan toxin and can be under RpoH activation. To study OrtT-Sal, we chemically synthesized and expressed in E. coli the whole toxin and its N- and C-terminal regions (OrtT-Sal1-29 and OrtT-Sal29-57, respectively). Our findings have shown that the overproduction of the polypeptides resulted in severe growth inhibition and cell lysis. Using circular dichroism, we found that OrtT-Sal, OrtT-Sal1-29, and OrtT-Sal29-57 form an alpha-helical structure in the presence of SDS micelles or TFE. Finally, using carboxyfluorescein-loaded lipid vesicles, we determined that the polypeptides damage lipid membrane directly.

Minerais séricos, características morfométricas ósseas e deposição de minerais ósseos de frangos de corte alimentados com dieta com inclusão de bentonita / Serum minerals, bone morphometric characteristics and deposition of bone minerals from broilers fed diet containing bentonite

O objetivo deste trabalho foi avaliar a concentração sérica de cálcio, cloretos, ferro, fósforo e magnésio, as características morfométricas ósseas e a deposição de cálcio e fósforo nas tíbias de frangos de corte recebendo dieta com zero, 0,25 ou 0,50% de bentonita. Um ensaio foi conduzido com 288 frangos de corte de 14 a 21 dias de idade, submetidos a três dietas experimentais: sem inclusão (0,0); com inclusão de 0,25 e com inclusão de 0,50% do adsorvente bentonita. Não foram observadas diferenças (P>0,05) no desempenho das aves, nos níveis séricos de cálcio, cloretos, ferro e magnésio, no entanto os níveis de fósforo foram reduzidos (P<0,05) nas aves que ingeriram dieta com 0,50% de bentonita. Em relação às tíbias, observou-se redução (P<0,05) na matéria mineral (g e %) e no teor de cálcio com a inclusão de 0,50% de bentonita. Houve redução (P<0,05) nos níveis de fósforo das tíbias com a inclusão de 0,25 e 0,50% de bentonita. Conclui-se que a inclusão de até 0,50% do adsorvente de micotoxinas bentonita na dieta de frangos de corte não altera o desempenho zootécnico das aves. A inclusão de 0,25% de bentonita, na dieta de frangos de corte, não altera a concentração dos minerais séricos e a deposição de minerais nas tíbias, entretanto a inclusão de 0,5% reduz os níveis séricos de fósforo, o teor de matéria mineral e a concentração de cálcio e fósforo ósseos, sem afetar as características morfométricas ósseas.(AU)

The aim of this study was to evaluate performance, serum concentration of calcium, chloride, iron, magnesium, phosphorus, and bone characteristics, ash, calcium, and phosphorus in tibias of broilers receiving diet with zero, 0.25 or 0.50% of bentonite. No differences were found on performance of poultry, on serum mineral calcium, chloride, iron, magnesium, however phosphorus levels of broilers fed on diets containing 0.5% bentonite was reduced. With respect to tibia, reduction was observed on mineral matter (g and %) and calcium levels with inclusion of 0.50% bentonite, and reduction on phosphorus levels with inclusion of 0.25 or 0.50% of bentonite on diet. We conclude that the inclusion of up to 0.50% of mycotoxin adsorbent bentonite in diet of broiler does not change broiler performance. The inclusion of 0.25% of bentonite in diet of broiler does not change serum mineral concentration and mineral deposition; however, the inclusion of 0.5% decrease serum levels of phosphorus, the content of bone mineral matter, with not effects on bone morphometric characteristics.(AU)

Minerais séricos, características morfométricas ósseas e deposição de minerais ósseos de frangos de corte alimentados com dieta com inclusão de bentonita / Serum minerals, bone morphometric characteristics and deposition of bone minerals from broilers fed diet containing bentonite

O objetivo deste trabalho foi avaliar a concentração sérica de cálcio, cloretos, ferro, fósforo e magnésio, as características morfométricas ósseas e a deposição de cálcio e fósforo nas tíbias de frangos de corte recebendo dieta com zero, 0,25 ou 0,50% de bentonita. Um ensaio foi conduzido com 288 frangos de corte de 14 a 21 dias de idade, submetidos a três dietas experimentais: sem inclusão (0,0); com inclusão de 0,25 e com inclusão de 0,50% do adsorvente bentonita. Não foram observadas diferenças (P>0,05) no desempenho das aves, nos níveis séricos de cálcio, cloretos, ferro e magnésio, no entanto os níveis de fósforo foram reduzidos (P<0,05) nas aves que ingeriram dieta com 0,50% de bentonita. Em relação às tíbias, observou-se redução (P<0,05) na matéria mineral (g e %) e no teor de cálcio com a inclusão de 0,50% de bentonita. Houve redução (P<0,05) nos níveis de fósforo das tíbias com a inclusão de 0,25 e 0,50% de bentonita. Conclui-se que a inclusão de até 0,50% do adsorvente de micotoxinas bentonita na dieta de frangos de corte não altera o desempenho zootécnico das aves. A inclusão de 0,25% de bentonita, na dieta de frangos de corte, não altera a concentração dos minerais séricos e a deposição de minerais nas tíbias, entretanto a inclusão de 0,5% reduz os níveis séricos de fósforo, o teor de matéria mineral e a concentração de cálcio e fósforo ósseos, sem afetar as características morfométricas ósseas.(AU)

The aim of this study was to evaluate performance, serum concentration of calcium, chloride, iron, magnesium, phosphorus, and bone characteristics, ash, calcium, and phosphorus in tibias of broilers receiving diet with zero, 0.25 or 0.50% of bentonite. No differences were found on performance of poultry, on serum mineral calcium, chloride, iron, magnesium, however phosphorus levels of broilers fed on diets containing 0.5% bentonite was reduced. With respect to tibia, reduction was observed on mineral matter (g and %) and calcium levels with inclusion of 0.50% bentonite, and reduction on phosphorus levels with inclusion of 0.25 or 0.50% of bentonite on diet. We conclude that the inclusion of up to 0.50% of mycotoxin adsorbent bentonite in diet of broiler does not change broiler performance. The inclusion of 0.25% of bentonite in diet of broiler does not change serum mineral concentration and mineral deposition; however, the inclusion of 0.5% decrease serum levels of phosphorus, the content of bone mineral matter, with not effects on bone morphometric characteristics.(AU)

Determining the Relative Binding Affinity of Ricin Toxin A Inhibitors by Using Molecular Docking and Nonequilibrium Work.

Ricin is a ribosome-inactivating protein (RIP type 2) consisting of two subunits, ricin toxin A (RTA) and ricin toxin B (RTB). Because of its cytotoxicity, ricin has worried world authorities for its potential use as a chemical weapon; therefore, its inhibition is of great biotechnological interest. RTA is the target for inhibitor synthesis, and pterin derivatives are promising candidates to inhibit it. In this study, we used a combination of the molecular docking approach and fast steered molecular dynamics (SMD) to assess the correlation between nonequilibrium work, ⟨ W⟩, and the IC50 for six RTA inhibitors. The results showed that molecular docking is a powerful tool to predict good bioactive poses of RTA inhibitors, and ⟨ W⟩ presented a strong correlation with IC50 ( R2 = 0.961). Such a profile ranked the RTA inhibitors better than the molecular docking approach. Therefore, the combination of docking and fast SMD simulation was shown to be a promising tool to distinguish RTA-active inhibitors from inactive ones and could be used as postdocking filtering approach.

Type C botulism in domestic chickens, dogs and black-pencilled marmoset (Callithrix penicillata) in Minas Gerais, Brazil.

Botulism is a well-known intoxication that affects humans and animals. The disease is endemic in cattle in Brazil and recently emerged as an important disease in commercial laying hens and broiler chickens in Europe. Dogs and other animal species can also be affected. Although antitoxins are commonly administered to humans diagnosed with botulism, in animals this is rarely the case and the treatment of botulism is still based only on support therapy. In the present work, we report an outbreak of type C botulism in Brazil that simultaneously affected domestic chickens, dogs and a black-pencilled marmoset (Callithrix penicillata). The successful use of Clostridium botulinum types C and D antitoxin for the treatment of an affected dog is also described.

Diseño de un programa de análisis de peligros y puntos críticos control (HACCP) para la producción de toxoide tetánico / Design of a program of hazard analysis and critical control point (HACCP) for production of Tetanus Toxoid

El toxoide tetánico es una neurotoxina modificada que induce la formación de una antitoxina protectora contra la enfermedad denominada tétanos. Este antígeno es obtenido a partir de procesos fermentativos con la bacteria anaerobia Clostridium tetani y es utilizado para la formulación de vacunas simples y combinadas inactivadas. Con el propósito de atender a las recomendaciones y regulaciones de la Organización Mundial de la Salud (OMS), el objetivo de este trabajo fue diseñar un Programa de Análisis de Peligros y Puntos Críticos de Control (HACCP) en la producción del antígeno Toxoide Tetánico, desde la recepción de la cepa certificada en el área de producción hasta el almacenamiento del toxoide tetánico purificado. Para ello, inicialmente se evaluó el cumplimiento de los prerequisitos (BPM, POES, BPL). Posteriormente, se procedió al diseño del plan HACCP mediante la ejecución de las 5 tareas preliminares y la aplicación de los 7 principios, conforme a la metodología descrita por la OMS. A partir del análisis de peligros en todas las etapas del proceso de producción del toxoide tetánico se identificaron 3 puntos críticos de control: detoxificación, filtración estéril final y almacenamiento de toxoide tetánico purificado. Se establecieron los límites críticos, los procedimientos de vigilancia, las acciones correctivas, los procedimientos de verificación y de documentación. La propuesta tiene como fin garantizar la calidad e inocuidad del producto elaborado, la protección del personal involucrado en el proceso y del medio ambiente con miras a la obtención de la certificación como laboratorio productor de vacunas

Tetanus toxoid is a modified neurotoxin that induces the formation of protective antitoxin of the disease called tetanus. This antigen is obtained from fermentation processes with anaerobic bacteria Clostridium tetani and it is used to formulate simple and combined inactivated vaccines. In order to meet the recommendations and regulations of World Health Organization (WHO), the aim of this work was to design a Program of Hazard Analysis and Critical Control Points (HACCP) in the production of antigen Tetanus Toxoid, starting from the receipt of the certified strain in the production area through the storage of purified tetanus toxoid. For this, initially fulfilling the prerequisites (GMP, SSOP and GLP) was evaluated. Subsequently, we proceeded to design HACCP plan by running 5 preliminary tasks and application of the 7 principles, according to the methodology described by WHO. From the hazard analysis at all stages of the production process of tetanus toxoid three critical control points were identified: detoxification, final sterile filtration and storage of purified tetanus toxoid. Critical limits, monitoring procedures, corrective actions, verification and documentation procedures were established. The proposal aims to assure the quality and safety of the final product, the protection of personnel involved in the process and the environment, with a view to obtaining certification as a vaccine production laboratory

Quality of horse F(ab)2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p 0.05. Results Horse F(ab)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates...(AU)

Quality of horse F(ab)2 antitoxins and antirabies immunoglobulins: protein content and anticomplementary activity

Background Among other applications, immunotherapy is used for the post-exposure treatment and/or prophylaxis of important infectious diseases, such as botulism, diphtheria, tetanus and rabies. The effectiveness of serum therapy is widely proven, but improvements on the immunoglobulin purification process and on the quality control are necessary to reduce the amount of protein aggregates. These may trigger adverse reactions in patients by activating the complement system and inducing the generation of anaphylatoxins. Herein, we used immunochemical methods to predict the quality of horse F(ab)2 anti-botulinum AB, anti-diphtheric, antitetanic and anti-rabies immunoglobulins, in terms of amount of proteins and protein aggregates. Methods Samples were submitted to protein quantification, SDS-PAGE, Western blot analysis and molecular exclusion chromatography. The anticomplementary activity was determined in vitro by detecting the production of C5a/C5a desArg, the most potent anaphylatoxin. Data were analyzed by one-way ANOVA followed by Tukey's post-test, and differences were considered statistically significant when p 0.05. Results Horse F(ab)2 antitoxins and anti-rabies immunoglobulin preparations presented different amounts of protein. SDS-PAGE and Western blot analyses revealed the presence of protein aggregates, non-immunoglobulin contaminants and, unexpectedly, IgG whole molecules in the samples, indicating the non-complete digestion of immunoglobulins. The chromatographic profiles of antitoxins and anti-rabies immunoglobulins allowed to estimate the percentage of contaminants and aggregates in the samples. Although protein aggregates were present, the samples were not able to induce the generation of C5a/C5a desArg in vitro, indicating that they probably contain acceptable levels of aggregates...

Plasmid pUM505 encodes a Toxin-Antitoxin system conferring plasmid stability and increased Pseudomonas aeruginosa virulence.

Pseudomonas aeruginosa plasmid pUM505 possesses a pathogenicity island that contains the pumAB genes that encode products with sequence similarity to Toxin-Antitoxin (TA) modules. RT-PCR assays on the overlapping regions of the pumAB genes generated a bicistronic messenger RNA, suggesting that they form an operon. When the pumAB genes were cloned into the pJET vector, recombinant plasmid pJET-pumAB was maintained under nonselective conditions in Escherichia coli cells after six daily subcultures, whereas pJET without pumAB genes was lost. These data indicate that pumAB genes confer post-segregational plasmid stability. In addition, overexpression of the PumA protein in the E. coli BL21 strain resulted in a significant growth inhibition, while BL21 co-expressing the PumA and PumB proteins did not show growth inhibition. These results indicate that pumAB genes encode a TA system where the PumB protein counters the toxic effects of the PumA toxin. Furthermore, P. aeruginosa PAO1 transformants with the pumA gene increased Caenorhabditis elegans and mouse mortality rate and improved mouse organ invasion, effects neutralized by the PumB protein. Moreover, purified recombinant His-PumA protein decreased the viability of C. elegans, indicating that the PumA protein could acts as a toxin. These results indicate that PumA has the potential to promoter the PAO1 virulence against C. elegans and mice when is expressed in absence of PumB. This is the first description, to our knowledge, of a plasmid-encoded TA system that confers plasmid stability and encoded a toxin with the possible ability to increase the P. aeruginosa virulence.