Campylobacter coli in Swine Slaughtering Flowchart and Research of cdt Genes

Background: Campylobacter spp. are among the microorganisms most commonly associated with foodborne disease. Campylobacter spp. isolation from pigs during the slaughter and final products have been reported in several countries, including Brazil. However, very little is known about the sources of contamination in the slaughtering flowchart and how these microorganisms are spread in processing plants. Considering the possibility of the pigs carry Campylobacter spp. since the farm or its products are contaminated in the slaughterhouse, this study had as aim to track Campylobacter spp. in pig slaughtering flowchart to understand the behavior of these pathogens in the production line.Materials, Methods & Results: Forty animals of 10 lots, four from each lot, were followed during slaughter. Stool samples were collected from the floor of each enclosure where the pigs were housed on the farm and immediately after stunning on slaughterhouse. Samples from carcass surface were collected after removal of the animals from scrap machine, after evisceration and before the refrigeration chamber. It was also collected surface samples from jowls and samples from the scalding tank water before and after the passage of animals. The swabs containing samples were plated onto Columbia agar supplemented with activated charcoal, oxygen reduction solution and antibiotics supplement, and incubated at 42°C for 48 h under microaerobic conditions. The colonies which presented with a shiny and moist appearance were analyzed by Gram staining for identification of Campylobacter by morphology, and then tested for catalase and oxidase. The Campylobacter isolates were identified for species C. jejuni or C. coli by PCR. Bands profiles were determined by rep-PCR and used to compare the strains. Campylobacter was isolated from 19 (9.5%) of the 200 pig samples analyzed, seven (36.8%) of the rectum, seven (36.8%) after evisceration and five (26.3%) before the refrigeration chamber. Campylobacter was not isolated from jowls and from scalding tank water. All isolates were C. coliand cdtnegative.Persistence of strains originating from the farm and cross contaminations during the slaughtering flowchart was identified by the analysis of the bands profiles obtained by rep-PCR.Discussion: C. coli was the species of Campylobacter present in the swine intestinal tract and in the swine slaughterhouse. The animals, once contaminated, can carry the microorganism during the stages of the slaughtering flowchart. The farm where the animals came from is an important source of contamination during processing, however cross contamination also plays a relevant role. The evisceration was considered the most critical stage, due to the greater number of isolates obtained after this procedure, what emphasize the importance of the hygienic-sanitary management in this stage. Campylobacter spp. can survive, despite not being able to multiply, in foods at refrigeration temperatures (-1 to 5°C) for one to three weeks. Therefore, the high percentage of isolates obtained from the carcass before the refrigeration chamber may represent a problem, since the contamination of the carcasses that enter in this sector can be maintained until the food reaches the consumer. There was no similarity between strains isolated from different lots, indicating that there were no persistence of strains both in the farm and in the slaughterhouse

Isolamento de Leptospira em feto equino abortado/Isolation of Leptospira in an aborted equine fetus

Estudos sorológicos da leptospirose em equinos demonstram que a doença ocorre em todo o mundo. A frequência de aglutininas anti-Leptospira varia entre os países e existe uma grande susceptibilidade dos animais em relação aos sorovares predominantes. A maioria das infecções permanece assintomática, mas o aborto e a morte fetal são sequelas comuns da leptospirose equina. Este relato descreve um aborto em uma égua no município de Pelotas (RS), onde foi possível isolar leptospiras do feto, caracterizar a virulência do isolado em modelo animal e realizar uma caracterização genotípica preliminar da cepa isolada

Avaliação da função renal e hepática em hamsters infectados experimentalmente com Leptospira interrogans / Evaluation of renal and hepatic function in hamsters experimentally infected with Leptospira interrogans

Hamsters (Mesocricetus auratus) são amplamente usados como modelo animal experimental para o estudo da leptospirose. Apesar disso, informações sobre os parâmetros bioquímicos da função renal e hepática em animais menores de 6 semanas, assim como as alterações sofridas nesses níveis durante a infecção por leptospiras patogênicas, são escassas na literatura. Neste trabalho avaliamos os níveis de bilirrubina total (BT), alanina aminotransferase (ALT) e creatinina (CR) antes e depois da infecção por Leptospira interrogans. Em nosso estudo preliminar, descrevemos valores normais para parâmetros não encontrados em bibliografia. Porém, não foi encontrada nenhuma diferença estatística significativa entre os valores de BT, ALT e CR apresentados pelos grupos após a infecção. 

Isolamento e perfil de suscetibilidade a antimicrobianos de isolados de Salmonella obtidos durante o abate de suínos / Isolation and antimicrobial susceptibility pattern of Salmonella isolates recovered from pig slaughter

Este estudo foi realizado com o objetivo de isolar Salmonella em cinco diferentes pontos do fluxograma de abate de suínos, avaliando o padrão de suscetibilidade dos isolados a sete agentes antimicrobianos comumente usados na cadeia suinícola. Das 200 amostras coletadas, em 23 (11,5%) foi possível o isolamento de Salmonella spp. Destes isolados, todos apresentaram resistência a um ou mais dos antimicrobianos testados, sendo que 100% apresentaram resistência à tetraciclina e trimetoprim, 89,95% à ampicilina, 69,56% à gentamicina, 26,08% à norfloxacina, 65,21% à estreptomicina e 17,39% à neomicina. O isolamento de Salmonella durante o abate de suínos demonstra o alto risco de contaminação para os funcionários e consumidores. Medidas para o controle do uso de antimicrobianos na criação de suínos devem ser urgentemente implementadas

Potencial antigênico de proteínas recombinantes de Leptospira Interrogans / Antigenic potential of recombinant proteins from Leptospira Interrogans

A leptospirose é uma zoonose generalizada no mundo. Atualmente, as vacinas comerciais disponíveis não oferecem proteção heteróloga contra todos os sorovares leptospirais. Assim, é importante identificar alvos conservados e capazes de induzir imunidade protetora de amplo espectro. Nesse contexto, o objetivo deste estudo foi avaliar a antigenicidade de proteínas recombinantes de Leptospira interrogans sorovar Copenhageni, a fim de selecionar candidatos para o desenvolvimento de uma vacina recombinante contra a leptospirose. Após análise in silico, cinco lipoproteínas foram selecionadas (LIC10463, LIC10498, LIC10666, LIC11889 e LIC12666), clonadas e expressas em Escherichia coli e purificadas por cromatografia de afinidade. As proteínas recombinantes rLIC10463, rLIC10498, rLIC10666 e rLIC11889 obtidas demonstraram potencial antigênico e, portanto, podem ser utilizadas em ensaios de imunogenicidade e imunoproteção em modelo animal.

Isolamento de leptospiras em bovinos abatidos em frigoríficos de Pelotas/RS

Leptospirosis is caused by a variety of leptospiral serovars which are distributed worldwide. Bovine leptospirosis has economic importance to countries involved on dairy and beef raising, due the losses in milk yield of affected cows and in weight among beef cattle. In addition, inapparent infection in herds presents a serious problem in the recognition and spread of infection. Bovine leptospirosis is not only a financial hazard in endemic herds, but also an occupational risk factor for veterinarians, rural workers, and slaughterhouse employees. Furthermore, available vaccines for bovine leptospirosis are killed whole-cell preparations that protect against the serovars present, making imported vaccines unsatisfactory for the control of the disease in Brazilian herds. In this light, the present study´s objective was to obtain new isolates of leptospires from cattle slaughtered in abattoirs of the city of Pelotas, Brazil. Bovine kidneys were obtained from 250 animals at three abattoirs of the city of Pelotas. Each kidney was transported to the laboratory in individual tubes and processed for isolation within two hours after slaughter. Cultures were incubated at 30°C and examined weekly for ten weeks by darkfield microscopy to detect the presence of leptospires. In order to determine if the isolates would produce infection in laboratory animals, groups of two 28-day-old hamsters were inoculated intraperitoneally with 108 leptospires of each isolate. To definitely characterize the new isolates, bacterial culture and DNA were sent to Fundação Oswaldo Cruz for serogrouping and DNA sequencing. A total of three (1.2%) strains were isolated from the kidney tissue of adult male bovines from the Capão do Leão (n=1) and Pedro Osório (n=2) municipalities. The new strains were named BOV3, BOV14, and BOV15. The virulence test using young hamsters showed that the two strains (BOV3 and BOV15) were capable of reproducing the main clinical and pathological findings of experimental leptospirosis in hamsters. None of the animals demonstrated clinical signs of infection when infected with BOV14. This study reports the isolation of three strains of Leptospira from cattle and their preliminary virulence characterization. These results are demonstrating the risk of leptospirosis in farms and slaughterhouses in Southern Brazil, transmission to other livestock species, and exposure to humans. Additional tests are being employed for the classification of new isolates.A leptospirose é causada por uma variedade de sorovares leptospirais os quais são distribuídos amplamente no mundo. A leptospirose bovina possui importância econômica nos países envolvidos na produção leiteira e de carne, devido as perdas na produção de leite e no peso dos animais acometidos. Além disso, infecções inaparentes em rebanhos representam um problema sério para o reconhecimento e disseminação da enfermidade. A leptospirose bovina não é somente um problema econômico, mas também um fator de risco para veterinários, trabalhadores rurais e magarefes. Vacinas disponíveis para a leptospirose bovina são preparações de células inteiras inativadas que protegem contra os sorovares presentes, tornando as vacinas importadas insatisfatórias para o controle da enfermidade em rebanhos brasileiros. Dessa forma, o objetivo do presente estudo foi isolar leptospiras de bovinos abatidos em frigoríficos de Pelotas, Brasil. Rins bovinos foram obtidos de 250 animais de três frigoríficos de Pelotas. Cada rim foi transportado ao laboratório em tubos individuais e processados para o isolamento até duas horas após o abate. Culturas foram incubadas a 30°C e examinadas semanalmente através de microscopia de campo escuro para a detecção da presença de leptospiras. Para determinar se os isolados eram virulentos para animais de laboratório, grupos de dois hamsters com 28 dias de idade foram inoculados através da via intraperitonial com 108 leptospiras de cada uma dos isolados. Para a caracterização definitiva dos novos isolados, culturas bacterianas e DNA foram enviados para a Fundação Oswaldo Cruz para a realização de sorogrupagem e sequenciamento de DNA. Três (1,2%) cepas foram isoladas dos rins de três bovinos machos adultos dos municípios do Capão do Leão (n=1) e Pedro Osório (n=2). Os novos isolados receberam o nome de BOV3, BOV14 e BOV15. O teste de virulência usando hamsters jovens mostrou que duas cepas (BOV3 e BOV15) foram capazes de reproduzir os principais achados clínicos e patológicos da leptospirose experimental. Nenhum animal infectado com BOV14 demonstrou sinais clínicos da enfermidade. Assim, este estudo relata o isolamento e a caracterização preliminar da virulência de três cepas de Leptospira de bovinos. Estes resultados demonstram o risco para a leptospirose em fazendas, abatedouros no sul do Brasil, para a transmissão a outras espécies animais e humanos. Técnicas adicionais estão sendo empregadas para a classificação definitiva dos novos isolados

New approaches for the development of a vaccine against leptospirosis

Leptospirosis is a worldwide zoonosis, causing a major economic impact on the agricultural sector. Currently, the vaccines used to prevent disease in cattle are formulated with inactivated Leptospira whole-cell bacterins, which have limited success, since they induce an immunity of short duration and serovar-specific protection. Preliminary results from our group showed that recombinant fragments of Lig (leptospiral immunoglobulin-like) proteins are antigenic, immunogenic and confer full or partial protection against homologous challenge in hamster model. Thus, rLigs fragments are potential candidates for a subunit vaccine against human and animal leptospirosis. Therefore, the purpose of this study was to evaluate vaccine preparations with recombinant proteins LigA625-1224aa (rLigANI), LigB131-649aa (rLigBRep), LigB625-1044aa (rLigB7-11) and LigA131-1224aa (rLigAFull) when administered with Alum Hydroxide as adjuvant, either individually or combined, in order to identify formulations that are capable of inducing a protective response against homologous challenge. Male and female hamsters were immunized with the preparations by intramuscular injection on days 0 and 14, and the homologous challenge was performed using Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130, by intraperitoneal injection on day 28. Following expression and purification of the four proteins were obtained four batches with a purity of 90%. All four recombinant proteins showed antigenicity, reacting against human sera with convalescent leptospirosis. Immunization of hamsters with preparations containing rLigANI, rLigBRep, rLigB7-11, and rLigAFull conferred variable protection (0-100%) against homologous challenge. Although no preparation has induced a sterilizing protection, evidenced by the isolation and immunofluorescence techniques, the association between rLigANI and rLigBRep was able to confer significant protection (p <0.05) in all experiments. In contrast, the LigAFull as well as rLigB7-11, did not provide protection. Our study has proposed a new approach for the development of a recombinant vaccine against bovine leptospirosis. It is the first study that tested the rLigA in its entire form. Furthermore, it was possible to obtain expression in soluble form. Although the protein obtained has been recognized by sera from convalescent human in vitro, it was only capable of conferring significant levels of survival (p <0.05) to challenged animals but not in relation to mortality. Moreover, the association between rLigBRep and rLigANI, another novel approach, testing showed that even at different doses (40 g ou 60 g) of each target, both the analysis of the mortality and survival showed significant results (p <0.01) in three of the five experiments. The results obtained in our study represent an important contribution to the development of a vaccine against leptospirosis.Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul - FAPERGSA leptospirose é uma zoonose que possui distribuição mundial, causando um importante impacto econômico no setor agropecuário. Atualmente, as vacinas empregadas para a prevenção da enfermidade são formuladas com culturas de leptospiras inteiras inativadas, as quais possuem sucesso limitado, já que induzem uma imunidade de curta duração e proteção sorovar-específica. Resultados preliminares revelaram que fragmentos recombinantes das proteínas Ligs (Leptospiral immunoglobulin-like) são antigênicas, imunogênicas e conferem proteção total ou parcial contra o desafio homólogo em hamsters. Dessa forma, fragmentos rLigs são candidatos potenciais para uma vacina de subunidade contra a leptospirose humana e animal. Assim sendo, a proposta deste projeto foi avaliar preparações vacinais com as proteínas recombinantes LigA625-1224aa (rLigANI), LigB131-649aa (rLigBRep), LigB625-1044aa (rLigB7-11) e LigA131-1224aa (rLigAFull) quando administradas com Hidróxido de Alumínio como adjuvante, individualmente ou combinadas, a fim de identificar quais formulações serão capazes de induzir uma resposta protetora contra o desafio homólogo. Hamsters machos e fêmeas foram imunizados com as preparações através da via intramuscular, nos dias 0 e 14, e o desafio homólogo foi realizado através da via intraperitoneal, no dia 28, utilizando Leptospira interrogans sorovar Copenhageni cepa Fiocruz L1-130. Após a expressão e purificação das quatro proteínas, obteve-se quatro lotes com pureza de 90%. Todas as quatro proteínas recombinantes apresentaram antigenicidade, reagindo contra soro de humanos e animal convalescentes de leptospirose. A imunização dos hamsters contendo as preparações com rLigANI, rLigBRep, rLigB7-11 e rLigAFull conferiram proteções variáveis (0-100%) contra o desafio homólogo. Embora nenhuma preparação tenha induzido a uma proteção esterilizante, evidenciada através das técnicas de reisolamento e imunofluorescência, a associação entre rLigANI e rLigBRep foi capaz de conferir proteção significativa (p<0,05) em todos os experimentos. Em contrapartida, o fragmento inteiro de LigA, assim como rLigB7-11, não conferiram proteção. Nosso estudo propôs uma nova abordagem para o desenvolvimento de uma vacina recombinante contra a leptospirose. É o primeiro estudo que testou a proteína LigA recombinante em sua forma inteira. Além disso, foi possível obter a sua expressão na forma solúvel. Embora a proteína obtida tenha sido reconhecida por soros de humanos convalescentes in vitro, foi capaz de conferir apenas níveis de sobrevivência significativa (p<0,05) aos animais desafiados, mas não em relação à mortalidade. Por outro lado, a associação entre rLigBRep e rLigANI, outra abordagem inédita, demonstrou que mesmo testando-se em diferentes doses (40g ou 60g) de cada alvo, tanto a análise de mortalidade quanto a de sobrevivência revelou resultados significativos (p<0,01) em três dos cinco experimentos realizados. Os resultados obtidos em nosso estudo representam uma contribuição importante para o desenvolvimento de uma vacina contra a leptospirose

Avaliação da antigenicidade de três quimeras recombinantes de b. Henselae

Bartonella spp. são bactérias em formato de cocobacilo, gram-negativas, intracelulares facultativas, aeróbicas e oxidase negativas, possuem crescimento lento, sendo os únicos bacilos gram-negativos relatados como capazes de viver dentro dos eritrócitos (GARY, 1990; REGIER; ÓROURKE; KEMPF, 2016; VIEIRADAMIANI et al., 2015). Bartonella henselae é a espécie de maior importância clínica dentro do gênero, pois infecta tanto animais quanto humanos, sendo o agente etiológico da Doença da Arranhadura do Gato (Cat Scratch Disease - CSD). (BREITSCHWERDT, 2017; KSIAA et al., 2019). A transmissão para humanos decorre principalmente da arranhadura do gato, sendo os humanos hospedeiros acidentais e os felinos os hospedeiros definitivos (IANNINO et al., 2018; MALHEIROS et al., 2016).Sem bols

Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Guérin

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems

Campylobacter coli in Swine Slaughtering Flowchart and Research of cdt Genes

Background: Campylobacter spp. are among the microorganisms most commonly associated with foodborne disease. Campylobacter spp. isolation from pigs during the slaughter and final products have been reported in several countries, including Brazil. However, very little is known about the sources of contamination in the slaughtering flowchart and how these microorganisms are spread in processing plants. Considering the possibility of the pigs carry Campylobacter spp. since the farm or its products are contaminated in the slaughterhouse, this study had as aim to track Campylobacter spp. in pig slaughtering flowchart to understand the behavior of these pathogens in the production line.Materials, Methods &amp; Results: Forty animals of 10 lots, four from each lot, were followed during slaughter. Stool samples were collected from the floor of each enclosure where the pigs were housed on the farm and immediately after stunning on slaughterhouse. Samples from carcass surface were collected after removal of the animals from scrap machine, after evisceration and before the refrigeration chamber. It was also collected surface samples from jowls and samples from the scalding tank water before and after the passage of animals. The swabs containing samples were plated onto Columbia agar supplemented with activated charcoal, oxygen reduction solution and antibiotics supplement, and incubated at 42°C for 48 h under microaerobic conditions. The colonies which presented with a shiny and moist appearance were analyzed by Gram staining for identification of Campylobacter by morphology, and then tested for catalase and oxidase. The Campylobacter isolates were identified for species C. jejuni or C. coli by PCR. Bands profiles were determined by rep-PCR and used to compare the strains. Campylobacter was isolated from 19 (9.5%) of the 200 pig samples analyzed, seven (36.8%) of the rectum, seven (36.8%) after evisceration and five (26.3%) before the refrigeration chamber. Campylobacter was not isolated from jowls and from scalding tank water. All isolates were C. coliand cdtnegative.Persistence of strains originating from the farm and cross contaminations during the slaughtering flowchart was identified by the analysis of the bands profiles obtained by rep-PCR.Discussion: C. coli was the species of Campylobacter present in the swine intestinal tract and in the swine slaughterhouse. The animals, once contaminated, can carry the microorganism during the stages of the slaughtering flowchart. The farm where the animals came from is an important source of contamination during processing, however cross contamination also plays a relevant role. The evisceration was considered the most critical stage, due to the greater number of isolates obtained after this procedure, what emphasize the importance of the hygienic-sanitary management in this stage. Campylobacter spp. can survive, despite not being able to multiply, in foods at refrigeration temperatures (-1 to 5°C) for one to three weeks. Therefore, the high percentage of isolates obtained from the carcass before the refrigeration chamber may represent a problem, since the contamination of the carcasses that enter in this sector can be maintained until the food reaches the consumer. There was no similarity between strains isolated from different lots, indicating that there were no persistence of strains both in the farm and in the slaughterhouse