Placentitis and abortion caused by a multidrug resistant strain of Campylobacter fetus subspecies fetus in a sheep in Uruguay / Placentitis y aborto causado por una cepa de Campylobacter fetus subespecie fetus multirresistente a antibióticos en una oveja en Uruguay

Abstract Campylobacter fetus fetus (Cff) is a major infectious cause of abortion in sheep worldwide, and an opportunistic human pathogen. Information on Cff as an ovine abortifacient in South America is limited. We describe a case of abortion caused by a multidrug resistant strain of Cff in a sheep in Uruguay. In August 2017, 3/57 pregnant ewes (5.3%) aborted whithin one week. Histopathologic examination of the placenta of an aborted ewe revealed severe neutrophilic and fibrinonecrotizing placentitis with vasculitis and thrombosis of the chorionic arterioles. Cff was isolated on microaerobic culture in Skirrow agar, and further confirmed by 16S rDNA PCR amplification and sequencing, and endpoint and real time PCR assays. Antimicrobial sensitivity testing revealed resistance to tetracyclines, nalidixic acid, telithromycin and clindamycin. Other abortifacients were not detected. Further studies are necessary to determine the geographic distribution, ecology, epidemiology, economic impact, and antimicrobial resistance of Cff in sheep flocks in Uruguay.

Resumen Campylobacter fetus fetus (Cff) es una importante causa de abortos en ovinos y un patógeno oportunista en humanos. La información sobre Cff como abortifaciente en ovinos en Sudamérica es limitada. Describimos un caso de aborto causado por una cepa de Cff mul tirresistente a antibióticos en una oveja en Uruguay. En agosto de 2017, 3/57 ovejas prenadas (5,3%) abortaron en una semana. El examen histopatológico de la placenta de una de ellas reveló placentitis neutrofílica fibrinonecrosante severa, vasculitis y trombosis. Cff fue aislado en microaerobiosis en agar Skirrow, y confirmado mediante amplificación del ADNr 16S por PCR seguida de secuenciación, y por PCR punto final y qPCR. Las pruebas de sensibilidad antimicrobiana revelaron resistencia a tetraciclinas, ácido nalidíxico, telitromicina y clindamicina. No se detectaron otros abortifacientes. Son necesarios más estudios para determinar la distribución geográfica, ecología, epidemiología, el impacto económico y la resistencia antimicrobiana de Cff en majadas ovinas de Uruguay.

Campylobacter fetus in abomasal fluid from spontaneously aborted bovine and ovine fetuses

Pregnancy losses are a major concern in livestock industry due to their economic impact on producers. Campylobacter fetus subspecies fetus (Cff) and C. fetus subspecies venerealis (Cfv) are directly related to reproductive failures in ruminants. Cff colonizes the gastrointestinal tract of a wide range of hosts leading to abortion, while Cfv is restricted to genital tract being generally associated to infertility in bovine. Considering the great economic losses related to campylobacteriosis in cattle and ovine herds, this study aims to investigate the occurrence of C. fetus, considering Cff and Cfv subspecies, in bovine and ovine spontaneously aborted fetuses in state of Rio Grande do Sul, Brazil. In this study, samples of abomasal fluid collected from 30 spontaneously aborted bovine (n = 18) and ovine (n = 12) fetuses were investigated for the detection of Campylobacter fetus throughout conventional PCR. Positive fetuses for C. fetus presence were further analyzed by molecular assays for Cff and Cfv detection, in order to determine subspecies identification. When available, samples of the main organs of the thoracic and abdominal cavities, as well as the brain, skeletal muscle, eyelid, skin, and placenta were collected for further histopathological analyses and bacterial culture, aiming to assess the presence of infection lesions and pathogens in those sites, respectively. Additionally, RT-qPCR assays were also performed for the detection of ruminant pestivirus, in order to detect bovine viral diarrhea cases. Throughout the present methodology, C. fetus was detected in the abomasal fluid samples of 2 bovine fetuses, being both identified as Cfv subspecies by PCR. Histopathological analyses demonstrated that macroscopic and microscopic changes found in the Cfv-positive animals were not either specific or directly related to Campylobacter infections. Moreover, no significant bacterial growth was observed in microbiological culture from the collected tissues, and both fetuses were negative for ruminant pestivirus. Differently, there was no detection of C. fetus in any of the analyzed ovine fetuses. Considering that abortion diagnosis rates reported in cattle and sheep industry are highly variable among the published studies, and that abortion diagnoses are commonly inconclusive due to difficulties in sampling methodology and inadequate identification of the pathogen involved, it is important to investigate the etiological causes of abortion the herds for better understanding the causes of pregnancy issues and monitoring their occurrence. In addition, the absence of pathognomonic lesions in the tissues investigated in the histopathological analyses observed in this study strongly suggests that well-known etiological agents commonly associated to abortion, such as Leptospira spp., Toxoplasma spp., Chlamydia spp. and Neospora caninum, are unlikely to be the cause of infection of the analyzed fetuses. Taking this into account, the presence of C. fetus in the abomasal fluid samples from two bovine fetuses demonstrated in the present study suggests the possible association of Cfv not only with infertility, but also with cases of bovine abortion, highlighting the importance of investigating unusual causal agents of abortions in sheep and cattle. Overall, an adequate diagnosis is essential for establishing better prevention strategies to avoid the circulation of abortion-related infectious agents in the herds.(AU)

Selection and characterization of peptides mimetic to Campylobacter fetus subsp. venerealis using phage display / Seleção e caracterização de peptídeos miméticos a Campylobacter fetus subsp. venerealis usando phage display

ABSTRACT: Bovine genital campylobacteriosis (BGC) is a venereal and subclinical disease that affects the fertility of cattle herds, and it is caused by Campylobacter fetus subsp. venerealis . This study selected peptides mimetic to the BGC-causing agent from a phage library. Phage display is a technique that applies bacteriophage libraries that reveal peptides fused to the viral capsid in biological selections against target proteins. Biopannings were performed for biological selection in the phage library using rabbit hyperimmune serum and C. fetus subsp. venerealis protein extract. Five selected heptapeptides were considered mimetic to Cfv-NCTC 10354 based on the results of bioinformatics analysis and assays with hyperimmune serum and cervicovaginal mucus obtained from heifers. ALASLPL and LSYLFPP were the most reactive peptides and considered promising as possible mimetic immunogens for C. fetus subsp. venerealis.

RESUMO: Campilobacteriose Genital Bovina (CGB) é uma doença venérea e subclínica que causa problemas reprodutivos em rebanhos, causada por Campylobacter fetus subsp. venerealis. Este trabalho teve como objetivo selecionar peptídeos miméticos ao agente da CGB de uma biblioteca de fagos. Phage display é uma técnica que aplica bibliotecas de bacteriófagos que expõem peptídeos fusionados ao capsídeo viral em seleções biológicas contra proteínas alvo. Biopannings foram realizados para seleção biológica na biblioteca de fagos por meio de soro hiperimune de coelho e extrato proteico de C. fetus subsp. venerealis. Cinco heptapeptídeos selecionados foram considerados miméticos para Cfv-NCTC 10354 a partir de análises de bioinformática e ensaios com soro hiperimune e muco cérvico-vaginal de novilhas. ALASLPL e LSYLFPP foram os peptídeos mais reativos e considerados promissores como possíveis imunógenos miméticos para C. fetus subsp. venerealis.

Consumption of minas frescal cheese may be a source of human infection by Campylobacter jejuni / Consumo de queijo minas frescal pode ser fonte de infecção humana por Campylobacter jejuni

Campylobacter spp. is an emerging pathogen that causes gastroenteritis in humans and the consumption of dairy food can characterize sources of infection. We aimed to verify the viability and a presence of transcripts associated with characteristics of virulence and adaptation of C. jejuni isolated from Minas Frescal cheeses, produced with contaminated milk and stored under refrigeration for up to ten days. The samples were analyzed for bioindicators, Campylobacter spp., pH, acidity, moisture and sodium chloride. Campylobacter spp. recovered were evaluated for the production of transcripts of: ciaB, dnaJ, p19 and sodB. The results were correlated with the viability of C. jejuni and changes in their transcriptome. Storage at lowtemperatures reduced C. jejuni from the first to the fourth day. The variations in humidity, pH and acidity influenced the decreasing of C. jejuni. There was a reduction in transcripts' production of the four genes, more pronounced on the fourth day, indicating the inability of the microorganism to perform its metabolic activities, due to the conditions of injury. Despite the presence of mechanisms of virulence and adaptation, C. jejuni could not remain viable four days after production. However, consumption of fresh cheese contaminated with Campylobacter jejuni can be a source of infection when consumed up to four days after production.

Campylobacter spp. é um patógeno emergente que causa gastroenterite em seres humanos e o consumo de produtos lácteos pode caracterizar fontes de infecção. O objetivo deste estudo foi verificar a viabilidade e a presença de transcritos associadas a características de virulência e adaptação de C. jejuniisoladas de queijos frescos, produzidos com leite contaminado e mantidos refrigeradas por dez dias. Foram analisados bioindicadores, Campylobacter spp., pH, acidez, umidade e cloreto de sódio. Campylobacter spp. recuperados foram avaliados quanto à produção dos transcritos: ciaB, dnaJ, p19 e sodB. Os resultados foram correlacionados com a viabilidade de C. jejuni e alterações no transcriptoma. O armazenamento em baixas temperaturas reduziu C. jejuni do primeiro ao quarto dia. As variações na umidade, pH e acidez influenciaram a queda de C. jejuni. Houve uma redução na produção de transcritos dos quatro genes, mais pronunciada no quarto dia, indicando a incapacidade do micro-organismo em realizar suas atividades metabólicas, devido às condições de injúria. Apesar da presença de mecanismos de virulência e adaptação, C. jejuni não permaneceu viável quatro dias após a produção. Porém, o consumo de queijo fresco contaminado com Campylobacter jejunipode ser uma fonte de infecção quando consumido até quatro dias após a produção.

Detecção de Campylobacter jejuni em produtos de frango utilizando separação imunomagnética / Detection of Campylobacter jejuni in chicken products using immunomagnetic separation

Campylobacter jejuni é o principal causador de gastroenterite bacteriana aguda, e a carne de frango tem se mostrado uma importante fonte de transmissão. Este microrganismo é de difícil isolamento e os métodos convencionais muitas vezes não são eficientes, podendo levar a resultados errôneos. O objetivo deste trabalho foi desenvolver e testar a técnica de separação imunomagnética (IMS) na detecção de C. jejuni em produtos de frango. Micropartículas magnéticas ligadas a anticorpos policlonais anti-C. jejuni foram utilizadas para concentrar C. jejuni antes da semeadura em ágar. O protocolo foi comparado com o método convencional. C. jejuni foi recuperado do alimento experimentalmente contaminado por ambos os métodos, entretanto, quando foi usada a IMS, a presença de microrganismos contaminantes nos meios de cultura foi menor. C. jejuni foi isolado de 7% das amostras de alimento naturalmente contaminadas, usando IMS, e de 3% pelo método convencional. C. coli foi isolado de uma amostra pelo método convencional, mas não foi detectado pelo protocolo com IMS. A técnica de IMS pode ser usada para isolamento de C. jejuni de alimentos, oferecendo a vantagem de detectar em amostras o microrganismo cujo isolamento não é obtido por meio do método convencional.(AU)

Campylobacter jejuni is the main cause of acute bacterial gastroenteritis and chicken meat has shown to be an important source of infection. This microorganism is difficult to isolate and the conventional methods are often inefficient and may lead to erroneous results. This study aimed at developing and testing the technique of immunomagnetic separation (IMS) in the detection of C. jejuni in chicken products. Microparticles magnetically connected anti-C. jejuni polyclonal antibodies were used to concentrate C. jejuni before agar seeding. The protocol was compared with the conventional method. C. jejuni was recovered from experimentally contaminated food for both methods, however, when the IMS was used, the presence of contaminating microorganisms in the means of culture was smaller. C. jejuni was isolated from 7% of samples of food naturally contaminated, using IMS, and 3% by conventional method. C. coli was isolated from a sample by conventional method, but it was not detected by protocol with IMS. The IMS technique can be used for isolation of C. jejuni in food, offering the advantage of detecting the microorganism in samples from which the isolation is not obtained with the use of the conventional method.(AU)

Viability and gene transcription of Campylobacter jejuni in infant milk formulas / Viabilidade e transcrição genética de Campylobacter jejuni em fórmulas lácteas infantis

This study simulated the contamination of two varieties of infant milk formulas (homemade and commercial) with 103 and 104 CFU/mLof Campylobacter jejuni, that were kept under refrigeration (4-7ºC) for up to 48 hours. The aim of this study was to verify the maintenance of the viability and ability of Campylobacter jejuni to produce transcripts of virulence and resistance to stress conditions during periods of 0 (after preparation), 24 and 48 hours. C. jejuni remained viable during all analyzed stages and thepresence of coliforms was not detected. In general, the counts reduced 1 log cycle after 48 hours for all samples, except the 104 CFU/mL inoculum of commercial formula, which reduced 2 log cycles, indicating greater injury of C. jejuni in this food matrix. C. jejuni showed to be more adapted to homemade matrix, due to high transcription of the gene related to cell invasion, ciaB, and more susceptible in the commercial matrix, due to the high transcription of genes related to conditions of stress tolerance (dnaJ, p19, sodB). The low infective dose of C. jejuni coupled with greater vulnerability of children less than five years indicate the need for care in the preparation and maintenance of infant formulas, to prevent the use of contaminated raw material and cross-contamination, especially in homemade formulations

Este estudo simulou a contaminação de duas variedades de fórmulas de leite infantil com Campylobacter jejuni (caseiras e comerciais), que foram mantidas sob refrigeração (4-7ºC) por até 48 horas. O objetivo do estudo foi verificar a manutenção da viabilidade e capacidade de produzir transcritos de virulência e resistência a condições de estresse durante os períodos de 0 (após preparação), 24 e 48 horas. C. jejunipermaneceu viável durante todas as etapas analisadas e a presença de coliformes não foi detectada. Em geral, as contagens reduziram 1 ciclo log após 48 horas para todas as amostras, exceto o inóculo de 104 CFU/mL na fórmula comercial, que reduziu 2 ciclos logarítmicos, indicando maior lesão de C. jejuni nesta matriz alimentar. C. jejuni mostrou-se mais adaptado à matriz popular, devido à alta transcrição do gene relacionado à invasão celular, ciaB, e mais suscetível na matriz comercial, devido à alta transcrição de genes relacionados a tolerância a condições de estresse (dnaJ, p19, sodB). A baixa dose infectante de C. jejuni, juntamente com maior vulnerabilidade de crianças menores de cinco anos, indicam a necessidade de cuidados na preparação e manutenção de fórmulas infantis, para prevenir o uso de matéria-prima contaminada e contaminação cruzada, especialmente em formulações caseiras.

Campylobacter jejuni and Campylobacter coli originated from chicken carcasses modulate their transcriptome to translate virulence genes in human cells / Campylobacter jejuni e Campylobacter coli originadas de carcaças de frango modulam seu transcriptoma para traduzir genes de virulência em células humanas

The aim was to determine the spread of genetically similar profiles of Campylobacter in chicken carcasses and evaluate their ability to produce transcripts for ciaB, dnaJ, p19 and sodB genes, before and after cultivation in Caco-2 cells. The strains used were isolated from 420 samples of chicken carcasses chilled and frozen ready for marketing. The species were identified by PCR-multiplex, the phylogeny was determined by RAPD-PCR and the presence of transcripts was performed by RT-PCR. We identified 74 (17.6%) of Campylobacter strains, being 55 (74.3%) C. jejuni and 19 (25.7%) C. coli. The phylogenetic relationship demonstrated heterogeneity between isolates of the same species, with absence of clones, indicating the high level of diversity of circulating genotypes. The gene transcription showed conflicting results before and after the culture in Caco-2 cell, so that before cultivation isolates showed greater capacity to transcribe genes related to survival and after the interaction with human cells, the strains showed higher potential to transcribe genes associated with virulence. The result of this study contributes to the understanding of how these seemingly fragile microorganisms are the most prevalent bacterial agents in human gastroenteritis.(AU)

O objetivo foi determinar a disseminação de perfis geneticamente semelhantes de Campylobacter em carcaças de frango e avaliar sua capacidade de produzir transcritos para os genes ciaB, dnaJ, p19 e sodB, antes e após o cultivo em células Caco-2. As cepas utilizadas foram isoladas de 420 amostras de carcaças de frango resfriadas e congeladas prontas para comercialização. As espécies foram identificadas por PCR-multiplex, a filogenia foi determinada por RAPD-PCR e a presença de transcritos foi realizada por RT-PCR. Identificamos 74 (17,6%) das cepas de Campylobacter, sendo 55 (74,3%) C. jejuni e 19 (25,7%) C. coli. A relação filogenética demonstrou heterogeneidade entre isolados da mesma espécie, com ausência de clones, indicando o alto nível de diversidade dos genótipos circulantes. A transcrição gênica mostrou resultados conflitantes antes e após a cultura em células Caco-2, de modo que, antes do cultivo, os isolados apresentaram maior capacidade de transcrever genes relacionados à sobrevivência e após a interação com células humanas, as linhagens apresentaram maior potencial para transcrever genes associados à virulência. O resultado deste estudo contribui para a compreensão de como esses microrganismos aparentemente frágeis são os agentes bacterianos mais prevalentes na gastroenterite humana.(AU)

The development of indirect and sandwich ELISA-based detection methods for the detection of Campylobacter jejuni using monoclonal and polyclonal antibody preparations

Aims@#The current gold standard method for the detection of Campylobacter jejuni is the culturing method followed up by immuno-based detection method, of which, the ELISA is the most often used. Many commercial detection methods based on ELISA use monoclonal antibody preparations although polyclonal antibody can be more sensitive and cheaper to produce. In this study, a comparison of indirect and sandwich ELISA-based detection methods for the detection of C. jejuni using a commercial monoclonal and polyclonal antibody preparations was explored. @*Methodology and results@#An indirect and sandwich ELISA-based methods for the detection of C. jejuni was carried out using the same concentration of antibody (5 μg/mL) and the same concentration of the bacterium at 1×109 CFU/mL. At the pre-screening for optimum concentration of antibody to be used for both assay formats, the commercial monoclonal preparation gave a poor absorbance value of about 0.112 compared to 1.582 for the polyclonal antibody preparation. Hence, the use of the monoclonal antibody was not pursued further. Using the polyclonal antibody, the calculated Limits of Detection (LOD) value obtained for the indirect and sandwich ELISA methods were at 1.6×104 CFU/mL and at 1.29×104 CFU/mL, respectively, which are more sensitive than commercially used methods. The results of the specificity test obtained from the developed polyclonal antibody were then tested against other common food borne bacterial pathogens such as Salmonella Typhimurium, Listeria monocytogenes and Escherichia coli tested using the sandwich ELISA format indicated that the responses by other bacterial genus were relatively low with the translated cross-reactivity percentages of 1.78, 2.36, and 6.87%, respectively. @*Conclusion, significance and impact of study@#The results indicated that the developed system using a polyclonal antibody preparation can be more sensitive than monoclonal preparation. In addition, it is also specific towards Campylobacter while the monoclonal antibody preparation fares poorly.

Ocorrência de ovinos portadores da infecção por Campylobacter spp. no estado de Pernambuco / Occurrence of sheep carrier of infection with Campylobacter spp. in the state of Pernambuco, Brazil

Objetivou-se com este estudo determinar a ocorrência e os fatores de risco associados à infecção por Campylobacter spp. em criações de ovinos no estado de Pernambuco, Brasil. Foram coletadas 421 amostras fecais de ovinos procedentes de 20 rebanhos para o isolamento de Campylobacter spp. As espécies Campylobacter fetus subsp. fetus e Campylobacter jejuni foram identificadas por Reação em Cadeia da Polimerase (PCR). Para análise dos fatores de risco foi realizada uma análise univariada e posteriormente regressão logística a partir de questionário com perguntas objetivas sobre o manejo higiênico-sanitário e reprodutivo. A ocorrência para Campylobacter spp. foi de 4,5% (19/421; I.C. 2,8% - 7,1%). Das 19 amostras positivas no cultivo, oito (1,9%; I.C. 0,9% - 3,9%) foram classificadas como C. fetus subsp. fetus e sete (1,7%; I.C. 0,7% - 3,6%) como C. jejuni, com co-infecção em quatro amostras (0,95%). O número de focos identificados foi de 35,0% (7/20) das criações de ovinos que apresentavam pelo menos um animal positivo. Na análise de regressão logística não foi identificada nenhuma das variáveis como fator de risco. Este é o primeiro registro da infecção por Campylobacter spp. em rebanhos ovinos no Nordeste do Brasil, concluindo-se que a infecção ocorre nesses rebanhos. Dessa forma, se faz necessário à implementação de medidas de controle e prevenção, para impedir a propagação do agente entre as criações, evitando prejuízos para ovinocultura e riscos para saúde pública, uma vez que a campilobacteriose é considerada uma zoonose emergente.(AU)

The objective of this study was to determine the occurrence and risk factors associated with Campylobacter spp. infection in sheep in the State of Pernambuco, Brazil. A total of 421 fecal samples were collected from 20 herds for the isolation of Campylobacter spp. The species Campylobacter fetus and Campylobacter jejuni were identified by Polymerase Chain Reaction (PCR). To analyze the risk factors, logistic regression was conducted through a questionnaire about the hygienic-sanitary and reproductive management. The occurrence of Campylobacter spp. was 4.5% (19/421; C.I. 2.8 to 7.1%). Of the 19 positive samples isolated, eight (1.9% CI 0.9 to 3.9%) were classified as C. fetus subsp. fetus and seven (1.7% CI 0.7 to 3.5%) as C. jejuni, with co-infection in four samples (0.95%). The number of identified focuses was 35.0% (7/20) of the sheep herds that had at least one positive animal. The logistic regression analysis did not identify any of the variables as a risk factor. This appears to be the first report of infection with Campylobacter spp. in sheep herds in northeastern Brazil. Thus it is necessary to implement measures for control and prevention avoid damage to sheep production and risk to public health, since campylobacteriosis is considered an emerging zoonosis.(AU)

Seletividade e produtividade de meios de cultura para isolamento de Campylobacter spp / Selectivity and productivity of culture media for isolation of Campylobacter spp

[{"text": "A campilobacteriose é uma zoonose\r\nemergente de origem alimentar\r\ncausada por bactérias do gênero\r\nCampylobacter. Vários fatores dificultam\r\no isolamento deste patógeno\r\nem amostras naturalmente contaminadas,\r\npor isso devem ser utilizadas\r\nmetodologias normalizadas bem\r\ncomo meios de cultura com desempenho\r\nadequado, prevenindo a ocorrência\r\nde resultados falso negativos.\r\nAssim, avaliou-se a performance\r\nde meios de cultura recomendados\r\npelas ISO 10272-1 para detecção\r\nde Campylobacterspp. com testes\r\nde seletividade e produtividade em\r\nculturas puras e o desempenho destes\r\nmeios em amostras de carne de\r\nfrango artificialmente contaminadas.\r\nCepas ATCC de C. coli e C. jejuni e\r\ndos interferentes S. aureus, E. coli e\r\nProteusmirabilis foram inoculadas\r\nnos meios indicados pelas normas\r\noficiais e posteriormente inoculados\r\nem amostras fortificadas. Os meios\r\ntestados, tanto em culturas puras\r\nquanto em amostras fortificadas,\r\ntiveram desempenho satisfatório,\r\nmostrando boa seletividade e produtividade,\r\npermitindo que os laboratórios\r\noptem pela combinação de\r\nmeios com melhor performance para\r\nisolamento e identificação de Campylobacter\r\nspp. em amostras naturalmente\r\ncontaminadas.(AU)", "_i": "pt"}]

Ocorrência da infecção por Campylobacter fetus subsp. venerealis e Tritrichomonas foetus em búfalos no estado de Pernambuco, Brasil / Infection occurrence of Campylobacter fetus subsp. venerealis and Tritrichomonas foetus in buffaloes in the state of Pernambuco, Brazil

Objetivou-se com estudo determinar a ocorrência da infecção por Campylobacter fetus subsp. venerealis e Tritrichomonas foetus em búfalos no estado de Pernambuco, Brasil. Foram coletadas 133 amostras biológicas (muco cervicovaginal e raspado prepucial) de animais, procedentes de oito propriedades, de diferentes regiões do estado. O material biológico coletado foi transferido para solução salina tamponada (PBS) e, posteriormente, inoculado em meios de transporte específicos, Lander para diagnóstico de C. fetus subsp. venerealis e Diamond para T. foetus. Para o diagnóstico das infecções por Campylobacter fetus subsp. venerealis e Tritrichomonas foetus, as amostras foram submetidas à reação em cadeia da polimerase (PCR) e cultivadas em meio ágar Columbia acrescido de antibiótico e Diamond, respectivamente. Para pesquisa de C. fetus subsp. venerealis, observou-se uma ocorrência de 1,8% (2/113) de animais positivos no exame microbiológico com confirmação pela PCR. Em relação à procedência, observou-se que 100% das amostras positivas pertenciam a dois machos do mesmo rebanho. Nenhum animal foi positivo na pesquisa de T. foetus. Este é o primeiro registro da infecção por C. fetus subsp. venerealis em búfalos no Brasil. Apesar da baixa ocorrência, recomenda-se adoção de medidas de controle, com o intuito de se evitar a disseminação do agente para outros rebanhos.(AU)

The objective this study was to determine the occurrence of infection with Campylobacter fetus subsp. venerealis and Tritrichomonas foetus in buffaloes in the State of Pernambuco, Brazil. Biological samples were collected (cervico vaginal mucus and shaved prepucial) of 113 animals, coming from 8 properties in different regions of the state. The biological material collected was transferred into phosphate buffered saline (PBS) and inoculated in the specific transport, Lander for diagnosis of C. fetus subsp. venerealis and Diamond for T. fetus subsequently. For the diagnosis of infection by Campylobacter fetus subsp. venrealis and Tritrichomonas foetus the samples were submitted to Polymerase Chain Reaction (PCR) grown in Columbia agar plus antibiotics and Diamond, respectively. There was an occurrence of 1.8% (2/113) of positive animals in the microbiological examination with confirmation by PCR, for C. fetus subsp. venerealis. We observed that 100% of positive samples were from two (2) males from the same herd. No animals were positive for T. foetus. This is the first report of infection with C. fetus subsp. venerealis in buffaloes in Brazil. Despite rare occurrence, control measures are recommended in order to prevent the spread of the agent to other herds.(AU)

Campylobacter Data in Childhood from a University Hospital.

Background and Objectives: Due to underdiagnosis because of the technical difficulties plus inadequacy of laboratories, actual incidence of campylobacteriosis may substantially be greater than the reported incidence in many countries including Turkey. The purpose of this study was to evaluate and emphasize the diagnostic methods of campylobacteriosis, and the clinical and laboratory data of children with Campylobacterial gastroenteritis. Methods: This study was conducted in Yeditepe University Hospital, Istanbul, Turkey. Clinical (demographical data, symptoms and findings) and laboratory (stool microscopy, rapid antigen tests, culture, and multiplex PCR and blood test results) variables of children with Campylobacter infection between January 2010 and October 2012 were evaluated retrospectively from the hospital database. Results: Out of 1275 stool cultures, Campylobacter spp. was detected in 90 of them (7%). The diagnosis was made by positive stool culture (n = 87) and/or multiplex polymerase chain reaction (PCR) test (n = 8, whereas 3 of them were culture negative). The distribution of Campylobacter isolates were; C. jejuni (85.5%), C. upsaliensis (8.9%), C. coli (1.1%), and others (4.5%). The presenting symptoms were diarrhea (100%), fever (68.9%), abdominal pain (34.4%), dehydration (27.8%), vomiting (25.5%), bloody diarrhea (5.6%), and convulsion (1%). Hospitalization was required in 25.5% of patients. Conclusions: Although stool culture is a reference method in diagnosis, the PCR test can be used in culture negative patients with clinical manifestations. Diarrhea, fever, abdominal pain, and vomiting were most commonly encountered symptoms whereas bloody diarrhea and convulsion were rarely seen in campylobacteriosis. Also antibiotherapy and hospitalisation were not commonly required.

Occurrence of Campylobacter jejuni and C. coli on broiler carcasses after chilling in southern Brazil / Campylobacter jejuni e C. coli em carcaças de frangos após a refrigeração por imersão no sul do Brasil

Campylobacter jejuni and C. coli have been associated with gastrointestinal disorders in human beings, due mainly to the consumption of chicken meat. Despite control measures for reducing contamination by these bacteria, the detection of Campylobacter in carcasses after chilling remains high. A total of 105 carcasses were assessed by the horizontal detection method in five federally inspected slaughterhouses in southern Brazil in 2012 and in the first three months of 2013. Campylobacter was isolated in 37.1% of the carcasses, of which 97.5% contained C. jejuni and 2.5% were infected by C. coli. The rate of positive carcasses across the slaughterhouses ranged from 0 to 71.4%. Determining the occurrence of Campylobacter among flocks is crucial for estimating the microbial load at specific points along the slaughtering process and for minimizing the risk of contamination of end products by Campylobacter.(AU)

Campylobacter jejuni e C. coli têm sido associados a problemas gastroentéricos em seres humanos principalmente devido ao consumo de carne de frango. Embora medidas de controle sejam realizadas para reduzir a contaminação por estas bactérias, a identificação de Campylobacter em carcaças após a refrigeração por imersão é alto. Foram analisadas 105 carcaças pelo método de detecção horizontal em cinco abatedouros sob Inspeção Federal no sul do Brasil em 2012 e nos três primeiros meses de 2013. Campylobacter foi isolada em 37,1% das carcaças analisadas, as quais 97,5% foram identificados como C. jejuni e 2,5% como C. coli. A ocorrência de carcaças positivas entre matadouros variou de zero a 71,4%. O conhecimento sobre a ocorrência de Campylobacter entre os lotes é fundamental para estimar a extensão da carga microbiana em pontos específicos do abate e consequentemente minimizar o risco de contaminação por Campylobacter em produtos finais de frangos.(AU)

Campylobacter fetus em bovinos no estado do Rio Grande do Sul / Campylobacter fetus in cattle from Rio Grande do Sul state, Brazil

A campilobacteriose genital bovina (CGB) é uma doença infectocontagiosa causada por Campylobacter fetus, determina infertilidade temporária, endometrite leve e aborto em fêmeas, além de aumentar o intervalo entre partos. A ocorrência de CGB entre rebanhos no Brasil tem variado muito entre as diferentes regiões. Com isso, o objetivo deste trabalho foi identificar, por meio da reação em cadeia da polimerase (PCR), a ocorrência de amostras positivas para C. fetus, oriundas de bovinos, no período de 1999 a 2010, no Rio Grande do Sul, e analisar a positividade em machos e fêmeas. Foram utilizadas 816 amostras procedentes de 37 municípios, localizados predominantemente nas mesorregiões sudoeste e centro ocidental rio-grandense, das quais 480 aspirados prepuciais (92 provenientes de duas centrais de inseminação artificial e 388 de estabelecimentos de criação - monta natural), 324 aspirados cervicais e conteúdo abomasal de 12 fetos bovinos abortados. Como resultado, 10,9% das amostras (89/816) foram positivas para C. fetus. Quando analisados os resultados em relação à origem das amostras, 6,5% (6/92) das coletadas de machos de centrais de inseminação foram positivas, e das obtidas de touros utilizados em monta natural, 9% (35/388). Já entre as fêmeas, esse percentual foi de 13,6% (44/324) e, nas amostras obtidas de fetos abortados, 33,3% (4/12) foram positivas. Quando analisados os 91 estabelecimentos de criação com monta natural e os 37 municípios, foram positivos 44,0% (40/91) e 63,2% (24/37), respectivamente. Com isso, foi demonstrada a importância da CGB para os rebanhos bovinos, e uma maior ocorrência de amostras positivas em fêmeas, quando comparadas às amostras provenientes de machos.

Bovine genital campylobacteriosis (BGC) is an infectious disease caused by Campylobacter fetus, which determines temporary infertility, mild endometritis, miscarriage in females and also increases the calving interval. The occurrence of BGC in the Brazilian herds has varied widely among regions. The aim of this study was to identify by polymerase chain reaction (PCR) the occurrence of C. fetus in bovines from Rio Grande do Sul (RS), Brazil using samples collected from1999 to 2010. A total of 816 samples from 37 counties localized predominantly in the Southwest and Central Western regions of the RS state were analyzed. Four hundred eighty preputial aspirated samples (92 from artificial insemination centers and 388 from farms that use natural mating) and 324 cervical aspirates and abomasal contents of 12 aborted fetuses were analyzed. As result, 10.9% (89/816) were positive for C. fetus. When the results were analyzed in relation to its origin, 6.5% (6/92) of the males samples from insemination centers were positive, and the ones from natural mating 9% (35/388) were positives. For the females, this percentage was 13.6% (44/324) of positivity, and the samples from the aborted fetuses 33.3% (4/12) were positive. When the 91 farms that used natural mating and the 37 counties were analyzed, it showed a positivity rate of 44.0% (40/91) and 63.2% (24/37), respectively. This study shows the importance of BGC for bovine herds, and a larger occurrence of positive samples among females when compared to male.

Campylobacter jejuni in commercial eggs

This study evaluated the ability of Campylobacter jejuni to penetrate through the pores of the shells of commercial eggs and colonize the interior of these eggs, which may become a risk factor for human infection. Furthermore, this study assessed the survival and viability of the bacteria in commercial eggs. The eggs were placed in contact with wood shavings infected with C. jejuni to check the passage of the bacteria. In parallel, the bacteria were inoculated directly into the air chamber to assess the viability in the egg yolk. To determine whether the albumen and egg fertility interferes with the entry and survival of bacteria, we used varying concentrations of albumen and SPF and commercial eggs. C. jejuni was recovered in SPF eggs (fertile) after three hours in contact with contaminated wood shavings but not in infertile commercial eggs. The colonies isolated in the SPF eggs were identified by multiplex PCR and the similarity between strains verified by RAPD-PCR. The bacteria grew in different concentrations of albumen in commercial and SPF eggs. We did not find C. jejuni in commercial eggs inoculated directly into the air chamber, but the bacteria were viable during all periods tested in the wood shavings. This study shows that consumption of commercial eggs infected with C. jejuni does not represent a potential risk to human health.

In vitro development of murine embryos in presence of campylobacter fetus / Desarrollo de embriones murinos in vitro en presencia de campylobacter fetus

Bovine campylobacteriosis caused by Campylobacter fetus is associated with reproductive losses. The knowledge about the mechanisms of bacterial pathogenesis is limited, then a murine experimental model is proposed. BALB/c females and males were used. Two-cell embryos were cultured in Ham-F10 as control group (CG). Treatment groups were constituted by the addition of Cfv 1 and 3, or Cff 2 and 5. Morulae were placed in Ham-F10 (CG); treatment groups were constituted by the addition of Cfv27, CFF (cell-free filtrate) and Brucella broth (BB). Blastocysts were cultured in MEM (CG); challenge group were constituted by the addition of Cfv 27. Differentiation, hatching, hatched, adhesion and expansion were evaluated. Results were analyzed by Chi2 test. In two-cell embryo, the differentiation rate was not modified when the study strains were added (p > 0.05). The differentiation rate at 24 h for embryos at the morula stage was lower for BB, Cfv, and CFF, compared with CG (p < 0.05). After 48 h culture, no differences were observed in blastocyst formation for Cfv and BB, compared to CG (p > 0.05). However, the differentiation rate for the CFF group was lower than for CG (p < 0.05). At 48 and 72 h, the hatching rate was higher in CFF and Cfv groups than in CG (p < 0.05). Differences were not detected in blastocyst cultures. In conclusion, under these experimental conditions, Cf was not detrimental to the development of murine embryos. Efforts will be intensified to establish in vitro infection models that reproduce their pathogenicity.

La campilobacteriosis bovina caudada por Campylobacter fetus produce pérdidas reproductivas existiendo poca información de los mecanismos de patogenicidad de dicha bacteria, por lo cual se propone un modelo utilizando ratones BALC/c. Embriones de dos células fueron cultivados en Ham-F10: grupo control (GC), los grupos experimentales fueron adicionados con las cepas Cfv 1, Cfv 3, Cff 2 y Cff 5. Mórulas fueron cultivadas en Ham-F10 (GC); los grupos tratados recibieron Cfv27, CFF (filtrado libre de células) y caldo Brucella (BB). Blastocistos fueron cultivados en MEM (GC) y MEM más Cfv 27 (grupo desafiado). Se evaluó: diferenciación, "hatching", "hatched", adhesión y expansión. Los resultados fueron analizados por Chi2. En embriones de dos células, la diferenciación no fue modificada por acción de las cepas evaluadas (p > 0,05). Para embriones en estadío de mórula, la diferenciación a las 24 h de cultivo fue menor para BB, Cfv, y CFF, comparado con el GC (p < 0,05). Luego de 48 h de cultivo, no hubo diferencias entre Cfv, BB, y CG (p > 0,05), no obstante para el grupo CFF la diferenciación fue menor al CG (p < 0,05). El porcentaje de "hatching" (48 y 72 h de cultivo), fue mayor en los grupos CFF y Cfv comparado con el GC (p < 0,05). La adición de Cfv 27 no modificó el desarrollo de blastocistos. En el modelo propuesto, Cf no afectó negativamente el desarrollo embrionario. Futuros trabajos serán necesarios para establecer un modelo de infección in vitro en pos de reproducir su patogenicidad.

Production and characterization of monoclonal antibodies against Campylobacter fetus subsp. venerealis / Produção e caracterização de anticorpos monoespecíficos contra Campylobacter fetus subsp. venerealis

Myeloma cells Sp2/0-Ag14 and spleen cells from BALB/c mouse immunized with sonicated Campylobacter fetus subsp. venerealis NCTC 10354 were fused with polyethylene glycol (PEG) for the selection of clones producing antibodies. Clones were obtained by limiting dilution and screened for the production of specific antibodies to C. fetus subsp. venerealis NCTC 10354 by indirect ELISA and western blot against a panel of bacteria: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352, and Arcobacter skirrowii LMG 6621 for the ELISA and C. fetus subsp. venerealis NCTC 10354 and C. sputorum biovar sputorum LMG 6647 for the western blotting. Fifteen clones producing monoclonal antibodies (MAbs) anti-C. fetus subsp. venerealis of the IgM (1) and IgG (14) classes were further screened for species-specificity. Four clones of the 15 obtained were producers of species-specific monoclonal antibodies (MAbs): two were specific for C. fetus subsp. venerealis and two were specific for C. fetus subsp. fetus. None of the clones were reactive against C. sputorum biovar sputorum LMG 6647. All clones recognized a protein with molecular mass of approximately 148 kDa from lysed C. fetus subsp. venerealis NCTC 10354.

Para a produção de anticorpos monoclonais contra Campylobacter fetus subsp. venerealis foram utilizadas as linhagens de células de mieloma Sp2/0-Ag14 e células de baço de camundongos BALB/c imunizados com sonicado de C. fetus subsp. venerealis NCTC 10354. A detecção dos anticorpos monoclonais foi realizada por ELISA indireto utilizando antígeno sonicado de C. fetus subsp. venerealis NCTC 10354. A clonagem foi realizada por diluição limitante e os clones foram caracterizados por ELISA indireto utilizando um painel de bactérias escolhidas em função da prevalência e habitats: C. fetus subsp. venerealis NCTC 10354, C. fetus subsp. fetus ADRI 1812, C. sputorum biovar sputorum LMG 6647, C. lari NCTC 11352 e Arcobacter skirrowii LMG 6621; e no "western blotting" utilizando antígenos sonicados de C. fetus subsp. venerealis NCTC 10354 e C. sputorum biovar sputorum LMG 6647. Foram obtidos 15 clones produtores de anticorpos anti- C. fetus subsp. venerealis das classes IgM (1) e IgG (14). Quatro clones dentre os 15 clones obtidos foram produtores de anticorpos monoclonais espécie-específicos: dois clones reagiram com maior especificidade contra C. fetus subsp. venerealis NCTC 10354 e dois clones reagiram com maior especificidade contra C. fetus subsp. fetus ADRI 1812. Nenhum dos clones reagiu contra C. sputorum biovar sputorum LMG 6647, comprovando a especificidade dos anticorpos monoclonais testados. Todos os clones reconheceram uma proteína de massa molecular de aproximadamente 148 kDa no sonicado de C. fetus subsp. venerealis NCTC 10354.

Campilobacteriose genital bovina e tricomonose genital bovina: epidemiologia, diagnóstico e controle / Bovine genital campylobacteriosis and bovine genital trichomonosis: epidemiology, diagnosis and control

A presente atualização trata de duas das mais importantes doenças sexualmente transmitidas de bovinos, a campilobacteriose genital bovina e a tricomonose genital bovina. São abordados aspectos relacionados à epidemiologia destas doenças, principalmente em relação a sua distribuição no Brasil. Também são revisados aspectos importantes de diagnóstico, incluindo as técnicas e interpretação dos resultados, além de medidas de controle para ambas as doenças.

The present update deals with two of the most important sexually transmitted diseases of cattle: bovine genital campylobacteriosis and bovine genital trichomonosis. Epidemiological aspects, mainly their distribution in Brazil, alongside with their diagnosis in cattle are presented and commented. The main points in their diagnoses, including the description of the techniques and the interpretation of the results are also reviewed. Finally the control and prevention of both diseases are discussed.

Uso do ensaio imunoenzimático e imunoblotting para detecção de imunoglobulinas contra Campylobacter fetus em muco cérvico-vaginal de fêmeas bovinas / The use of enzyme-linked immunosorbent assay and immunoblotting for the detection of Campylobacter fetus immunoglobulins in the cervico-vaginal mucus of female cattle

Foi padronizado um ensaio imunoenzimático do tipo indireto para detecção de imunoglobulina A (ELISA IgA) anti- Campylobacter fetus subp. venerealis em muco cérvico- vaginal bovino utilizando um extrato protéico de Campylobacter fetus subsp. venerealis produzido pelo método de extração ácida pelo tampão de glicina (0,2M; pH2,2). A média dos valores de densidade ótica (DO450) foi de 0,143±0,09. As bandas protéicas dos antígenos de Campylobacter fetus subsp. venerealis e de Campylobacter fetus subsp. fetus melhor reconhecidas pela IgA do muco cérvico- vaginal migraram em 42,6 kDa mas a proteina evidenciada em 93 kDa foi reconhecida exclusivamente pelo Campylobacter fetus subsp. venerealis. Os anticorpos presentes na amostra de muco vaginal testada no “immunoblotting” que apresentou resultado positivo no ELISA IgA, reconheceu antígenos de C. jejuni subsp. jejuni e C. fetus subsp. fetus.

An indirect enzyme-linked immunosorbent assay was developed to detect antigenspecific secretory IgA antibodies to Campylobacter fetus subsp. venerealis in bovine vaginal mucus with a protein extract of the Campylobacter fetus subsp. venerealis by the acid glycine extraction method. Mean optical density measurement (λ=450 nm) was 0.143±0.9. The most immunoreactive protein bands of the Campylobacter fetus subsp. venerealis or Campylobacter fetus subsp. fetus recognized by IgA in immunoblotting, using bovine vaginal mucus samples, migrate at 42.6 kDa. The protein that migrates at 93 kDa was recognized exclusively for C. fetus subsp. venerealis. A positive vaginal mucus sample of a cow from negative herd recognized antigens of C. jejuni subsp. jejuni e C. fetus subsp. fetus.

Polymerase chain reaction for the diagnosis of bovine genital campylobacteriosis / Reação em cadeia da polimerase para o diagnóstico de campilobacteriose genital bovina

Bovine genital campylobacteriosis is a common venereal disease of cattle; the prevalence of this disease can be underestimated mostly because of the nature of the etiological agent, the microaerobic Campylobacter fetus subspecies venerealis. The purpose of the current study was to evaluate the utilization of polymerase chain reaction (PCR) in the diagnosis of genital campylobacteriosis in samples obtained from bull prepuce aspirate, cow cervical mucus, and abomasum contents of aborted fetuses, collected into enrichment medium. Five different DNA extraction protocols were tested: thermal extraction, lysis with proteinase K, lysis with guanidine isothiocyanate, lysis with DNAzol, and lysis with hexadecyltrimethylammonium bromide (CTAB). The specificity, sensitivity, and technical application of the PCR assay were also evaluated with clinical samples and compared to bacterial isolation by standard culture. DNA extraction by the CTAB protocol provided better results in PCR, and it was able to detect 63 colony-forming units per ml of C. fetus. Out of 277 clinical samples tested, 68 (24 percent) were positive for Campylobacter fetus using PCR, while only 8 (2.8 percent) of the samples were positive by bacterial isolation in solid medium, proving the superiority of the PCR technique when compared to the standard isolation method, and providing evidence for its usefulness as a better screening test in cattle for the diagnosis of bovine genital campylobacteriosis.

Campilobacteriose genital bovina é uma doença venérea comum em bovinos. A prevalência desta doença pode ser subestimada na maioria das vezes pela natureza microaeróbica do agente etiológico, Campylobacter fetus subspecies venerealis. O propósito do presente estudo foi avaliar a utilização da reação de polimerase em cadeia (PCR) no diagnóstico de campilobacteriose genital em amostras obtidas de aspirado prepucial de touros, muco cervical de vacas e conteúdo abomasal de fetos abortados, coletados em meio enriquecido. Cinco protocolos diferentes de extração de DNA foram testados: termo extração, lise com proteinase K, lise com guanidine isothiocyanate, lise com DNAzol e lise com hexadeciltrimetilamônio brometo (CTAB). A especificidade, sensibilidade e a aplicação da técnica da PCR foram também avaliadas com amostras clínicas e comparadas com bactérias isoladas por cultura padrão. DNA extraído pelo protocolo de CTAB demonstrou os melhores resultados na PCR, e foi capaz de detectar 63 unidades formadoras de colônias de C. fetus por ml de meio. Das 277 amostras clínicas testadas, 68 (24 por cento) foram positivas para Campylobacter fetus pela PCR, enquanto 8 (2,8 por cento) das amostras foram positivas por isolamento bacteriológico, provando a superioridade da técnica de PCR quando comparada com métodos padrão de isolamento, e fornecendo evidências de sua utilização como um teste de melhor projeção para diagnóstico em campilobacteriose genital bovina.