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Diagnosis of Brachyspira Pilosicoli, Brachyspira Hyodysenteriae And

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Diagnosis of Brachyspira Pilosicoli, Brachyspira Hyodysenteriae And Pesq. Vet. Bras. 39(7):476-480, July 2019 DOI: 10.1590/1678-5150-PVB-6030 Original Article Livestock Diseases ISSN 0100-736X (Print) ISSN 1678-5150 (Online) PVB-6030 LD Diagnosis of Brachyspira pilosicoli, Brachyspira hyodysenteriae and Brachyspira intermedia in hens and laying hens in the western region of Paraná through bacterial isolation and identification in qPCR1 Thiago Goulart2, Leonardo Gruchouskei2 , Jessica Gonçalves2, 2, Monica R. Matos2 , Mayane Faccin2 and Aline de M. Viott2* ABSTRACT.-João Pedro Cavasin Goulart T., Gruchouskei L., Gonçalves J., Cavasin J.P., Matos M.R., Faccin M & Viott A.M. 2019. Diagnosis of Brachyspira pilosicoli, Brachyspira hyodysenteriae and Brachyspira intermedia in hens and laying hens in the western region of Paraná Diagnosis of Brachyspira pilosicoli, Brachyspira through bacterial isolation and identification in qPCR. Pesquisa Veterinária Brasileira 39(7):476-480. Laboratório de Patologia Veterinária, Universidade Federal do Paraná, Setor Palotina, hyodysenteriae and Brachyspira intermedia Av. Pioneiro 2153, Palotina, PR 85450-000, Brazil. E-mail: [email protected] in hens and laying hens in the western region Bacteria of the genus Brachyspira can cause enteric diseases in poultry causing a decrease in of Paraná through bacterial isolation and such as Australia, Italy, and the United States, but in Brazil, until now, epidemiological studies identification in qPCR productivity.about Brachyspira The occurrence sp. frequency of this were disease only carried in chickens out on has pig already farms. Thebeen objective verified ofin thiscountries study was to evaluate the presence of bacteria of the genus Brachyspira sp. through isolation and Brachyspira pilosicoli, Brachyspira hyodysenteriae and Brachyspira [Diagnóstico de Brachyspira pilosicoli, Brachyspira intermedia using the qPCR technique. Samples from 110 hens aged from 35 to 82 weeks hyodysenteriae e Brachyspira intermedia em aves de confirmationwere collected, of 40the were species from commercial egg farms and 70 were from laying hens matrices. were submitted to qPCR to identify the three species proposed. Cecum fragments of the birds postura e matrizes de corte na região oeste do ParanáqPCR]. For the first evaluation, bacterial isolation was performed from the feces. Positive samples através do isolamento bacteriano e identificação na cells. Of the 110 samples, 48 characteristic isolates of Brachyspira (43.6%) were obtained Gruchouskei L., Goulart T., Gonçalves J., Cavasin J., Matos were collected and fixed in formaldehydeB. hyodysenteriae for histological (11.8%)evaluation and and 5 countingall from the of goblet same M.R., Faccin M & Viott A.M. 476- farm as Brachyspira intermedia (4.5%), 2 samples were positive for both agents (1.8%) and 480 and28 were of these not characterized in qPCR 13 identified by qPCR (25.5%).as None histopathological lesions were observed goblet cells of the positive hens. It can be evidenced by the occurrence of Brachyspira sp. in inlaying the chickenfarms and cecum hens and in Brazil, no significant with special statistical relevance difference to Brachyspira was noticed intermedia in the countthat can of be potentially pathogenic for these animals. INDEX TERMS: Brachyspira pilosicoli, Brachyspira hyodysenteriae, Brachyspira intermedia, hens, laying hens, Paraná, bacterial isolation, qPCR, commercial birds, frequency, enteropathogens, spirochetosis, bacterioses. RESUMO.- [Diagnóstico de Brachyspira pilosicoli, Brachyspira entéricas em aves acarretando a queda de produtividade. hyodysenteriae e Brachyspira intermedia em aves de postura e matrizes de corte na região oeste do Paraná em países como a Austrália, Itália e Estados Unidos, porém através do isolamento bacteriano e identificação na qPCR.] noA ocorrência Brasil, até destao momento, enfermidade trabalhos em epidemiológicosgalinhas já foi verificada sobre a Bactérias do gênero Brachyspira podem ocasionar enfermidades frequencia de Brachyspira sp. só foram realizados em granjas de suínos. O objetivo deste estudo foi avaliar a presença de 1 Received on December 9, 2018. bactérias do gênero Brachyspira sp. através do isolamento e Accepted for publication on January 31, 2019. 2 Laboratório de Patologia Veterinária, Universidade Federal do Paraná Brachyspira pilosicoli, Brachyspira (UFPR), Setor Palotina, Av. Pioneiro 2153, Palotina, PR 85450-000, Brazil. hyodysenteriae e Brachyspira intermedia utilizando a técnica *Corresponding author: [email protected] deconfirmação qPCR. Foram das coletadasespécies amostras de 110 aves com idade 476 Diagnosis of Brachyspira pilosicoli, Brachyspira hyodysenteriae and Brachyspira intermedia in hens and laying hens in the western region of Paraná 477 entre 35 e 82 semanas, sendo 40 de granjas de postura was 72.4% in laying granges and 31% of these samples were pathogenic (Bano et al. 2008). In Australia the prevalence primeiramente procedeu-se o isolamento bacteriano a partir found was 42% in broilers and 68% in laying hens (Stephens comercialdas fezes. eAs 70 amostras de granjas positivas de matrizes foram de corte.submetidas Para avaliação a qPCR & Hampson 2002); and recently in Argentina, 44% positivity was found in the evaluated laying hens (Illanes et al. 2016). Due to the lack of data on the occurrence of AIS in Brazil, para identificação das três espécies propostas. Fragmentos deDas ceco 110 amostrasdas aves foram obtidos coletados 48 isolamentose fixados em característicos formol para Brachyspira sp. in commercial birds and broiler breeders in avaliaçãode Brachyspira histológica e contagem de células caliciformes. westernthis study Paraná evaluated State, forthrough the first selective time theanaerobic occurrence culture, of como B. hyodysenteriae (11,8%) e 5 sendo todas da mesma granja (4,5%) como (43,6%) B. intermedia e destes, 2na amostras qPCR 13 foram identificadas positivas B. pilosicoli, B. intermedia and B. hyodysenteriae species. and isolates were identified using the qPCR technique for MATERIALS AND METHODS para ambos os agentes (1,8%) e 28 não foram caracterizadas atravéscontagem da de qPCR células (25,5%). caliciformes Não foram das aves observadas positivas. alteraçõesConclui-se Samples. This study was approved by the Animal Use Ethics histopatológicasque a Brachyspira no sp. ceco é frequente e diferença em estatística granjas designificativa poedeiras na e Committee of the Pallottine Sector of the “Universidade Federal do matrizes de corte no Brasil, com especial relevância para a Paraná” (UFPR) under the protocol CEUA-Palotina 06/2017. One B. intermedia que possui potência patogênico para estas aves. hundred and ten samples were collected, all in farms located in western Paraná. Forty samples of hens kept in cage were collected TERMOS DE INDEXAÇÂO: Brachyspira pilosicoli, Brachyspira from two commercial poultry farms located in the district of Céu hyodysenteriae, Brachyspira intermedia, aves de postura, matrizes de corte, Paraná, isolamento bacteriano, qPCR, aves comerciais, of cages and the birds were 82 weeks old, the other farm had no frequência, enteropatogenos, espiroquetose, bacterioses. Azul, of the Californian style. One of the properties had four floors This last property had also a pig grange. The other 70 samples were fences, only one floor of cages and birds were aged 35 and 40 weeks. INTRODUCTION from broiler breeders coming from two breeders that had a rigorous biosafety scheme with separate nuclei and climate-controlled houses Bacteria of the Brachyspira species can cause enteric diseases, with a density of seven birds per m2. Of these, 20 samples were from especially in birds and swine, besides that, they have zoonotic the Assis Chateaubriand city broiler, divided between two nuclei aged feature. Four species of are considered pathogenic Brachyspira 47 and 59 weeks. The remaining 50 samples were from a Palotina to birds, being Brachyspira intermedia, Brachyspira pilosicoli, and and Brachyspira alvinipulli Brachyspira hyodysenteriae, one of the nuclei aged 60 weeks had a history of diarrhea at week one or more species can be concomitantly involved in the 46.city Thebreeder, birds divided were randomly into five nuclei selected aged throughout 46, 60, 65 theand house67 weeks; and development of diseases (Song & Hampson 2009, Mappley et al. then necropsied. Cecum segments were collected and immediately 2014). On hens, the Brachyspira sp. is associated with a clinical condition known as avian intestinal spirochetosis (AIS) packed in an ice-cold isotherm box and sent to the laboratory for commonly observed in older birds (Phillips et al. 2005, embeddingbacterial isolation. technique Cecum recommended fragments wereby Tolosa collected et al. and (2003) fixed for in Medhanie et al. 2013, Mappley et al. 2014). B. intermedia and 10% buffered formalin and further processed by the routine paraffin B. pilosicoli are the main causers (Stephens & Hampson 2002, Bacterial isolation. The protocol for bacterial isolation followed Bano et al. 2008), and B. alvinipulli appears in lower frequency periodic acid-Schiff (PAS) and hematoxylin-eosin (HE) staining. (Phillips et al. 2006, Feberwee et al. 2008). The clinical signs the recommendations by Neves (2012). Feces samples were seeded include chronic diarrhea, weight loss, low egg productivity, by plating with Brachyspira sp. (anaerobiosis agar (Neogen Co, MI, and eggs dirty with feces (Medhanie et al. 2013). Burch et al. (2006) compared the performance of lots with AIS and the USA), 5% sheep blood, 6.25mg/μl rifampicin (Sigma-Aldrich Co, standard
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