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Treponema Denticola in Microflora of Bovine Periodontitis1

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Treponema Denticola in Microflora of Bovine Periodontitis1 Pesq. Vet. Bras. 35(3):237-240, março 2015 DOI: 10.1590/S0100-736X2015000300005 Treponema denticola in microflora of bovine periodontitis1 2 3 4 5* Ana Carolina Borsanelli , Elerson Gaetti-Jardim Júnior , Jürgen Döbereiner ABSTRACT.- and Iveraldo S. Dutra Trepo- nema denticola in the microflora of bovine periodontitis. Pesquisa Veterinária Brasileira 35(3):237-240.Borsanelli A.C., Gaetti-Jardim Júnior E., Döbereiner J. & Dutra I.S. 2015. Departamento de Apoio, Produção e Saúde Animal, Faculdade de Medicina Veterinária de Araçatuba, Universidade Estadual Paulista, Rua Clóvis Pestana 793, Cx. Postal 533, Jardim Dona Amélia, Araçatuba, SP 16050-680, Brazil. E-mail: [email protected] Periodontitis in cattle is an infectious purulent progressiveTreponema disease species associated in periodontal with strict anaerobic subgingival biofilm and is epidemiologically related to soil management at several locations of Brazil. This study aimed to detect pockets of cattle with lesions deeper than 5mm in the gingival sulcus of 6 to 24-month-old animals considered periodontally healthy. WeT. amylovorumused paper conesT. denticola to collectT. maltophilum the materials,T. mediumafter removal and T. of vincentii supragingival plaques, and kept frozen (at -80°C) up to DNA extraction and polymerase chain reactionTreponema (PCR) amylovorum using , , T. denticola, T. primers. maltophilum In periodontal pocket, it was possible to identify by PCR directly, the presenceT. amylovorum of T. denticola in 73% of animals (19/26),T. maltophilum in 42.3% (11/26)Treponema and medium and in T. 54% vincentii (14/26). Among the 25 healthy sites, it was possible to identify Treponema in 18 amylovorum (72%), T. maltophilum in two (8%) and in eight (32%). T. denticola were not detected over all 51 evaluated samples. The presence of , and, in particular, the widely recognized in subgingival microflora brings an original and potencially Treponema denticolaimportant contribution in studies of the bovine periodontitis. INDEX TERMS: Bovine periodontitis, periodontal disease, subgingival microflora, RESUMO.- [Treponema .denticola na microbiota da pe- - riodontite bovina.] A periodontite bovina é um processo tar espécies de Treponema presentes na bolsa periodontal infeccioso purulento e progressivo associado à presença degeográficas bovinos comdo Brasil. lesões O de trabalho profundidade teve por maior objetivo que 5mmdetec e - do sulco gengival de animais com idade de 6 a 24 meses camente relacionada ao manejo do solo em amplas áreas - de biofilme subgengival anaeróbio estrito e epidemiologi e considerados periodontalmente sadios. Os materiais fo 1 ram colhidos por meio de cones de papel, após a remoção 2 Received on January 5, 2015. cadeiado biofilme da polimerase supragengival, (PCR) ecom mantidos o emprego sob decongelamento iniciadores Accepted for publication on March 16, 2015. de(-80°C) até a extração do DNA e realização da reação eme Programa de Pós-Graduação em Medicina Veterinária, Faculdade de T. amylovorum T. denticola T. maltophilum T. medium Cências Agrárias e Veterinárias, Universidade Estadual Paulista (Unesp), T. vincentii - Via3 de Acesso Professor Paulo Donato Castellane s/n, Jaboticabal, SP- mais , , , - 14664-900, Brazil. E-mail: [email protected] ça de Treponema. Na bolsa amylovorum periodontalde de 73% ((19/26) dosT. denti ani- Departamento de Patologia e Propedêutica Clínica, Faculdade de Od cola foi possível ( detectar) T. maltophilum diretamente, pela PCR,sítios a presen sadios ontologia4 de Araçatuba, Unesp, Rua José Bonifácio 1193, Araçatuba, SP- em , 42,3% T. amylovorum11/26) em dois 16015-050, Brazil. E-mail: [email protected] e T.de denticola 54% 14 /26e em oito T. maltophilumDos 25 Trepo-, Ex-Pesquisador da Empresa Brasileira de Pesquisa Agropecuária (Em nema18 medium(72%) foi e possívelT. vincentii identificar n , nas brapa),5 General Editor of “Pesquisa Veterinária Brasileira”, Seropédica, RJ- 23897-970, Brazil. E-mail: [email protected] amostras(8%) avaliadas A presença (32%) de Treponema amylovorum. Departamento de Apoio, Produção e Saúde Animal, Faculdadeisdutra@ de Me T. maltophilum na microbiota subgengivalão foram detectados, e em especial 51do dicina Veterinária de Araçatuba, Unesp, Rua Clóvis Pestana 793, Jardim . T. denticola,, Dona Amélia, Araçatuba, SP 16050-680. *Corresponding author: fmva.unesp.br amplamente reconhecido periodontopatógeno 237 238 Ana Carolina Borsanelli et al. MATERIALS AND METHODS Periodontitis clinical characterization and sample collection traz uma contribuição original de importância potencial - nos estudos da periodontite bovina. Treponema denticola TERMOS DE INDEXAÇÃO: Periodontite bovina, doença periodon Clinical status of 6 to 24-month-old cattle was established tal, microbiota subgengival, . after intra-oral and periodontal evaluation, considering during INTRODUCTION all stages the Ethics Committee on Animal Experiment criteria- Cara inchada - (Process FOA nº 2013-01402). The indicators for periodontal lesion identification were the same as those observed by Döbe “ ” in cattle is a purulent progressive perio reiner et al. (2000) that consist of dental arch visible aspects, dontitis associated with strict anaerobic Gram-negative which was performed by animal containment and with the aid microorganisms. The disease of peculiar epidemiological pocketof a mouth (n=26) opener, and andfrom probing gingival to sulcus measure of animalsperiodontal considered pocket characteristics had great economic and health importance- depth. Samples were obtained from injured bovine periodontal in Brazilian cattle breeding from the 1960s to the 1980s.- periodontally healthy (n=25) from farms considered endemic or- Initially, the condition was associated with new large pas- harmless for the disease. Gingival sulcus sampling was carried- ture areas in Southeastern, Midwestern and Northern Bra out from cattle with periodontal pockets deeper than 5mm be zil (Döbereiner et al. 2000). The disease recurs in appa tween the palatal medial edge of the second and third jaw pre rent clinical manifestation in herds after grazing reform or molar tooth. when cattle in dentition stage are fed with forage grown in samplingPeriodontal procedures pocket of gingivalsampling sulcus was madeor periodontal after food pocket removal, ma- an endemic area (Dutra etcara al. 1993,inchada Döbereiner et al. 2004). when needed. Gaetti-Jardim Jr et al. (2012) have described the Pathogenic microorganisms in periodontal pocket of Bacteroidescalves is a constant Fusobacteriumin “ ” cultivation through- terial. After supragingival bacterial biofilm removal with a sterile conventional culture media, especially black-pigmented gauze pad, samples were collected by paper cone, which was left species, spp. and other anaero for about 60 seconds. Then, the cone was transferred to a tube bic Gram-negative bacteria (Blobel et al. 1984, Dutra et al.- containing 1ml of sterile ultrapure water, and stored at -80°C until DNA extraction. mless1986, onesBotteon results et al. in 1993). spontaneous In this clinicalcontext, remission the transfer of pe of- Bacterial identification by polymerase chain reaction (PCR) affected animals from periodontitis endemic areas to har Bacteroides Each sample bacterial DNA detection in sterile ultrapure riodontal pocket microflora process and its modification, water was priory performed by commercial DNATreponema extraction amy kit- especially the black-pigmented (Dutra et al.- lovorum(GenEluteT. Mammaliandenticola T. Genomicmaltophilum DNAT. Miniprep medium andKit, T.Sigma). vincentii In 2000). addition, specific primers were used to identify Throughout several clinical forms of periodontal dise , , , l volumes containing ase in humans, spirochetes in microflora are associated (Table 1). ++ - with high risk of developing specific site injury (SocranskyTreponema Amplifications were performed in 25μ denticola& Haffajee 2010). Regarded as an important periodontal- 11.9μl water for PCR, 5μl PCR/Mg buffer (Boehringer Man pathogen and part of Socransky’s red complex, nheim, Indianapolis, IN, USA), 1μl dNTP (Pharmacia Biotech,- (Socransky et al. 1998) is more common in perio Piscataway, NJ, USA), 0.1μl Taq DNA polymerase (Invitrogen do dontal disease sites than in healthy ones, and more often Brasil, São Paulo, SP, Brazil), 0.2μl of each primer pair (Invit rogen do Brasil) and 5μl of the sample. This amplification was found in subgingival than in supragingival plaques (Riviere performed in a PCR apparatus (Perkin Elmer GeneAmp PCR et al.In 1992, order Haffajeeto increase et al. knowledge 1998, Ximénez-Fyvie on bovine periodontitis et al. 2000, System 9700, Norwalk, CT, USA) programmed for one cycle at Avila-Campos & Velásquez-Melendéz 2002). - 94°C (5min), and 30 to 36 cycles at 94°C (1min). The annealing- temperature of each primer was programmed for a time ranging Treponemamicroflora, this study aimed to identify by means of poly- from 30 seconds to one minute, 2min at 72°C and a final exten merase chain reaction (PCR), spirochete species from the sion of 5min at 72°C. PCR amplification products were subjected genus in subgingival biofilm samples from cat to electrophoresis on 1% agarose gel and staining with ethidium tle with and without periodontitis.Table 1. Polymerase chain reaction (PCR) primersbromide used (0.5mg/ml). to identify Treponema spp. genus species within subgingival microflora of cattle with periodontitis and healthy sites of animals without clinical evidence of the disease Treponema
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