Pesq. Vet. Bras. 33(7):840-846, julho 2013 Torque teno sus virus (TTSuV) infection at different stages of pig production cycle1 Raquel de A. Leme2 2 2* ABSTRACT.- , Alice F. Alfieri and AmauriTorque tenoA. Alfieri sus virus (TTSuV) infection at different stages of pig production cycle. Pesquisa Veterinária Brasileira 33(7):840-846.Leme Laboratório R.A., Alfieri de VirologiaA.F. & Alfieri Animal, A.A. Departamento2013. de Medicina Veterinária Preventiva, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Campus Univer- sitário, Cx. Postal Londrina, PR 86057-970, Brazil E-mail: Torque teno sus virus (TTSuV) infection is present in pig herds worldwide. It has been demonstrated that10011, TTSuV might increase the severity of other [email protected] viral diseases with economic and public health impacts. At present, there is no information on the age distribution of pigs infected with TTSuV in Brazilian herds. This study evaluated the fre- quency of TTSuV infection in pigs at different stages of production. Fecal samples (n =190) from pigs at 1 to 24 weeks of age and from breeders at 6 farrow-to-weaning (up to 8 weeks of age) and 9 grower-to-finish (9 weeks of age onwards) farms in the western region of- Paraná state, Brazil, were evaluated by PCR. Fragments of the 5’ UTRs of TTSuV1 and/or infectionsTTSuVk2 DNAsby both were genera identified between in 126 and (66.3%) within theof the different fecal samples. pig production Significant stages. differen Fecal ces were found with the percentages of positive samples for TTSuV1, TTSuVk2, and mixed samples from the grower-to-finish farms had TTSuV detection rates (90.1%; 64/71) that inwere the significantly animals from (p<0.05) the other higher stages. than The those UTR fromnucleotide the farrow-to-weaning sequences in this farms study (52.1%;presen- 62/119). TTSuV detection was significantly (p<0.05) more frequent in finisher pigs than productionted higher similaritiesstages and thatto strains the viral from infection Norway rate (96%, increases TTSuV1), with andthe ageArgentina of the animals. and China In (97.1%, TTSuVk2). These results suggest that TTSuV infection has spread to pigs of all the western region of Paraná state, Brazil, TTSuV1 and TTSuVk2-induced infections were inmore pig frequentlyherds of Brazil. observed in suckling piglets and finisher pigs, respectively. Phylogenetic analysis pointed out the possibility of different strains of TTSuV1 and TTSuVk2 circulating Torque teno sus virus, Iotatorquevirus, Kappatorquevirus distribution, PCR, swine. INDEX TERMS: , TTSuV1 and TTSuVk2, age RESUMO.- [Infecção pelo Torque teno sus virus (TTSuV) há informações sobre a distribuição da infecção pelo TT- em diferentes categorias do ciclo de produção de su- SuV, de acordo com a faixa etária, em rebanhos suinícolas ínos.] A infecção pelo Torque teno sus virus (TTSuV) está brasileiros. Este estudo avaliou a frequência da infecção presente em rebanhos suinícolas em todo o mundo. Tem pelo TTSuV nas diferentes categorias de produção de suí- sido demonstrado que a infecção pelo TTSuV pode aumen- nos. Amostras fecais (n - tar a gravidade de outras importantes doenças virais com nas de idade e de reprodutores provenientes 6 unidades impactos econômicos e na saúde pública. Atualmente não produtoras de leitão (até=190) 8 semanas de suínos de idade) com 1 e a9 24unidades sema de terminação (9 semanas de idade em diante) da região oeste do Paraná, Brasil, foram avaliadas pela técnica de 1 2 LaboratórioReceived on deFebruary Virologia 7, 2013.Animal, Departamento de Medicina Veteriná- ria Preventiva,Accepted for Universidade publication on Estadual April 11, de 2013. Londrina (UEL), Rodovia Celso PCR. Fragmentos da região 5’ UTR do DNA do TTSuV1 e/- 970, Brazil. *Corresponding author: ou TTSuVk2 foram identificados em 126 (66,3%) amostras Garcia Cid, Campus Universitário, Cx. Postal 10011, Londrina, PR 86057- fecais. Diferenças significativas foram encontradas em rela [email protected] ção às porcentagens de amostras positivas para o TTSuV1, 840 Torque teno sus virus (TTSuV) infection at different stages of pig production cycle 841 - - tra categorias. Amostras fecais provenientes de unidades Leme et deTTSuVk2 terminação e infecção apresentaram mista por taxasambos de os detecção gêneros deinter TTSuV e in systems). are specific research tools that are not well estab lishedStudies for this have virus demonstrated (Kekarainen the & Segaléspresence 2009, of TTSuV in que aquelas provenientes de unidades produtoras de leitão al. 2012 - (90.1%; 64/71) significativamente (p<0.05) mais altas do- increasesthe serum with and theorgans age ofof thepigs animals of different (Sibila ages et al. (Kekara 2009b, que(52.1%; nos 62/119).suínos de A outras detecção categorias. do TTSuV As emsequências animais de ternu- inen & Segalés 2012) and have shown that the prevalence cleotídeosminação foi da significativamente UTR deste estudo (p<0.05) apresentaram mais frequente maior simi do- - Aramouni et al. 2010). In Brazil, TTSuV infection has been identified in suckling piglets (Leme et al. 2012) and slaugh alaridade infecção com pelo cepas TTSuV da Noruegaencontra-se (96%, disseminada TTSuV1) e emArgentina suínos theseter-age studies, pigs (Leme no information et al. 2013), is available and in relativethe reproductive to the age dee China todas (97,1%, as categorias TTSuVk2). de produção Estes resultados e que a taxasugerem da infec que- distributiontracts of boars of andTTSuV sows infection (Ritterbusch throughout et al. 2012). the Brazilian Despite pig production system. região oeste do estado do Paraná, infecções induzidas pelo The aims of this study were to evaluate natural infection ção viral aumenta de acordo com a idade dos animais. Na- das em leitões de maternidade e suínos de terminação, evaluate the frequency of TTSuV infection at different sta- TTSuV1 e TTSuVk2 foram mais frequentemente observa- gesby TTSuV of pig production.in 1 to 24 week-old pigs and in breeders, and to emrespectivamente. rebanhos suinícolas A análise brasileiros. filogenética indicou a possibili MATERIALS AND METHODS dade de diferentes cepas de TTSuV1 e TTSuVk2 circulando Torque teno sus virus, Iotatorquevirus, One hundred and ninety fecal samples of pigs from the western Kappatorquevirus region of Paraná state, Brazil, were included in this study. The PCR,TERMOS suínos. DE INDEXAÇÃO: samples were selected independent of their consistency (diar- , TTSuV1 e TTSuVk2, distribuição por idade, INTRODUCTION Six farrow-to-weaning farms, which include breeder sows and bo- rheic or not), collected between 2008 and 2011, and stored at 4°C. Torque teno virus (TTV) is a non-enveloped virus with a - ars, suckling piglets, and weaned pigs up to 8 weeks of age, and nine grower-to-finish farms, where 9-week-old pigs are housed circular negative-sense single-stranded DNA (ssDNA) ge and fed until they reach 24 weeks of age, were evaluated. A total of nome. This virus was first isolated from a Japanese man 119Fecal fecal samplessamples fromfor each farrow-to-weaning of the pig production farms and stages 71 samples were with non A-E post-transfusional hepatitis (Nishizawa et al. from grower-to-finish farms were selected. n - 1997). Since then, TTV has been shown to infect humans, n non-humanTorque teno primates, sus virus and farm(TTSuV) animals infection (Leary in et pigs al. 1999, was nanalyzed: suckling pigletsn (1 to 3 weeks old, =35), weaned pi Okamoto et al. 2000, 2001, 2002, Brassard et al. 2008). glets (4 to 8 weeks old, =43), finisher pigs (9 to 24 weeks old, =71), and breeders ( =41). 5,000Fecal x g suspensions were prepared at 10 to 20% (w/v) in 0.01- first described by Leary et al. (1999). The first full genome and the differentiation of TTSuV into separate species was tion.M phosphate-buffered saline (PBS), pH 7.2, and centrifuged at analysis of TTSuV was completed by Okamoto et al. (2002),- for 3 min. The supernatants were used for DNA extrac ly Anelloviridae performed by Niel et al. (2005). TTSuV belongs to the fami- To determine the frequency of TTSuV infection, viral ssDNA and is classified into theIotatorquevirus species TTSuV1a, and was extracted by using a combination of the phenol/chloroform/- (represented by Sd-TTV31 strain) and TTSuV1b (represen mediatelyisoamyl alcohol submitted (25:24:1) to a polymerase and silica/guanidinium chain reaction isothiocyanate (PCR) assay. Kappatorquevirusted by Sd-TTV1p strain) in the genus nucleic acid extraction methods (Alfieri et al. 2006) and was im TTSuVk2TTSuV (representedis widely distributed by Sd-TTV2p in pig strain) herds in thea number genus (Iotatorquevirus Kappatorquevirus) targeting the (ICTV 2012). non-codingSpecific PCRregion assays of the were viral performed genome, using and primersthe technique for TTSuV1 was of countries in Asia, Europe, and America (McKeown et al. ) and TTSuVk2 ( performed as described (Segalés et al. 2009), with modifications found2004, Nielin both et al. healthy 2005, andGallei diseased et al. 2010, pigs, Savicand a et number al. 2010, of (Leme et al. 2012). The amplification reaction was performed in studiesPérez et have al. 2011,been performedLeme et al. to 2012). determine TTSuV the infection importan is- a thermocycler (Swift™ MaxPro Thermal Cycler, Esco Healthcare ce of the virus and the role it plays in infectious diseases Pte, Singapore) at 94°C for 5 min for denaturation followed by 40 cycles of 94°C/1 min, 54°C/1 min, and 72°C/1 min and a final The detection of TTSuV infection is currently based on extensionFive at 72°C for 5 min. The expected sizes of the amplified- (Meng 2012). products were 305 and 252 bp for TTSuV1 and k2, respectively. positive samples for TTSuV1 and TTSuVk2 were ran Western blot and indirect ELISA assays to detect TTSuV2- the amplicons obtained in this study. The amplicons were puri- domly selected for sequence analysis to confirm the specificity of conventional and real-time PCR assays.