Peer reviewed Brief communication Detection of Torque teno sus virus in diarrheic piglet fecal samples positive or negative for porcine group A rotavirus Raquel de Arruda Leme, DVM, MSc; Elis Lorenzetti, DVM, PhD; Alice F. Alfieri, DVM, PhD; Amauri A. Alfieri, DVM, PhD Summary Resumen - Detección del Torque teno sus Résumé - Détection du Torque teno Association of Torque teno sus virus virus en muestras fecales de lechones diar- sus virus à partir d’échantillons fécaux (TTSuV) and porcine group A rotavirus reicos positivas o negativas al rotavirus provenant de porcs diarrhéiques positifs (PoRVA) was evaluated in PoRVA-positive porcino grupo A ou négatifs pour le rotavirus porcin du groupe A or PoRVA-negative diarrheic piglet fecal Se evaluó la asociación del Torque teno samples. Molecular TTSuV detection was sus virus (TTSuV) y el rotavirus porcino L’association du Torque teno sus virus 40.4% (21/52) and 53.3% (49/92) in grupo A (PoRVA por sus siglas en inglés) en (TTSuV) et du rotavirus porcin de groupe PoRVA-positive and -negative fecal samples, muestras fecales de lechones diarreicos nega- A (PoRVA) fut évaluée dans des échantil- respectively. No association (P = .19) was tivos o positivos al PoRVA. La detección lons fécaux provenant de porcs diarrhéiques observed between TTSuV and PoRVA diar- molecular del TTSuV fue 40.4% (21/52) y PoRVA-positifs ou PoRVA-négatifs. La rhea. 53.3% (49/92) en muestras fecales positivas détection moléculaire de TTSuV était de Keywords: swine, intestinal health, diar- y negativas al PoRVA, respectivamente. No 40,4% (21/52) et 53,3% (49/92) dans les rhea, porcine enteric viruses, Torque teno se observó asociación (P = .19) entre TTSuV échantillons PoRVA-positifs et PoRVA- sus virus y PoRVA en las diarreas. négatifs, respectivement. Aucune association (P = 0,19) ne fut notée entre TTSuV et Received: November 25, 2013 PoRVA dans les diarrées. Accepted: April 14, 2014 orque teno virus (TTV), a member gross or histological lesions, and infection reaction (PCR) assay in pigs experimentally of the family Anelloviridae, is a is common in both healthy and diseased infected with CSFV, the TTSuV2 serum non-enveloped virus with a single- pigs.1 Studies have evaluated TTSuV infec- load was significantly larger in pigs with Tstranded, negative-sense, circular DNA tion as a contributor to the emergence or clinical signs of disease than in the healthy genome. Infection has been demonstrated worsening of other important viral diseases controls.6 in multiple species, including humans and of economic and public health impact. It Torque teno sus virus has been reported in swine.1 The virus genome can be detected is believed that TTSuV may contribute to bone marrow and peripheral blood mono- in various organs, secretions, and excretions these clinical syndromes as a co-infection nuclear cells from clinically healthy pigs and from both humans and animals.1,2 associated with porcine circovirus 2 (PCV2) in various fetal tissue samples.7 The presence and hepatitis E virus (HEV) infections.1,4 In pigs, the virus is named Torque teno sus of TTSuV DNA in intestinal samples8-10 Studies have evaluated TTSuV infection in virus (TTSuV) and is categorized into two and the high rates of TTSuV detection in association with porcine reproductive and genera. Genus Iotatorquevirus includes the fecal samples11-13 suggests that enterocytes respiratory syndrome virus (PRRSV) and species Torque teno sus virus 1a and Torque might be targets for virus replication. classical swine fever virus (CSFV); however, teno sus virus 1b (TTSuV1), and the genus no correlation between TTSuV, PRRSV, Maintenance of intestinal health is essential Kappatorquevirus includes the species Torque and CSFV clinical signs or diseases has to ensure pig productivity. Neonatal diarrhea teno sus virus k2 (TTSuV2).3 been identified.1,5 In contrast, when loads is one of the most economically important Torque teno sus virus has not been associ- of TTSuV DNA were evaluated by means syndromes affecting piglets worldwide.14 ated with specific clinical pathology or of a real-time quantitative polymerase chain Occurrence of diarrhea depends on several factors, including host immunity, manage- Laboratory of Animal Virology, Department of Veterinary Preventive Medicine, Universidade Estadual ment procedures, and infectious agents (bac- de Londrina, Londrina, Paraná, Brazil. teria, protozoa, and viruses). Microorgan- Corresponding author: Dr Amauri A. Alfieri, Laboratory of Animal Virology, Department of isms in single or mixed infections may be the Veterinary Preventive Medicine, Universidade Estadual de Londrina, Campus Universitário, PO Box determining factor for occurrence of neona- 10011, 86057-970, Londrina, Paraná, Brazil; Tel: +55 43 3371 5876; Fax: +55 43 33714485; tal diarrhea. The health or immunological E-mail: [email protected]. status of individual animals, environmental This article is available online at http://www.aasv.org/shap.html. conditions, and management procedures de Arruda Leme R, Lorenzetti E, Alfieri AF, et al. Detection of Torque teno sus virus in diarrheic piglet associated with concurrent infections may fecal samples positive or negative for porcine group A rotavirus. J Swine Health Prod. 2014;22(6): enhance the severity of clinical disease.14-16 287–290. Journal of Swine Health and Production — Volume 22, Number 6 287 Although TTSuV is excreted in diarrheic Fecal suspensions were prepared and the samples included in this analysis were diar- feces, to the knowledge of the authors, no supernatants were used for nucleic acid rheic, the study did not intend to evaluate studies have sought association of TTSuV extraction.17 Polymerase chain reaction TTSuV as a causative agent of diarrhea. For with important enteric virus infections. assays were performed using specific primers this, the presence of other enteric pathogens for TTSuV1 and TTSuV2 in a previously (bacteria, protozoa, and various viruses) Porcine group A rotavirus (PoRVA) is impli- described technique.12,18 Two positive should be investigated. cated in enteric diseases of pigs. It causes a samples for each TTSuV genus were ran- common health problem and is the most fre- Results based on each TTSuV genus in pig- domly selected by drawing lots for sequence quent viral etiological agent involved in the lets aged 1 to 3 weeks are in agreement with analysis to confirm the specificity of the pig neonatal diarrhea complex throughout a Brazilian study13 that evaluated TTSuV amplicons obtained. the world.16 Most studies on TTSuV infec- infection at various stages of the pig produc- tion in association with other viruses have Statistical analysis was performed with Epi tion cycle. The TTSuV1 genus was detected focused on hepatic, respiratory, reproduc- Info (http://wwwn.cdc.gov/epiinfo/) in fecal samples from suckling piglets more tive, or multisystemic diseases. The aim of using chi-square (χ²) analysis to compare frequently (P < .05) than the TTSuV2 genus this study was to determine the frequency of the percentages of positive samples for each or mixed infections of both genera. TTSuV DNA detection in feces of diarrheic TTSuV genus between and within both Piglet fecal samples included in this study piglets previously identified as PoRVA-pos- groups of fecal samples analyzed (PoRVA- were tested for PoRVA immediately after itive or PoRVA-negative by polyacrylamide positive and PoRVA-negative), and to collection. In acute infections, high loads gel electrophoresis (PAGE). determine whether detection of TTSuV was of PoRVA are shed in feces (1010-12 virus associated with PoRVA diarrhea. The confi- particles per gram of feces).9 This facilitates dence limit for the statistical tests was set at Materials and methods diagnosis by the PAGE technique, which 95% (P < .05). This study is in agreement with the ethical is considered of high specificity for PoRVA principles determined by the Brazilian Col- detection. The same does not apply, for lege of Animal Experimentation (COBEA) Results example, to the atypical rotaviruses, which and was approved by the Ethics Committee Of the 144 diarrheic suckling piglet fecal are eliminated in feces in smaller loads19 on Animal Experimentation of the Universi- samples included in this study, 48.6% (70) and cannot always be detected by the PAGE dade Estadual de Londrina. were positive for TTSuV. The specificity of technique. For this reason, and to maintain the amplicons obtained for each TTSuV In total, 144 piglet diarrheic fecal samples consistency since 2004, the PAGE technique genus was confirmed during sequence were included in this study. The samples were is considered a useful tool to screen fecal analysis. The detection rate for TTSuV1 derived from a collection of feces (2004 to samples for PoRVA. was higher (P < .05) than that for either 2012) that had been stored at 4°C. Fecal sam- TTSuV2 or a combination of both genera Fecal samples included in this study were ples were selected on the basis of the Brazilian in both groups evaluated (PoRVA-positive stored for diagnostic purposes at 4°C to state of origin (specifically, South, Midwest, and PoRVA-negative samples). However, avoid repeated freezing and thawing, which and Southeast areas of Brazil where commer- TTSuV1, TTSuV2, or co-infection detec- would accelerate degradation of nucleic acid cial swine production is concentrated), age of 20 tion rates did not differ between the in the samples. Molecular assays target- the animals, fecal consistency, and previous PoRVA-positive and PoRVA-negative groups ing