Viral Loads in Serum of Postweaning Multisystemic Wasting Syndrome (PMWS)-Affected and Healthy Pigs in Brazil

Research in Veterinary Science 101 (2015) 38–41

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Research in Veterinary Science

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Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) viral loads in serum of postweaning multisystemic wasting syndrome (PMWS)-affected and healthy pigs in Brazil

Thais Fumaco Teixeira a,b,⁎, Samuel Paulo Cibulski a,b, Helton Fernandes dos Santos a, Adriéli Wendlant a, Francisco Esmaile de Sales Lima b,CandiceSchmidta, Ana Cláudia Franco b, Paulo Michel Roehe a,b a Virology, FEPAGRO Animal Health — Instituto de Pesquisas Veterinárias Desidério Finamor (IPVDF), Estrada do Conde 6000, Eldorado do Sul, RS 92990-000, Brazil b Virology Laboratory, Department of Microbiology, Immunology and Parasitology, Instituto de Ciências Básicas da Saúde, UFRGS, Av. Sarmento Leite 500, sala 208, Porto Alegre, RS 90050-170, Brazil article info abstract

Article history: Associations between Torque teno sus viruses (TTSuVs) and the occurrence of postweaning multisystemic Received 25 March 2015 wasting syndrome (PMWS) have been reported with controversial results. Currently, no studies have been Accepted 30 May 2015 performed comparing simultaneously viral loads of TTSuVs and PCV2. To examine the role for TTSuVs in PMWS-affected animals, a SYBR Green-based quantitative PCR (qPCR) was designed to detect and quantify Keywords: TTSuV1, TTSuV2 and PCV2 genomes in swine sera. TTSuV1 genome loads were significantly higher in healthy Torque teno sus virus (TTSuV) adults than in young and SPF animals (p b 0.05) suggesting that the prevalence of TTSuV1 infection increases Anellovirus fi Postweaning multisystemic wasting syndrome with age and bears no association with PMWS. Regarding TTSuV2, no signi cant variation was detected in (PMWS) viral loads within any of the groups. As expected, PCV2 genome loads were higher in PMWS-affected swine Quantitative PCR (qPCR) than in healthy or SPF animals (p b 0.001). These findings provide clear evidence to indicate that neither TTSuV1 nor TTSuV2 viral loads have any correlation with the occurrence of PMWS. © 2015 Elsevier Ltd. All rights reserved.

Torque teno viruses (TTVs) are small, non-enveloped agents with nodes (Aramouni et al., 2010; Nieto et al., 2011; Teixeira et al., 2013; circular single stranded DNA genomes (Okamoto et al., 2002). The first Zhang et al., 2014). TTV genome identified was recovered from a Japanese patient with Recently, the International Committee on Taxonomy of Viruses post-transfusion hepatitis of unknown aetiology (Nishizawa et al., (ICTV, 2014) recognized two genera within the Anelloviridae family, 1997). Since then, a large number of TTVs infecting humans, non- due to their high genetic divergence: Iotatorquevirus, which includes human primates, tupayas, cats, dogs, swine, bats, sea lions, and mosqui- TTSuV1, and Kappatorquevirus, where TTSuV2 is classified. TTSuV infec- toes have been identified (Cibulski et al., 2014; Kekarainen and Segales, tion seems ubiquitous in both healthy and diseased swine and, so far, no 2009; Ng et al., 2011; Okamoto et al., 2002). association with disease has been demonstrated (Aramouni et al., 2010; In swine and wild boars, two genetically distinct TTVs, named Gallei et al., 2010; Teixeira et al., 2013; Zhang et al., 2014). It has been Torque teno sus viruses 1 (TTSuV1) and 2 (TTSuV2) have been identi- suggested that TTSuV infections could enhance the pathogenicity of fied (Martinez et al., 2006; Niel et al., 2005). The first description of porcine circovirus type 2 (PCV2), a virus which is considered essential, TTSuV was published by Leary et al. (1999), although evidence for though not sufficient, to induce postweaning multisystemic wasting TTSuV infections in Spanish pig farms (Segales et al., 2009) and in cell syndrome (PMWS) (Segales et al., 2005). Torque teno sus virus 1 cultures dates as early as 1985 (Teixeira et al., 2011). TTSuVs have was suggested as a potential cofactor in PMWS development in gno- been found in biological fluids such as colostrum and semen (Martinez- tobiotic pigs (Ellis et al., 2008).Torquetenosus2wasdetectedto Guino et al., 2009), as well as in tissues such as: lungs, kidneys, liver, significantly higher incidence in PMWS-affected pigs than in healthy spleen, heart, bone marrow, and mediastinal and mesenteric lymph ones (Aramouni et al., 2011; Kekarainen et al., 2006; Nieto et al., 2011). However, no significant differences were detected in TTSuV genome loads and the occurrence of PMWS (Lee et al., 2010). In view of such controversial results, this study was conducted ⁎ Corresponding author at: Virology, FEPAGRO Animal Health — Instituto de Pesquisas to quantitatively examine possible associations between the detection Veterinárias Desidério Finamor (IPVDF), Estrada do Conde 6000, Eldorado do Sul, RS 92990-000, Brazil. of TTSuV genomes and the occurrence of PMWS. To accomplish this E-mail address: [email protected] (T.F. Teixeira). task, a SYBR Green real-time PCR was designed to quantitate TTSuV1,

TTSuV2 and PCV2 DNA viral loads and to determine genome prevalence tested (Fig. 1). Likewise, no significant statistical difference was ob- in sera of PMWS-affected pigs and healthy animals. served between TTSuV1 and TTSuV2 genome loads in PMWS-affected Serum samples (181) were sent to laboratory from pig farms in the animals. state of Rio Grande do Sul, Brazil. Forty-nine sera from PMWS-affected In relation to PCV2, the MVL in PMWS-affected pigs was pigs (1–4 months old), plus 50 serum samples from healthy young pigs 3.2 × 107 copies/mL (SD ± 1.0 × 107) while in young healthy ani- (1–4 months old), as well as from 50 healthy adult pigs (6–19 months mals (1–4 months old) the MVL recorded was 5.1 × 105 copies/mL old) were used. Additionally, 32 serum samples from SPF pigs (6–- (SD ± 2.1 × 105). In adult healthy animals (6–19 months old) the 19 months old) were examined. PMWS was diagnosed with basis MVL was 2.7 × 105 copies/mL (SD ± 1.5 × 105), whereas in SPF animals on clinical signs, histopathological lesions and identification of PCV2 in the MVL was 1.0 × 103 copies/mL (SD ± 3.1 × 102). These findings tissues by conventional PCR as reported (Segales et al., 2005). DNA was reveal that the PCV2 MVL was higher in PMWS-affected swine than in extracted from 500 μL of serum with a standard phenol–chloroform healthy and SPF animals (p b 0.001). In SPF animals, unlike in the protocol (Sambrook and Russel, 2001). Primers used for the quantita- other groups of animals, the PCV2 MVL was significantly lower than tive TTSuV1, TTSuV2 and PCV2 SYBR Green real-time PCR (qPCR) are TTSuV1 and TTSuV2 MVLs (p b 0.001). shown in Supplemental Table 1. Positive controls used to construct The role for TTSuVs as a causative agent of disease in pigs has be- the standard curve are described in Supplemental methods (Dezen et come a matter of debate since its discovery (Niel et al., 2005; Okamoto al., 2011). Descriptive statistics and the Kruskal–Wallis test (with et al., 2002). In particular, associations between TTSuV and PMWS Dunn's as posttest) were performed using the GraphPad Prism5 soft- have been investigated, since relationships between the latter and ware. Differences were considered significant when p ≤ 0.05. other pathogens have been confirmed in a number of occasions (Ellis The standard curve obtained with the TTSuV1 plasmid control et al., 2000; Pogranichniy et al., 2002). Previous studies have searched (pCR2.1 + TTSuV1) showed an efficiency of ~99.4%. The detection for TTSuVs without simultaneously monitoring PCV2 viral loads. How- limit of the assay was 10 copies of plasmid DNA per reaction (Supple- ever, it is mandatory to examine PCV2 viral loads since MVLs N107/mL mental Fig. 1). For TTSuV2 qPCR, the standard curve with the TTSuV2 is a hallmark in establishing PMWS diagnosis (Brunborg et al., 2004). control (pCR2.1 + TTSuV2) showed an efficiency of ~99.8%, with a Thus, to define potential associations with disease, it is of importance detection limit of 100 copies of plasmid DNA per reaction (Supplemen- to examine not only the viral loads of TTSuVs, but also PCV2 viral tal Fig. 2). The regression coefficients (R2)were≥0.999 on both curves. loads. Although several studies have reported on TTSuV viral loads The observed temperature of dissociation for TTSuV1 and TTSuV2 qPCR (Aramouni et al., 2013; Brassard et al., 2010, 2013; Leblanc et al., was 78.75 °C and 77.41 °C, respectively. Negative controls did not give 2014; Lee et al., 2012; Nieto et al., 2012; Zhang et al., 2014), the high- rise to any amplification product, as revealed by the absence of spurious light of the present study is, for the first time, to compare simulta- peaks on the dissociation curve on both reactions. neously viral loads of both PCV2 and TTSuVs in healthy and PMWS- Frequencies of detection of TTSuV1, TTSuV2 and PCV2 genomes in affected pigs. In order to address this issue, here, highly sensitive the four groups of pigs in this study are summarized in Table 1. Both qPCRs were developed to detect and quantify TTSuV1, TTSuV2 and TTSuV species and PCV2 were detected at high frequencies in sampled PCV2 viral loads. The results obtained showed no correlation be- animals in all groups, and no significantly difference was observed tween TTSuV1 and TTSuV2 viral loads and the occurrence of PMWS. (p N 0.05). Four serum samples were negative for both TTSuV1 and Genomes of both TTSuV1 and TTSuV2 were detected in all groups TTSuV2 (4/181, 2.2%); one of these was from a PMWS-affected pig, of animals tested. Additionally, a significant finding was that older two were from healthy young pigs and the remainder from a SPF pig. animals tend to have higher TTSuV1 MVLs, regardless of their PMWS Regarding PCV2, only 17 sera were negative; five of those from adult status. pigs and twelve from SPF pigs. Some studies have detected a significant increase in TTSuV2 viral Mean viral loads (MVLs) of TTSuV1 and TTSuV2 genomes were loads in PMWS-affected pigs (Aramouni et al., 2011). However, the find- quantified and compared in all groups (Fig. 1). For TTSuV1, the MVL ings reported here are in agreement with other researchers, albeit in PMWS-affected pigs was 2.3 × 106 copies/mL (SD ± 9.8 × 105), employing different approaches, no correlation between TTSuVs and whereas in young healthy animals (1–4 months old) the MVL occurrence of PMWS could be established (Blomstrom et al., 2010; Lee was 1.6 × 105 copies/mL (SD ± 5.5 × 104). In adult healthy animals et al., 2010). In addition, TTSuV1 viral loads in adult animals were signif- (6–19 months old) the TTSuV1 MVL was 8.0 × 105 copies/mL icantly higher than those in healthy young and SPF animals; this is in (SD ± 4.0 × 105). In SPF animals, the MVL was 5.9 × 104 copies/mL accordance with previous reports (Martinez-Guino et al., 2009; Sibila (SD ± 1.4 × 104). TTSuV1 loads in healthy adults were significantly et al., 2009a, 2009b) where TTSuV prevalence in serum was found to higher than those in healthy young and SPF animals (p b 0.05). increase with ageing. For TTSuV2, the MVL in PMWS-affected pigs was 2.84 × Regarding the SPF animals, the PCV2 viral load was much lower than 106 copies/mL (SD ± 1.4 × 106) whereas in young healthy animals (1– those of TTSuV1 and TTSuV2. Clearly, these were not expected to be in- 4 months old) it was 5.2 × 106 copies/mL (SD ± 3.5 × 106). In adult fected with such viruses; however, to the knowledge of the authors, healthy animals (6–19 mouths old) the MVL was 2.4 ×106 copies/mL TTSuV testing is not included in screening test protocols for SPF animals. (SD ± 1.5 × 106) and in SPF animals the MVL was 7.6 × 105 copies/mL Therefore, before conducting experiments with SPF pigs, it might be of (SD ± 2.6 × 105). These findings reveal no significant differences interest to investigate the animal status in relation to PCV2 and TTSuVs (p N 0.05) among TTSuV2 viral loads in any of the different groups to prevent misleading results.

Table 1 Frequency of detection (%) of TTSuV1, TTSuV2 and PCV2 in serum samples from PMWS-affected pigs, healthy young and adult pigs and in speciﬁc pathogen free (SPF) animals.

PMWS-affected pigs Healthy young pigs Healthy adult pigs SPF pigs % (n/total) % (n/total) % (n/total) % (n/total)

TTSuV1 89.8 (44/49) 90.0 (45/50) 100 (50/50) 87.5 (28/32) TTSuV2 89.8 (44/49) 80.0 (40/50) 72.0 (36/50) 68.7 (22/32) TTSuV1/TTSuV2 81.6 (40/49) 74.0 (37/50) 72.0 (36/50) 59.4 (19/32) PCV2 100 (49/49) 100 (50/50) 90.0 (45/50) 62.5 (20/32) PCV2/TTSuV1 89.8 (44/49) 90.0 (45/50) 86.0 (43/50) 56.2 (18/32) PCV2/TTSuV2 89.8 (44/49) 80.0 (40/50) 64.0 (32/50) 40.6 (13/32) PCV2/TTSuV1/TTSuV2 81.6 (40/49) 74.0 (37/50) 62.0 (31/50) 37.5 (12/32) 40 T.F. Teixeira et al. / Research in Veterinary Science 101 (2015) 38–41

Fig. 1. Mean genome loads for TTSuV1, TTSuV2 and PCV2 in serum of PMWS-affected pigs and healthy control groups (young, adult and SPF pigs). Bars represent standard deviations. Signiﬁcant differences among groups are indicated: *(p b 0.05) and ***(p b 0.001).

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