• View in gallery

    Agarose gels revealing detection of g2-TTV DNA sequence amplicons in various Mycoplasma hyopneumoniae bacterins after PCR assay. Lanes 1, 2, and 3 contain DNA extracts from bacterins (products 2,b 3,c and 6,f respectively) that have negative results for g2-TTV DNA, whereas lanes 5, 9, and 11 contain DNA extracts from bacterins (products 1,a 8,h and 4,d respectively) that have positive results for g2-TTV DNA. Lanes 4, 6, 8, and 10 (dashed lines) contain water as negative control samples, and lane 7 contains pig serum that has negative results for TTV DNA.

  • View in gallery

    Agarose gels of nPCR assay of g2-TTV DNA for an M hyopneumoniae bacterin (product 5e) that was diluted with another M hyopneumoniae bacterin (product 2b [A]) or with water (B). Contents of the lanes were product 5 alone (plus sign); water alone (negative sign); and product 5 diluted with the other bacterin (panel A) or water (panel B) at dilutions of 2:1, 3:1, 4:1, 5:1, and 6:1 (from left to right, respectively). Notice in panel A that g2-TTV DNA was not detected until a ratio of 6:1 (product 2: product 5) was achieved, which indicated a material or materials contained in product 2 inhibited g2-TTV contained in product 5.

  • 1.

    Kyriakis SC, Saoulidis K, Lekkas S, et al. The effects of immuno-modulation on the clinical and pathological expression of postweaning multisystemic wasting syndrome. J Comp Pathol 2002;126:3846.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Allan GM, McNeilly F, Ellis JA, et al. Neonatal vaccination for Mycoplasma hyopneumoniae and postweaning multisystemic wasting syndrome: a field trial. Pig J 2001;48:3441.

    • Search Google Scholar
    • Export Citation
  • 3.

    Krakowka S, Ellis J, McNeilly F, et al. Mycoplasma hyopneumoniae bacterins and porcine circovirus type 2 (PCV2) infection: induction of postweaning multisystemic wasting syndrome (PMWS) in the gnotobiotic swine model of PCV2-associated disease. Can Vet J 2007;48:716724.

    • Search Google Scholar
    • Export Citation
  • 4.

    Irshad M, Joshi YK, Sharma Y, et al. Transfusion transmitted virus: a review on its molecular characteristics and role in medicine. World J Gasteroenterol 2006;12:51225134.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Hino S, Miyata H. Torque teno virus (TTV): current status. Rev Med Virol 2007;17:4557.

  • 6.

    Okamoto H, Takahashi M, Nishizawa T, et al. Genomic characterization of TT viruses (TTVs) in pigs, cats and dogs and their relatedness with species-specific TTVs in primates and tupaias. J Gen Virol 2002;83:12911297.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Simmonds P, Prescott LE, Logur C, et al. TT virus—part of the normal human flora? J Infect Dis 1999;180:17481750.

  • 8.

    Zein NN. TT virus infection: an emerging pathogen in search of its identity. J Pediatr 2000;136:573575.

  • 9.

    Leary TP, Erker JC, Chalmers ML, et al. Improved detection systems for TT virus reveal high prevalence in humans, non-human primates and farm animals. J Gen Virol 1999;80:21152120.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    McKeown NE, Fenaux M, Halbur PG, et al. Molecular characterization of porcine TT virus, an orphan virus, in pigs from six different countries. Vet Microbiol 2004;104:113117.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Niel C, Diniz-Mendes L, Devalle S. Rolling-circle amplification of torque teno virus (TTV) complete genomes from human and swine sera and identification of a novel swine TTV genogroup. J Gen Virol 2005;86:13431347.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Bigarre L, Beven V, de Boisseson C, et al. Pig anelloviruses are highly prevalent in swine herds in France. J Gen Virol 2005;86:631635.

  • 13.

    Kekarainen T, Sibila M, Segales J. Prevalence of swine torque teno virus in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs in Spain. J Gen Virol 2006;87:833837.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Martinez L, Kekarainen T, Sibila M, et al. Torque teno virus (TTV) is highly prevalent in the European wild boar (Sus scrofa). Vet Microbiol 2006;20:223229.

    • Search Google Scholar
    • Export Citation
  • 15.

    Gagnon CA, Tremblay D, Tijssen P, et al. The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada. Can Vet J 2007;48:811819.

    • Search Google Scholar
    • Export Citation
  • 16.

    Quintana J, Segalés J, Calsamiglia M, et al. Detection of porcine circovirus type 1 in commercial pig vaccines using polymerase chain reaction. Vet J 2006;171:570573.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Fenaux M, Opriessnig T, Halbur PG, et al. Detection and in vitro and in vivo characteristics of porcine circovirus DNA from a porcine-derived commercial pepsin product. J Gen Virol 2004;85:33773382.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Tischer I, Rasch R, Tochtermann G. Characterization of papova-virus- and picornavirus-like particles in permanent piglet kidney cell lines. Zentrabl Bakertiol [Orig A] 1974;226:153167.

    • Search Google Scholar
    • Export Citation
  • 19.

    Dulac GC, Afshar A. Porcine circovirus antigens in PK-15 cell line (ATCC CCL-33) and evidence of antibodies in Canadian pigs. Can J Vet Res 1989;53:431433.

    • Search Google Scholar
    • Export Citation
  • 20.

    Royer RL, Nawagitgul P, Halbur PG, et al. Susceptibility of porcine circovirus type 2 to commercial and laboratory disinfectants. J Swine Health Prod 2001;9:281284.

    • Search Google Scholar
    • Export Citation
  • 21.

    Allan GM, Ellis JA. Porcine circoviruses: a review. J Vet Diagn Invest 2000;12:314.

  • 22.

    Segales J, Domingo M. Postweaning multisystemic wasting syndrome (PMWS) in pigs. A review. Vet Q 2002;24:109124.

  • 23.

    Ellis JA, Krakowka S, Lairmore M, et al. Reproduction of lesions of post-weaning multisystemic wasting syndrome in gnotobiotic piglets. J Vet Diag Invest 1999;11:314.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Krakowka S, Ellis JA, Meehan B, et al. Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome by co-infection with porcine circovirus-2 (PCV 2) and porcine parvovirus (PPV). Vet Pathol 2000;37:254263.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Allan GM, Kennedy S, McNeilly F, et al. Experimental reproduction of wasting disease and death by co-infection of piglets with porcine circovirus and porcine parvovirus. J Comp Pathol 1999;121:111.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Allan GM, McNeilly F, Ellis JA, et al. Experimental infection of colostrum-deprived piglets with porcine circovirus type 2 (PCV-2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV-2 replication. Arch Virol 2000;145:24212429.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Harms PA, Sorden S, Halbur PG, et al. Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine respiratory and reproductive syndrome virus. Vet Pathol 2001;38:528539.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Opriessnig T, Thacker EL, Yu S, et al. Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2. Vet Pathol 2004;41:624640.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Krakowka S, Ellis JA, Meehan B, et al. In vivo immune activation is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2). Vet Pathol 2001;38:3142.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Opriessnig T, Yu S, Gallup JM, et al. Effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus. Vet Pathol 2003;40:521529.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Krakowka S, Allen G, Ellis JA. Evaluation of the effects of porcine genogroup 1 torque teno virus in gnotobiotic swine. Am J Vet Res 2008;69:16151622.

    • Search Google Scholar
    • Export Citation
  • 32.

    Ellis JA, Krakowka S. Effect of coinfection with genogroup 1 porcine torque teno virus on porcine circovirus type 2–associated postweaning multisystemic wasting syndrome in gnotobiotic pigs. Am J Vet Res 2008;69:16081614.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    Krakowka S, Hartunian C, Hamberg A, et al. Evaluation of induction of porcine dermatitis and nephropathy syndrome in gnotobiotic pigs with negative results for porcine circovirus type 2. Am J Vet Res 2008;69:16231629.

    • Search Google Scholar
    • Export Citation

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Evaluation of Mycoplasma hyopneumoniae bacterins for porcine torque teno virus DNAs

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  • 1 Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210
  • | 2 Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210
  • | 3 Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210
  • | 4 Queens University Belfast and Agri-Food and Biosciences Institute, Belfast, BT4 3SD, Northern Ireland
  • | 5 Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada
  • | 6 Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210
  • | 7 Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210
  • | 8 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210
  • | 9 Queens University Belfast and Agri-Food and Biosciences Institute, Belfast, BT4 3SD, Northern Ireland
  • | 10 Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK S7N 5B4, Canada

Abstract

Objective—To determine whether commercial Mycoplasma hyopneumoniae bacterins sold for use in swine contain porcine torque teno virus (TTV).

Sample Population—22 commercially available M hyopneumoniae bacterins.

Procedures—Direct and nested PCR assays for genogroup-specific TTV DNAs were performed on serials of M hyopneumoniae bacterins by use of published and custom-designed primer pairs at 3 laboratories in North America and Europe.

Results—Of the 22 bacterins tested by use of direct and nested PCR assays, 7 of 9 from the United States, 2 of 5 from Canada, and 4 of 8 from Europe contained genogroup 1– and genogroup 2–TTV DNAs. In some bacterins, the TTV DNAs were readily detected by use of direct PCR assays.

Conclusions and Clinical Relevance—Analysis of these data indicated that many of the commercially available M hyopneumoniae bacterins were contaminated with TTV DNA. It is possible that some of these bacterins could inadvertently transmit porcine TTV infection to TTV-naïve swine.

Abstract

Objective—To determine whether commercial Mycoplasma hyopneumoniae bacterins sold for use in swine contain porcine torque teno virus (TTV).

Sample Population—22 commercially available M hyopneumoniae bacterins.

Procedures—Direct and nested PCR assays for genogroup-specific TTV DNAs were performed on serials of M hyopneumoniae bacterins by use of published and custom-designed primer pairs at 3 laboratories in North America and Europe.

Results—Of the 22 bacterins tested by use of direct and nested PCR assays, 7 of 9 from the United States, 2 of 5 from Canada, and 4 of 8 from Europe contained genogroup 1– and genogroup 2–TTV DNAs. In some bacterins, the TTV DNAs were readily detected by use of direct PCR assays.

Conclusions and Clinical Relevance—Analysis of these data indicated that many of the commercially available M hyopneumoniae bacterins were contaminated with TTV DNA. It is possible that some of these bacterins could inadvertently transmit porcine TTV infection to TTV-naïve swine.

Bacterins are widely used in the swine industry as aids for prevention of bacterial infectious diseases. Of the bacterins currently recommended for use in swine, Mycoplasma hyopneumoniae products are one of the elements widely used in preventive measures for the control of PRDC. There is little doubt that these products, when used properly, will induce a protective immune response in inoculated swine, and their use to control PRDC certainly reduces economic losses associated with Mycoplasma-induced pneumonia. However, certain commercially available bacterins can potentiate the effects of PCV2 and promote the development of PMWS in PCV2-infected swine.1–3

The TTVs are recently discovered, single-stranded, circularized DNA viruses currently classified in the Circoviridae family as the genus Anellovirus.4,5 Species-specific Anelloviruses are widely distributed in human and domestic animal populations, and infected humans may remain viremic with TTV DNA for many years.4–6 At least 5 distinct clusters of human TTVs have been identified, but no positive associations have been found between TTV infection or viremia with TTV DNA and clinically relevant diseases.4,5 It is the general consensus of virologists who study TTV that the agents are orphan viruses and are a part of the normal microbial flora of humans.4–9

Swine are also infected with species-specific TTVs.9–15 Two clusters (g1-TTV and g2-TTV) have been identified in porcine serum samples.11 Similar to their human counterparts, porcine TTVs appear to be widely distributed in pigs throughout the world, and aside from a strong statistical association between viremia attributable to g2-TTV and PCV2-associated PMWS in European swine,13 the porcine TTVs are also believed to be nonpathogenic. Swine populations have been surveyed for evidence of TTV,10 and results of those efforts suggest it is possible that certain bacterins for use in pigs may harbor occult TTV. However, this possibility has not been formally examined. In support of this hypothesis, porcine circovirus type 1 DNAs have been detected in several European bacterins, which again suggests that products may contain unwanted adventitial agents.16 In the study reported here, commercially available Mycoplasma bacterins licensed in North America and Europe for use in pigs were screened for g1- and g2-TTV DNA11,13 by use of direct PCR and nPCR assays at 3 research laboratories.

Materials and Methods

Sample population—Sets of M hyopneumoniae bacterins licensed for use and commercially available in Europe, Canada, and the United States were purchased from distributors as multiple-use vials that contained 50 to 100 vaccine doses/vial. At The Ohio State University, 9 North American bacterins (products 1 through 9, respectively)a–i were examined. Aliquots from the first set of US products were used to determine whether these bacterins potentiated PCV2-associated PMWS when given to PCV2-infected gnotobiotic swine.3 A second set of bacterins was purchased from the manufacturers and stored until used for examination of TTV DNAs. At the University of Saskatchewan Western College of Veterinary Medicine, 5 Canadian bacterins (products 10 through 14, respectively)e,j–m were examined. At Queens University Belfast, 8 European bacterins (products 15 through 22, respectively)o–u were examined. Serial lot numbers and expiration dates of each bacterin were recorded.

PCR assay for TTVs—Primer pairs for g1- and g2-TTV were prepared from published sequences,13 and nPCR assays for specific TTV DNAs were performed essentially as described elsewhere.13 At the 2 research laboratories in North America, a direct PCR assay was developed to amplify g1-TTV signals from the bacterins. The direct assay used g1-TTV primers11,13 and also a primer pair specifically designed to be 100% homologous to g1-TTV identified in sera obtained from swine in Ohio; the US and Canadian bacterins evaluated were also tested by use of this PCR reagent. In addition to conventional water-only negative control samples for the experiments conducted at the laboratory in the United States, sera from gnotobiotic pigs known to have negative results for g1-TTV DNA and conventional and gnotobiotic pig serum known to contain only g1-TTV DNAs were used as positive control samples. At the other 2 laboratories, negative control samples consisted of multiple samples of water and sera obtained from Cesarean-derived, colostrum-deprived swine that were known to have negative results for TTV DNA; positive control samples at those 2 laboratories consisted of g1- or g2-TTV DNA (or both) from conventional swine. The PCR reactions were performed in triplicate and included water-only control samples between each bacterin DNA sample. The nPCR assays were considered valid only when all negative control samples had negative results for the assay. As an additional technical control sample, the US bacterins were shipped to the laboratory in Europe for testing with primer pairs and conditions used in that laboratory.

In each laboratory, bacterins were removed from the sealed vials with a syringe, and total DNA was extracted from each by use of a blood DNA extraction kitv in accordance with the manufacturer's protocol for blood. The Taq master mixturew contained Taq polymerase at a final concentration in 25μL of 1.25 U of Taq/μL, 1.5mM MgCl2, and 200μM of each deoxynucleoside triphosphate. To this, 0.5μM of each primer and 5.0 μL of template were added. A typical reaction mixture for direct PCR assay contained 12.5 μL of Taq master mixture,w 1.25 μL of forward primer (10.0μM), 1.25 μL of reverse primer (10.0M-), 5.0 μL of water, and 5.0 μL of template. Cycling conditions at the US laboratory were as follows: 40 cycles of 95°C for 15 minutes, 94°C for 30 seconds, 55°C (g1-TTV) or 54.4°C (g2-TTV) for 30 seconds, and 72°C for 60 seconds; and then 72°C for 10 minutes. For all reactions, PCR-amplified products were resolved in 1.20% (wt:vol) agarose gels in Tris-borate-EDTA buffer at 100 V for 75 minutes. The primer pairs used for the g1-TTV direct PCR assay were as follows: forward, 5′–G CGG TCA AAA TGG CGG AAG G–3′ and reverse, 5′–GGA CTT GAG CTC CCG ACC AA–3′.

For the nPCR assays, primer sequences specific for porcine g1- and g2-TTV were used.13 For the first round of PCR amplification, the same conditions as those used for the direct PCR assay for g1-TTV were used. An aliquot (4.0 μL) of product from the first round of amplification was then used in the second round of amplification. The cycling conditions used differed slightly from those used for the direct PCR assay for g1-TTV; conditions were as follows: 40 cycles of 95°C for 9 minutes, 94°C for 30 seconds, 49.7° to 52°C for 20 seconds, and 72°C for 30 seconds; and then 72°C for 7 minutes. For the g1-TTV nPCR assay, primers in the first round of amplification consisted of the following: forward, 5′–TA CAC TTC CGG GTT CAG GAG GCT– 3′ and reverse, 5′–A CTC AGC CAT TCG GAA CCT CAC–3′. For the second round of amplification, the primers used were as follows: forward, 5′–C AAT TTG GCT CGC TTC GCT CGC–3′ and reverse, 5′–TAC TTA TAT TCG CTT TCG TGG GAA C–3′. For the g2-TTV nPCR assay, primers for the first round of amplification were as follows: forward, 5′–AG TTA CAC ATA ACC ACC AAA CC–3′ and reverse, 5′–ATT ACC GCC TGC CCG ATA GGC–3′. Primers used for the second round of amplification were as follows: forward, 5′–CCA AAC CAC AGG AAA CTG TGC–3′ and reverse, 5′–CTT GAC TCC GCT CTC AGG AG–3′.

The amplified DNA products from all samples with positive results (g1-TTV, 260 to 290 bases for the nPCR assay and 120 bases for the direct PCR assay; g2-TTV, 230 bases for the nPCR assay) were recovered from the gels and independently sequenced in commercial laboratories by use of standard automated methods. Sequences for all amplicons were then compared with GenBank references for swine g1-TTV (accession No. DQ229865) and g2-TTV (accession No. DQ229860) sequences.

Results

North American bacterins—Of the 9 bacterins tested at the US laboratory, 6 (products 1, 4, 5, 7, 8, and 9) contained g1-TTV DNAs, as indicated by the fact that amplicons were recovered for both nPCR and direct PCR reactions. Three bacterins (products, 1, 4, and 8) contained g2-TTV DNAs (Figure 1). One lot of bacterin 1 had negative results when tested with published11,13 g1-TTV primers but positive results when tested by use of the direct PCR assay with the specially designed g1-TTV primers (Table 1).

Table 1—

Results* for direct PCR and nPCR assays conducted at a US laboratory to detect g1- and g2-TTV DNAs in commercially available Mycoplasma hyopneumoniae bacterins commonly used in swine in the United States.

ProductSerial No.nPCRDirect PCR
g1-TTVg2-TTVg1-TTVg2-TTV
1a271-130NDNA
271-150++NA
2b1619100ANDNA
1619112ANA
3c1151179ANDNA
1152343ANA
4dA472 266+++NA
A609 632++NA
5eA606 545Trace++NA
6f05434NDNA
95489NA
7g201 009+ND+NA
8h143-056+++NA
9i97883903A+ND+NA
Water§NANDNA
g1-TTV tissue homogenateNA+ND+NA

Results for the PCR-amplified sequence for TTV DNAs (g1-TTV, 260 to 290 bases for the nPCR assay and 120 bases for the direct PCR assay; g2-TTV, 230 bases for the nPCR assay) were scored as negative (negative sign), trace, or positive (positive sign).

Assay performed with primers specific for g1- and g2-TTVs.11,13

Primer pairs were designed from sequences amplified to be 100% homologous with the sequence for the g1-TTV recovered from swine in Ohio.

Negative control sample.

Positive control sample.

ND = Not determined. NA= Not applicable.

Figure 1—
Figure 1—

Agarose gels revealing detection of g2-TTV DNA sequence amplicons in various Mycoplasma hyopneumoniae bacterins after PCR assay. Lanes 1, 2, and 3 contain DNA extracts from bacterins (products 2,b 3,c and 6,f respectively) that have negative results for g2-TTV DNA, whereas lanes 5, 9, and 11 contain DNA extracts from bacterins (products 1,a 8,h and 4,d respectively) that have positive results for g2-TTV DNA. Lanes 4, 6, 8, and 10 (dashed lines) contain water as negative control samples, and lane 7 contains pig serum that has negative results for TTV DNA.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1601

Findings for TTV DNAs in Canadian bacterins in assays performed at the laboratory in Canada were summarized (Table 2). Two Canadian products (products 10 and 12) had positive results for both g1- and g2-TTV DNA. The remaining bacterins, which were tested by use of both the US and European laboratory extraction and hybridization protocols, had negative results for TTV DNA.

Table 2—

Results* for nPCR assays conducted at a Canadian laboratory to detect g1- and g2-TTV DNAs in commercially available M hyopneumoniae bacterins commonly used in swine in Canada.

ProductSerial No.g1-TTVg2-TTV
10i271-149++
11k1619110A
12eA606995++
13l05477A
14m07165904A
WaterNA
g1-TTV serum§NA+
g2-TTV serum§NA+

Assays performed with primers specific for g1-TTVs.11,13

Neg-ative control sample.

Positive control sample.

See Table 1 for remainder of key.

Data obtained for the European bacterins at the laboratory in Europe were summarized (Table 3). Of the 8 bacterins tested, 5 (products 15, 18, 19, 21, and 22) had positive results for both g1- and g2-TTV DNAs.

Table 3—

Results* for nPCR assays conducted at a European laboratory to detect g1- and g2-TTV DNAs in commercially available M hyopneumoniae bacterins commonly used in swine in Europe.

ProductSerial No.g1-TTVg2-TTV
15n271-356++
16o1522354 A
17p1542167
18qL55192++
19rL62832++
20sA47502 A
21t7547A02++
22uA180381M
WaterNA
g1-TTV serum§NA+
g2-TTV serum§NA+

Assays performed with primers specific for g1- and g2-TTVs.11, 13

Negative control sample.

Positive control sample.

See Table 1 for remainder of key.

As an additional quality-control procedure for the PCR assays, bacterins purchased in the United States were tested at both the US and European laboratories; the PCR assays were performed by use of local conditions for each laboratory (Table 4). All bacterins that had positive results when tested for g1-TTV by use of the nPCR assays at the US laboratory also had positive results when tested at the European laboratory. However, one of the bacterins (product 1) had negative results when tested at the US laboratory but positive results when tested at the European laboratory.

Table 4—

Results* for direct PCR and nPCR assays conducted at a European and a US laboratory to detect g1-and g2-TTV DNAs in commercially available M hyopneumoniae bacterins commonly used in swine in North America.

ProductSerial No.g1-TTVg2-TTV
Direct PCRnPCRDirect PCRnPCR
EuropeUSEuropeUSEuropeUSEuropeUS
1a271-150+++ND+ND
2b1619111BNDND
3c1151179ANDND
4dA600 651++++ND+Trace
5eA605 489TraceTrace+++++
6f05489NDND
8h143-056++++ND+Trace
WaterNANDND

The direct PCR assay for g1-TTV was performed at the European laboratory with primers for g1-TTV developed atthe US laboratory.

Assays performed with primers specific for g1-and g2-TTVs.11,13

Negative control sample.

§The primer pairs for g2-TTV were also used as a direct PCR method to detect g2-TTV DNAs.

See Table 1 for remainder of key.

PCR inhibitors—To determine whether a component or components in a product with negative results for TTV DNA may contain PCR inhibitory substances, samples of a bacterin (product 5) that had positive results for TTV DNA were diluted in a bacterin (product 2) that had negative results for TTV DNA and then retested for detection of g2-TTV DNAs by use of nPCR assays. The results revealed that the TTV-negative bacterin (product 2) contained material or materials that inhibited the g2-TTV DNA PCR signal in the TTV-positive bacterin (product 5; Figure 2). Thus, the TTV DNA status of the test-negative bacterins (predominately the various formulations of products 2, 3, and 6) could not be definitively determined because of contamination with a PCR inhibitor or inhibitors in the bacterins.

Figure 2—
Figure 2—

Agarose gels of nPCR assay of g2-TTV DNA for an M hyopneumoniae bacterin (product 5e) that was diluted with another M hyopneumoniae bacterin (product 2b [A]) or with water (B). Contents of the lanes were product 5 alone (plus sign); water alone (negative sign); and product 5 diluted with the other bacterin (panel A) or water (panel B) at dilutions of 2:1, 3:1, 4:1, 5:1, and 6:1 (from left to right, respectively). Notice in panel A that g2-TTV DNA was not detected until a ratio of 6:1 (product 2: product 5) was achieved, which indicated a material or materials contained in product 2 inhibited g2-TTV contained in product 5.

Citation: American Journal of Veterinary Research 69, 12; 10.2460/ajvr.69.12.1601

Sequence analysis of amplicons—At the US laboratory, amplicons from 4 bacterins with positive results for g1-TTV DNAs were sequenced and compared with the reported g1-TTV sequence (GenBank accession No. DQ229865). All had sequences > 90% identical to the sequence for DQ229865; however, each contained multiple unique single base substitutions, which indicated that laboratory contamination of these bacterins with 1 or more TTV DNA could not account for the positive TTV signals detected (data not shown). Similar results were obtained with the 2 bacterins that had positive results for both g1- and g2-TTV at the Canadian laboratory, except that sequence identity for the g1- and g2-TTV amplicons recovered from the bacterins was even higher (95% to 98%) than the identity for those obtained at the US laboratory. The situation at the European laboratory was similar in that bacterin sequences aligned (r 90% identical) with published g1- and g2-TTV sequences. Overall, sequencing of amplicons in all 3 laboratories revealed that the primers used resulted in amplification of g1- and g2-TTVs and not spurious DNA sequences contained in the bacterins.

Discussion

The study reported here revealed that many serials of M hyopneumoniae bacterins licensed and marketed in North America and Europe contained TTV DNAs, as detected by direct PCR or nPCR assays. Critical to these experiments, particularly for the use of PCR techniques, was reproducibility within each laboratory as well as among laboratories. These data were generated in 3 separate laboratories on 2 continents and used a common experimental design that enhanced and reinforced each of the data sets. As well, sequence analysis of amplicons identified by PCR assay confirmed that all were homologous to g1- or g2-TTV. Finally, alignment and homology analyses revealed that the amplicons recovered from each bacterin were unique, which indicated that laboratory contamination of bacterins by a common source of TTV DNAs cannot account for the TTV DNAs detected in these bacterins. By inference, these data also suggested that other bacterins manufactured for use in swine (or other species) that contain swine sera may also be inadvertently contaminated with TTV DNA sequences.

The source or sources of TTV contamination were not determined, but it is probable that locally obtained bacterin components used in the in vitro culture of M hyopneumoniae organisms were the most likely immediate source of porcine TTV DNAs. Certainly, this scenario could account for the differences between products with positive and negative results that were produced by the same manufacturer in different locations, presumably under identical production conditions. On the basis of the frequent detection of TTV in swine throughout the world, the pooled porcine sera that are used in vaccine production are likely to contain TTV DNAs.10,12–15 Although the specifics of each manufacturer's processes are proprietary and thus not available for examination, it is possible that swine serum is a key growth supplement needed for the successful propagation of M hyopneumoniae. This is the most logical source of TTV, although other sources (such as porcineorigin pepsin17 or porcine kidney cell monolayers18,19) cannot be excluded as possible sources of TTV.

Contamination of serial lots of these bacterins with TTV DNAs may be problematic for the biologics industry in general and the swine biologics industry in particular. In contrast to other killed or inactivated products, the mycoplasmal component in the bacterins described here was subjected to comparatively gentle inactivation procedures because harsh inactivation conditions are likely to damage immunoreactive epitopes contained in or on microbial plasma membranes. The TTVs, similar to other members of the Circoviridae family (including PCV220), are resistant to disinfectants and inactivation by mild fixatives. Thus, in contrast to the situation of contamination of several bacterins with porcine circovirus type 1 in which strong inactivation conditions were used,16 there is some likelihood that the Mycoplasma bacterins that had positive results when tested for TTV DNAs may still have contained infectious TTVs.

The larger issue is the unanswered question of an increased risk of disease if TTV-naïve pigs are inadvertently infected with TTV through the practice of routine inoculation with TTV-contaminated Mycoplasma bacterins. It is clear that a positive DNA signal obtained from a biological sample is not synonymous with the presence of infectious virus, and as such, these preliminary data should not be interpreted to mean that some or all of these bacterins contained infectious encapsulated virus or infectious DNAs. However, on the basis of the infectious nature of naked PCV2 DNA (as determined by the experience of one of the authors [SK]), it is likely that DNA from the biologically similar TTVs may also be infective without the absolute requirement for encapsulated DNA or intact virions. This issue (infectivity of TTVs contained in bacterins) is currently being investigated. In preliminary studies conducted by one of the authors (SK), multiple species of both g1- and g2-TTV DNAs were recovered from multiple tissues, including bone marrow of inoculated gnotobiotic swine. If these results are confirmed in additional experiments, then these data indicate that there is infectious TTV contained in at least some of the bacterins tested in the study reported here. Currently, we are not aware of any in vitro culture system for recovery of infectious TTVs in any species, including swine.

It is increasingly clear from studies with other members of the Circoviridae (ie, PCV2) that infection of the relevant host species with these viruses alone is not sufficient to induce clinical disease.21,22 However, coinfections of PCV2-infected swine with other pathogens (such as porcine parvovirus,15,21–25 porcine respiratory and reproductive syndrome virus,26,27 and M hyopneumoniae28) and immunizations29,30 may potentiate scenarios in which the porcine circovirus–associated disease is more severe. Currently, questions regarding infection and increased risks cannot be answered directly because specific inoculation and challenge-exposure experiments with TTV-positive bacterins alone or in conjunction with other infectious agents must be performed for conditions in which environmental contamination as a nonvaccine source of TTVs or challenge agents can be excluded.

The study described here revealed that both g1- and g2-TTV DNAs were detected in many commercially available swine Mycoplasma bacterins available in Europe and North America. Additional studies31–33 to determine the pathogenicity of g1-TTV have been competed, and further studies on the potential impact of TTV contamination of vaccines on health and disease of pigs are being conducted.

ABBREVIATIONS

g1-TTV

Genogroup 1 torque teno virus

g2-TTV

Genogroup 2 torque teno virus

nPCR

Nested PCR

PCV2

Porcine circovirus type 2

PMWS

Postweaning multisystemic wasting syndrome

PRDC

Porcine respiratory disease complex

TTV

Torque teno virus

a.

Ingelvac M hyo, Boehringer-Ingelheim Vetmedica Inc, St Joseph, Mo.

b.

Suvaxyn MH-One, Fort Dodge Animal Health, Fort Dodge, Iowa.

c.

Suvaxyn Respifend MH/HPS, Fort Dodge Animal Health, Fort Dodge, Iowa.

d.

RespiSure One, Pfizer Animal Health Inc, Exton, Pa.

e.

Respisure, Pfizer Animal Health Inc, Exton, Pa.

f.

M+PAC, Schering Plough Animal Health Corp, Omaha, Neb.

g.

PneumoSTAR Myco, Novartis Animal Health, Larchwood, Iowa.

h.

Myco Shield, Novartis Animal Health, Larchwood, Iowa.

i.

Myco Silencer ONCE, Intervet Inc, Millsboro, Del.

j.

Ingelvac M hyo, Boehringer-Ingelheim Vetmedica Inc, Burlington, ON, Canada.

k.

Suvaxyn MH-One, Wyeth Animal Health, Guelph, ON, Canada.

l.

M+PAC, Schering Canada Ltd, Pointe-Claire, QC, Canada.

m.

Myco Silencer BPM, Intervet Inc, Millsboro, Del.

n.

Ingelvac M hyo, Boehringer Ingelheim, Ingelheim am Rhein, Germany.

o.

M hyo Suvaxyn, Fort Dodge Animal Health, Southampton, England.

p.

M hyo Suvaxyn parasuis, Fort Dodge Animal Health, Southampton, England.

q.

Stellamune, Pfizer Animal Health Inc, Louvain-la-Neuve, Belgium.

r.

Stellamune ONCE, Pfizer Animal Health Inc, Louvain-la-Neuve, Belgium.

s.

M+PAC, SP Essex Animal Health (Burgwedel), Burgwedel, Germany

t.

Porcilis M hyo, Intervet International BV, Boxmeer, The Netherlands.

u.

Hyoresp, Merial Animal Health, Harlow, Essex, England.

v.

QIAamp DNA mini kit, Quiagen, Valencia, Calif.

w.

Qiagen Hot Star Taq polymerase, Quiagen, Valencia, Calif.

References

  • 1.

    Kyriakis SC, Saoulidis K, Lekkas S, et al. The effects of immuno-modulation on the clinical and pathological expression of postweaning multisystemic wasting syndrome. J Comp Pathol 2002;126:3846.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2.

    Allan GM, McNeilly F, Ellis JA, et al. Neonatal vaccination for Mycoplasma hyopneumoniae and postweaning multisystemic wasting syndrome: a field trial. Pig J 2001;48:3441.

    • Search Google Scholar
    • Export Citation
  • 3.

    Krakowka S, Ellis J, McNeilly F, et al. Mycoplasma hyopneumoniae bacterins and porcine circovirus type 2 (PCV2) infection: induction of postweaning multisystemic wasting syndrome (PMWS) in the gnotobiotic swine model of PCV2-associated disease. Can Vet J 2007;48:716724.

    • Search Google Scholar
    • Export Citation
  • 4.

    Irshad M, Joshi YK, Sharma Y, et al. Transfusion transmitted virus: a review on its molecular characteristics and role in medicine. World J Gasteroenterol 2006;12:51225134.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5.

    Hino S, Miyata H. Torque teno virus (TTV): current status. Rev Med Virol 2007;17:4557.

  • 6.

    Okamoto H, Takahashi M, Nishizawa T, et al. Genomic characterization of TT viruses (TTVs) in pigs, cats and dogs and their relatedness with species-specific TTVs in primates and tupaias. J Gen Virol 2002;83:12911297.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7.

    Simmonds P, Prescott LE, Logur C, et al. TT virus—part of the normal human flora? J Infect Dis 1999;180:17481750.

  • 8.

    Zein NN. TT virus infection: an emerging pathogen in search of its identity. J Pediatr 2000;136:573575.

  • 9.

    Leary TP, Erker JC, Chalmers ML, et al. Improved detection systems for TT virus reveal high prevalence in humans, non-human primates and farm animals. J Gen Virol 1999;80:21152120.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    McKeown NE, Fenaux M, Halbur PG, et al. Molecular characterization of porcine TT virus, an orphan virus, in pigs from six different countries. Vet Microbiol 2004;104:113117.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Niel C, Diniz-Mendes L, Devalle S. Rolling-circle amplification of torque teno virus (TTV) complete genomes from human and swine sera and identification of a novel swine TTV genogroup. J Gen Virol 2005;86:13431347.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Bigarre L, Beven V, de Boisseson C, et al. Pig anelloviruses are highly prevalent in swine herds in France. J Gen Virol 2005;86:631635.

  • 13.

    Kekarainen T, Sibila M, Segales J. Prevalence of swine torque teno virus in post-weaning multisystemic wasting syndrome (PMWS)-affected and non-PMWS-affected pigs in Spain. J Gen Virol 2006;87:833837.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 14.

    Martinez L, Kekarainen T, Sibila M, et al. Torque teno virus (TTV) is highly prevalent in the European wild boar (Sus scrofa). Vet Microbiol 2006;20:223229.

    • Search Google Scholar
    • Export Citation
  • 15.

    Gagnon CA, Tremblay D, Tijssen P, et al. The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada. Can Vet J 2007;48:811819.

    • Search Google Scholar
    • Export Citation
  • 16.

    Quintana J, Segalés J, Calsamiglia M, et al. Detection of porcine circovirus type 1 in commercial pig vaccines using polymerase chain reaction. Vet J 2006;171:570573.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 17.

    Fenaux M, Opriessnig T, Halbur PG, et al. Detection and in vitro and in vivo characteristics of porcine circovirus DNA from a porcine-derived commercial pepsin product. J Gen Virol 2004;85:33773382.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 18.

    Tischer I, Rasch R, Tochtermann G. Characterization of papova-virus- and picornavirus-like particles in permanent piglet kidney cell lines. Zentrabl Bakertiol [Orig A] 1974;226:153167.

    • Search Google Scholar
    • Export Citation
  • 19.

    Dulac GC, Afshar A. Porcine circovirus antigens in PK-15 cell line (ATCC CCL-33) and evidence of antibodies in Canadian pigs. Can J Vet Res 1989;53:431433.

    • Search Google Scholar
    • Export Citation
  • 20.

    Royer RL, Nawagitgul P, Halbur PG, et al. Susceptibility of porcine circovirus type 2 to commercial and laboratory disinfectants. J Swine Health Prod 2001;9:281284.

    • Search Google Scholar
    • Export Citation
  • 21.

    Allan GM, Ellis JA. Porcine circoviruses: a review. J Vet Diagn Invest 2000;12:314.

  • 22.

    Segales J, Domingo M. Postweaning multisystemic wasting syndrome (PMWS) in pigs. A review. Vet Q 2002;24:109124.

  • 23.

    Ellis JA, Krakowka S, Lairmore M, et al. Reproduction of lesions of post-weaning multisystemic wasting syndrome in gnotobiotic piglets. J Vet Diag Invest 1999;11:314.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 24.

    Krakowka S, Ellis JA, Meehan B, et al. Viral wasting syndrome of swine: experimental reproduction of postweaning multisystemic wasting syndrome by co-infection with porcine circovirus-2 (PCV 2) and porcine parvovirus (PPV). Vet Pathol 2000;37:254263.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 25.

    Allan GM, Kennedy S, McNeilly F, et al. Experimental reproduction of wasting disease and death by co-infection of piglets with porcine circovirus and porcine parvovirus. J Comp Pathol 1999;121:111.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 26.

    Allan GM, McNeilly F, Ellis JA, et al. Experimental infection of colostrum-deprived piglets with porcine circovirus type 2 (PCV-2) and porcine reproductive and respiratory syndrome virus (PRRSV) potentiates PCV-2 replication. Arch Virol 2000;145:24212429.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 27.

    Harms PA, Sorden S, Halbur PG, et al. Experimental reproduction of severe disease in CD/CD pigs concurrently infected with type 2 porcine circovirus and porcine respiratory and reproductive syndrome virus. Vet Pathol 2001;38:528539.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 28.

    Opriessnig T, Thacker EL, Yu S, et al. Experimental reproduction of postweaning multisystemic wasting syndrome in pigs by dual infection with Mycoplasma hyopneumoniae and porcine circovirus type 2. Vet Pathol 2004;41:624640.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 29.

    Krakowka S, Ellis JA, Meehan B, et al. In vivo immune activation is the pivotal event in the production of wasting disease in pigs infected with porcine circovirus-2 (PCV-2). Vet Pathol 2001;38:3142.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 30.

    Opriessnig T, Yu S, Gallup JM, et al. Effect of vaccination with selective bacterins on conventional pigs infected with type 2 porcine circovirus. Vet Pathol 2003;40:521529.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 31.

    Krakowka S, Allen G, Ellis JA. Evaluation of the effects of porcine genogroup 1 torque teno virus in gnotobiotic swine. Am J Vet Res 2008;69:16151622.

    • Search Google Scholar
    • Export Citation
  • 32.

    Ellis JA, Krakowka S. Effect of coinfection with genogroup 1 porcine torque teno virus on porcine circovirus type 2–associated postweaning multisystemic wasting syndrome in gnotobiotic pigs. Am J Vet Res 2008;69:16081614.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 33.

    Krakowka S, Hartunian C, Hamberg A, et al. Evaluation of induction of porcine dermatitis and nephropathy syndrome in gnotobiotic pigs with negative results for porcine circovirus type 2. Am J Vet Res 2008;69:16231629.

    • Search Google Scholar
    • Export Citation

Contributor Notes

Supported in part by a grant from the National Institutes of Health, Public Health Service (R01 A1053120).

The authors thank Judith Dubena, Amy Davis, and Sara Schmitz for technical assistance with the gnotobiotic baby pigs.

Address correspondence to Dr. Ellis.