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 primers^11,13 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 kit^v in accordance with the manufacturer's protocol for blood. The Taq master mixture^w 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 published^11,13 g1-TTV primers but positive results when tested by use of the direct PCR assay with the specially designed g1-TTV primers (Table 1).

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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

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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.

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.

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.

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.

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Agarose gels of nPCR assay of g2-TTV DNA for an M hyopneumoniae bacterin (product 5^e) that was diluted with another M hyopneumoniae bacterin (product 2^b [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

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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 monolayers^18,19) 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 immunizations^29,30 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 studies^31–33 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.