A quantitative TaqMan PCR assay for the ... - Wiley Online Library

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

A quantitative TaqMan PCR assay for the detection of Mycoplasma suis A.M.S. Guimaraes1,2, R.F.C. Vieira3, R. Poletto4, R. Vemulapalli1, A.P. Santos1, W. de Moraes5, Z.S. Cubas5, L.C. Santos5, J.N. Marchant-Forde4, J. Timenetsky6, A.W. Biondo3,* and J.B. Messick1 1 2 3 4 5 6

Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayette, IN, USA CAPES-Fulbright Program, Ministe´rio da Educaçaõ, Brası´lia, DF, Brazil Departamento de Medicina Veterina´ria, Universidade Federal do Parana´, Curitiba, PR, Brazil USDA-ARS Livestock Behavior Research Unit, West Lafayette, IN, USA Bela Vista Biological Sanctuary, Itaipu Binacional, Foz do Iguaçu, PR, Brazil Departamento de Microbiologia, Instituto de Cieˆncias Biome´dias, Universidade de Saõ Paulo, Sao Paulo, SP, Brazil

Keywords Mycoplasma suis, peccaries, pigs, quantitative PCR, real-time PCR, swine. Correspondence Joanne B. Messick, Department of Comparative Pathobiology, 625 Harrison Street, School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA. E-mail: [email protected] *Dr Biondo is also a visiting professor at Purdue University.

2011 ⁄ 0201: received 2 February 2011, revised 18 April 2011 and accepted 10 May 2011 doi:10.1111/j.1365-2672.2011.05053.x

Abstract Aim: To develop a TaqMan probe-based, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma suis in the blood of pigs. Methods and Results: Primers and probes specific to Myc. suis 16S rRNA gene were designed. The qPCR assay’s specificity, detection limit, intra- and interassay variability were evaluated and its performance was compared with a Myc. suis conventional PCR assay (cPCR). Blood of two experimentally infected pigs, 40 Indiana pigs, 40 Brazilian sows and 28 peccaries were tested. The assay detected as few as ten copies of Myc. suis plasmids and was 100-fold more sensitive than the cPCR. No cross-reactivity with nontarget pig mycoplasmas was observed. An average of 1Æ62 · 1011 and 2Æ75 · 108 target copies ml)1 of blood were detected in the acutely and chronically infected pigs, respectively. Three (7Æ5%) pigs and 32 (80Æ0%) sows were positive while all peccaries were negative for Myc. suis. Conclusion: The developed qPCR assay is highly sensitive and specific for Myc. suis detection and quantification. Significance and Impact of the Study: TaqMan qPCR is an accurate and quick test for detection of Myc. suis infected pigs, which can be used on varied instrumentation platforms.

Introduction Mycoplasma suis, formerly known as Eperythrozoon suis, is the causative agent of eperythrozoonosis in domestic pigs. This organism tightly adheres to the porcine erythrocytes and may cause acute disease with severe anaemia or chronic infection without haematological abnormalities. In the field, Myc. suis is most likely transmitted through the use of contaminated needles and surgical instruments or ingestion of contaminated blood (cannibalism). Arthropod vectors, such as Stomoxys calcitrans and Aedes aegypti, also have been implicated in the transmission of this pathogen (Prullage et al. 1993). Infected pigs, partic-

ularly those chronically infected, constitute the main source of infection within a herd. In addition, chronically infected animals may develop the acute form of the disease following immunosuppressive events (i.e. prepartum, weaning, environmental stress) or splenectomy (Messick 2004). Although previous studies have shown that Myc. suis significantly increases mortality of acutely infected pigs, impairs reproductive performance in chronically infected sows and decreases weight gain in feeder pigs (Henry 1979; Brownback 1981; Zinn et al. 1983; Oberst et al. 1993; Henderson et al. 1997; Messick 2004; Wu et al. 2006), its economic impact remains to be fully elucidated. Highly sensitive and specific diagnostic

ª 2011 The Authors Journal of Applied Microbiology 111, 417–425 ª 2011 The Society for Applied Microbiology

417

Development of a Myc. suis qPCR assay

A.M.S. Guimaraes et al.

techniques can be employed toward a better understanding of the actual consequences of the infection, particularly in chronically infected animals. Mycoplasma suis has never been successfully cultured in vitro. The diagnosis of the infection relies on clinical signs and the observation of organisms attached to erythrocytes in blood smears and, more recently, on detection of the organisms in blood samples using molecular methods (Messick 2004). Whereas the blood smear evaluation is neither sensitive nor specific for the detection of infection, molecular techniques, such as polymerase chain reaction (PCR) assays, are considered the gold standard for identification of infected animals (Messick 2004). As bacteremia may be low in chronically infected pigs, PCR analysis followed by the detection of specific PCR products by Southern blotting has been used to increase the sensitivity of Myc. suis detection. However, the later step is not used as a routine diagnostic assay (Guimaraes et al. 2007a). A LightCycler real-time PCR assay targeting the protein-coding msg1 gene of Myc. suis was previously described (Hoelzle et al. 2007). The assay is based on the principle of fluorescence resonance energy transfer between fluorescein- and LC Red 640-labelled hybridization probes. A major limitation for many laboratories to use this assay for Myc. suis detection is that only a few commercially available instrumentation platforms, such as LightCycler, can support this type of assay format. In contrast, assays based on TaqMan probes can be performed using any of the commercially available realtime PCR instruments. A real-time quantitative PCR (qPCR) assay based on TaqMan probe, however, has never been reported for the detection of Myc. suis. Therefore, the aim of this study was to develop a sensitive and specific TaqMan probe-based qPCR assay targeting the 16S ribosomal RNA (16S rRNA) gene of Myc. suis for detection and quantification of the organism in pig blood samples and to compare it with a conventional Myc. suis PCR (cPCR) assay. Material and methods Bacterial strains and culture conditions The E. suis Illinois strain (Myc. suis) (Messick et al. 1999) was used as positive control. Porcine mucosal mycoplasmas (Mycoplasma hyopneumoniae, Mycoplasma hyosynoviae, Mycoplasma hyopharyngis, Mycoplasma flocullare, Mycoplasma hyorhinis) were obtained from the Purdue University Mycoplasma Collection and grown in specific Mycoplasma media (SP4 and Friis) at 37C for 5–7 days. DNA was extracted from log phase cultures by a boiling method described elsewhere (Fan et al. 1995). 418

Experimental study Two random-source mixed-breed pigs (females, 45 dayold; pigs 1 and 2) were used in this study. Animals were housed at the Purdue University Animal Care and Use Facilities and provided food and water ad libitum. These pigs originated from a herd previously identified as having Myc. suis infected animals based on a speciesspecific, conventional PCR (cPCR) assay (Messick et al. 1998). Pig 1 was negative by cPCR for Myc. suis infection in three separate blood samples collected on days 0 (arrival day at the housing facility), 4 and 7; pig 2, sampled on days 0 and 4, was positive on both days. On day 7, pig 1 was splenectomized, whereas pig 2, considered to be chronically infected, was not splenectomized. Clinical signs were monitored daily and PCR performed at various time intervals (described below) on both pigs. Mycoplasma suis infection following splenectomy is known to result in acute disease (see Results). Thus, bacterial loads in this acutely infected pig (pig 1) were followed and compared with that of the chronically infected pig (pig 2). EDTA blood samples used for Myc. suis detection by qPCR were collected via venipuncture of the caudal vena cava; a total of ten blood samples were collected from pig 1 over a period of 80 days (days 0, 4, 7, 14, 35, 46, 53, 60, 71, 80) which included two peaks of Myc. suis bacteremia. Seven blood samples were collected from pig 2 during the same 80-day period (days 4, 16, 21, 35, 60, 71, 80). Packed cell volume (PCV), total plasma protein and blood smear evaluations (Modified Wright stain) were performed on all samples. A total of 100 ll of each sample was subjected to DNA extraction using a commercially available kit following the manufacturer’s instructions (Quick-gDNA Miniprep; Zymo Research Corporation, Orange, CA, USA). To control bacteremia and febrile episodes during acute infection, pig 1 was treated at peaks of Myc. suis bacteremia with oxytetracycline (20 mg kg)1, intramuscularly, twice daily) and flunixin meglumine (2Æ2 mg kg)1, intramuscularly, once every 24 h). The animal experimental protocols were approved by the Purdue University Animal Care and Use Committee (protocol number 06-100) and the experiments were performed according to our institutional ethical rules and laws. Field samples Two groups of pigs (Sus scrofa, females, total number: 40 animals) from a farm in Indiana, USA, were randomly selected from a previous, unrelated study. Group 1 (G1) included ten animals with blood samples collected at 10 days, 3 months and 6 months of age. Group 2 (G2)



included 30 animals between 3 and 6 months of age. A total of 100 ll of each sample was subjected to DNA extraction using a commercially available kit following the manufacturer’s instructions (Quick-gDNA Miniprep; Zymo Research Corporation). A total of 40 DNA samples extracted from blood of domestic sows from Brazilian farms used in a previous study (Guimaraes et al. 2007a) were randomly selected. These samples were previously tested using the same cPCR described herein with gels subsequently subjected to a Southern blot protocol (Guimaraes et al. 2007a). Additional epidemiological and clinical evaluations of these animals were described by Guimaraes et al. (2007a). Samples were stored at )20C until analysed in this study. Results obtained in this study were compared with both the cPCR and Southern blot analyses from Guimaraes et al. (2007a). Blood DNA samples from 22 captive collared peccaries (Tayassu tajacu) and six white-lipped peccaries (Tayassu pecari) that were previously negative by a cPCR for Myc. suis (Vieira et al. 2011) were also analysed. These samples were obtained from animals housed at the Bela Vista Biological Sanctuary, Foz do Iguaçu and at the Curitiba Zoo, Curitiba, both in Southern Brazil. Animal procedures were performed under approved regulations of the Brazilian Institute for the Environment and the Renewable Resources (IBAMA). Samples were stored in )20C until analysed in this study. Cloning of Mycoplasma suis 16S rRNA gene into pGEM-T easy Universal bacterial PCR primers (Messick et al. 1998) were used to amplify 1346 bp of the 16S rRNA gene of the E. suis Illinois strain (Myc. suis) (Messick et al. 1999). The PCR product was purified from the agarose gel using a commercially available kit (Zymoclean Gel DNA Recovery kit; Zymo Research Corporation) and cloned into a plasmid vector (pGEM-T easy; Promega, Madison, WI, USA) following the manufacturers’ protocols. This plasmid was used as a positive control for the Myc. suis PCR assays. Conventional PCR assays A cPCR assay for the detection of the beta-actin gene (GenBank accession number AY550069.1) of domestic pigs was performed to verify the presence of amplifiable DNA in all extractions (Guimaraes et al. 2007a). In addition, a cPCR for the detection of Myc. suis in pig blood DNA samples was used as previously described (Messick et al. 1998). Nuclease-free water was used as negative control in both assays. The recombinant plasmid contain-


ing the Myc. suis 16S rRNA gene as an insert was used as positive control for the Myc. suis cPCR assay. A DNA sample extracted from a random domestic pig was used as a positive control for the beta-actin PCR assay. PCR products were separated by electrophoresis on a 1–2% agarose gel stained with ethidium bromide. The gels were imaged under ultraviolet light using the VisionWorksls Analysis Software (Epi Chemi II Darkroom, UVP Inc., Upland, CA, USA). Design of primers and probe for quantitative PCR Primers and probes were designed based on the 16S rRNA gene of the Myc. suis. Briefly, 16S rRNA gene sequences from hemoplasmas and other porcine mycoplasmas were aligned using the ClustalW algorithm of the mega 4 software. Variable regions were manually selected for the design of primers and probes used in this assay. Oligonucleotides properties were further analysed using the Primer Express Software ver. 2.0 (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA, USA) and Oligo Analyzer (IDT Technologies, Coralville, IA). Primers and probe specificity were tested in silico using Blast algorithms and by individual global alignments using the GeneStream software (Pearson et al. 1997). The following forward and reverse primer sequences were selected to amplify a 157 bp fragment of the Myc. suis 16S rRNA gene: RTsuisF 5¢-CCC TGA TTG TAC TAA TTG AAT AAG-3¢ and RTsuisR 5¢-GCG AAC ACT TGT TAA GCA AG-3¢. The TaqMan probe specific to the amplicon was designed to contain the fluorescent dye 6-carboxyfluorescein (FAM) at the 5¢-end and a minor groove binding (MGB) nonfluorescent quencher (NFQ) at the 3¢-end (MGBsuis2: 5¢FAM- TGR ATA CAC AYT TCA G -MGBNFQ 3¢). The probe was custom synthesized at a commercial source (Applied Biosystems, Life Technologies Corporation). Quantitative PCR Absolute quantification assays were performed using a 7300 Real-Time PCR System (Applied Biosystems, Life Technologies Corporation). The 25 ll reaction-mixture contained 1· of PCR Buffer (500 mmol l)1 of KCl, 100 mmol l)1 of Tris–HCl pH 9Æ0, 1% Triton-X), 0Æ4 lmol l)1 of ROX (6-carboxy-X-rhodamine; Roche Diagnostics, Mannheim, Germany), 4Æ0 mmol l)1 of MgCl2, 0Æ2 mmol l)1 of each dNTP (dATP, dCTP, dGTP, dTTP), 0Æ45 lmol l)1 of each primer, 0Æ3 lmol l)1 of probe, 1 U of GoTaq (Promega) and 5 ll of DNA template. The cycling conditions consisted of: 95C for 10 min, followed by 40 cycles at 95C for 15 s, 58Æ5C for 45 s and


419



72C for 30 s. Nuclease-free water was used as negative control, while the recombinant plasmid containing the Myc. suis 16S rRNA gene as an insert was used as positive control. The data were acquired during the annealing step and analysed using the 7300 System sds software (Applied Biosystems, Life Technologies Corporation). The threshold was manually adjusted within the logarithmic curve, above background level and below the linear and plateau phase. The threshold cycle (Ct) of the plasmid dilutions was plotted against the log number of plasmid copies and used as input to create the standard curve. Linear regression analyses were then applied to calculate the r2 and slope values. Assuming 100% efficiency if DNA template is doubled in each cycle, the PCR efficiency was calculated as E = 10()1 ⁄ slope) ) 1, where E is PCR efficiency. qPCR specificity, detection limit and comparison to cPCR The DNA of the following organisms was used to test specificity of the qPCR assays: Myc. flocullare, Myc. hyopneumoniae, Myc. hyopharyngis, Myc. hyorhinis, Myc. hyosynoviae and a novel pig hemoplasma previously described (Messick et al. 2007). The detection limit of the assay was measured by testing tenfold dilutions of the recombinant plasmid containing the 16S rRNA gene of Myc. suis (from 109 to 1 copy of plasmid ⁄ reaction). Briefly, the plasmid concentration was quantified using a spectrophotometer (NanoDrop 2000; Thermo Scientific, Wilmington, DE, USA) and the copy number was calculated as described elsewhere (Applied Biosystems, 2003). Plasmid copy number was adjusted and serially diluted in both TE (10 mmol l)1 Tris–HCl, 0Æ1 mmol l)1 EDTA, pH 8Æ0) alone and TE buffer combined with 30 lg ml)1 of herring sperm DNA (Sigma-Aldrich, St Louis, MO, USA) to mock the presence of host DNA in the sample. The number of target copies ml)1 of blood was calculated based on 100% DNA extraction efficiency and considering one copy of the 16S rRNA gene per organism; thus, one target copy corresponds to one organism. Our laboratory has recently sequenced the entire Myc. suis genome (Guimaraes et al., 2011; GenBank accession number: ADWK00000000), and only one copy of the 16S rRNA gene was found in the genome, supporting this assumption. To compare the detection limit between cPCR and qPCR, the same plasmid dilutions described above (in 1· TE and in 1· TE with herring sperm) were evaluated using the Myc. suis cPCR assay. In addition, a DNA sample extracted from blood of a pig at peak of bacteremia (pig 1) was tenfold serially diluted in 1· TE and evaluated by both the cPCR and the qPCR assays. 420

Intra- and inter-assay repeatability The intra-assay repeatability was determined by running five replicates of the plasmid dilutions (109 to 1 copy of plasmid ⁄ reaction) in the same run. The standard curve was then generated using the 7300 System sds software (Applied Biosystems, Life Technologies Corporation). The inter-assay variability was determined by running triplicates of the same plasmid dilution in five different runs, in separate days. These replicates were used to determine the mean, standard deviation and coefficient of variation in Ct values for each plasmid dilution. DNA sequencing Samples that were positive for Myc. suis using the developed qPCR but negative using the cPCR method were selected for confirmation by DNA sequencing. To accomplish this, qPCR products were separated on a 2% agarose gel and then extracted from the gel using a commercially available kit (Zymoclean Gel DNA Recovery kit; Zymo Research Corporation). The final product was then directly sequenced in both directions (forward and reverse) at the Purdue University Genomics Core Facility, West Lafayette, IN. Results The qPCR assay had a reaction efficiency of E = 98Æ01% (r2 = 0Æ993) and consistently detected as few as ten copies of plasmid ⁄ reaction when using the plasmid standards diluted in TE (Fig. 1). When the plasmid dilutions contained 30 lg ml)1 of herring sperm, the efficiency was E = 101Æ39% (r2 = 0Æ998) and the assay also detected as few as ten copies of plasmid ⁄ reaction in 14 of 17 (82Æ4%) testing tubes at this dilution (Fig. 1). For plasmid diluted in either 1· TE or herring sperm, the limit of detection for the cPCR assay was 103 copies. The Myc. suis positive sample that was serially diluted and used as template for both assays had 1Æ62 · 1011 target copies ml)1 based on the plasmid-generated standard curve in the qPCR. The newly developed qPCR was able to detect as few as 8Æ1 · 102 target copies per 5 ll of extracted DNA (up to 10)6 dilution or 1Æ62 · 105 target copies ml)1 of blood), whereas the cPCR detected only 8Æ1 · 104 target copies per 5 ll of extracted DNA (up to 10)4 dilution or 1Æ62 · 107 target copies ml)1 of blood). Thus, the qPCR was generally 2 logs (100-fold) more sensitive than the cPCR for the detection of Myc. suis target copies. The intra-assay and inter-assay repeatability of the qPCR are shown in Table 1. No cross-reactivity was detected when DNA from nontarget mycoplasmas was used as the template.




(a)

Standard curve 44

Detector: TaqMan Slope: –3·370411

40

Intercept: 43·549465 R2: 0·993377

36

Ct

32 28 24 20 16 12

1

2

3

4

(b) 40

5 Log C0

6

7

8

9

Standard curve Detector: TaqMan Slope: –3·288935 Intercept: 42·407902

36

R2: 0·998243

32

28 Ct

Figure 1 Standard curves of serial dilutions of Mycoplasma suis positive controls calculated by 7300 System sds software (Applied Biosystems, Life Technologies Corporation). (a) Plasmid with Myc. suis 16S rRNA gene insert diluted in 1· TE. (b) Plasmid with Myc. suis 16S rRNA gene insert diluted 1· TE combined with 30 lg ml)1 of herring sperm DNA (Sigma Aldrich).

24

20

16

12

1

2

All DNA samples amplified the predicted product for the beta-actin gene, which indicates a successful DNA extraction. Pig 1 was negative for Myc. suis infection in the three samples (days 0, 4 and 7) collected prior to the splenectomy and in one sample (day 14) collected following this procedure. However, by day 35 (28 days following splenectomy) in the absence of experimental infection, pig 1 became positive for Myc. suis by qPCR and acute disease ensued by day 46 (Fig. 2). It is speculated that pig 2, or another infected animal housed in the same pen, was the source of this infection. The average load of Myc. suis target copies measured by qPCR for pig 1 (acutely infected animal, days 35–80) was 1Æ62 · 1011 target copies ml)1 of blood (range: 8Æ76 · 105–6Æ57 · 1011 target copies ml)1 of blood), whereas the average load of organisms in pig 2 (chronically infected animal, days 0–80) was 2Æ75 · 108 target copies ml)1 of blood (range: 2Æ25 · 105–8Æ23 · 108 target copies ml)1 of blood). Figure 2 shows the concentration of the target copies in the blood of both pigs during the course of the experiment. Tetracycline treatment administered to control the bacteremia in pig 1 reduced Myc suis target copies to an average of 7Æ41 · 107 ml)1 of blood (days 53–70, range: 8Æ76 · 105–2Æ2 · 108 target copies ml)1 of blood); no organisms were observed in

3

4

5

6

7

8

9

Log C0

blood smear preparations on these days. As anticipated, Myc. suis infection was not completely eliminated with the antibiotic treatment and by day 80, a second peak of bacteremia was detected. On blood smear evaluation, organisms were seen attached to the erythrocytes only at peaks of Myc. suis bacteremia of pig 1, when the average load was 4Æ86 · 1011 target copies ml)1 of blood (peak 1 = 3Æ16 · 1011 ml)1 and peak 2 = 6Æ57 · 1011 ml)1). For the Indiana pigs in G1 group (multiple blood collections per pig), two animals (2 ⁄ 10, 20%) were positive at least in one sampling time point. One pig was consistently positive in all three blood samples collected at 10 days, 3 and 6 months of age (8Æ80 · 104, 1 · 107 and 9Æ5 · 107 target copies ml)1 of blood, respectively), whereas the second pig was positive only at 10 days of age (6Æ70 · 104 target copies ml)1 of blood). In the G2 group (single blood collection time point from 3 to 6 months old pigs), only one pig (1 ⁄ 30, 3Æ33%) was positive for Myc. suis having 2Æ1 · 108 target copies ml)1 of blood. Combining both groups, three of 40 (7Æ5%) domestic pigs from the Indiana farm were positive by qPCR. The sample having the lowest number of target copies ml)1 of blood (6Æ70 · 104) was negative by cPCR. This diminished sensitivity of the cPCR was a trend observed for both experimental and field samples; thus, in


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Table 1 Intra- and inter-assay repeatability of the qPCR assay Intra-assay repeatability

Inter-assay repeatability

Plasmid diluted in 1· TE

Plasmid diluted in DNA*

Plasmid diluted in 1· TE

Plasmid diluted in DNA*

Plasmid copies

Mean-crossing point (Ct ± SD)

CV (%)


CV (%)


CV (%)


CV (%)

10 102 103 104 105 106 107 108 109

38Æ31 37Æ44 34Æ02 30Æ56 27Æ07 23Æ28 19Æ97 16Æ28 12Æ92

0Æ52 1Æ31 0Æ50 0Æ99 0Æ47 0Æ99 0Æ86 1Æ05 1Æ38

38Æ84 35Æ93 32Æ92 29Æ18 26Æ18 22Æ54 19Æ16 16Æ03 12Æ87

2Æ04 0Æ55 0Æ90 1Æ20 0Æ84 0Æ89 1Æ11 1Æ68 1Æ93

38Æ92 37Æ27 34Æ01 30Æ34 26Æ90 23Æ24 19Æ48 15Æ90 12Æ40

2Æ20 1Æ21 0Æ64 0Æ67 0Æ99 0Æ98 1Æ86 1Æ89 2Æ91

38Æ73 36Æ00 32Æ99 29Æ42 26Æ20 22Æ68 19Æ29 16Æ14 13Æ25

1Æ82 1Æ26 1Æ12 1Æ17 0Æ91 0Æ90 1Æ84 1Æ84 2Æ42

± ± ± ± ± ± ± ± ±

0Æ19 0Æ48 0Æ17 0Æ30 0Æ13 0Æ23 0Æ17 0Æ17 0Æ18

± ± ± ± ± ± ± ± ±

0Æ79 0Æ19 0Æ29 0Æ35 0Æ17 0Æ20 0Æ21 0Æ27 0Æ25

± ± ± ± ± ± ± ± ±

0Æ86 0Æ45 0Æ22 0Æ20 0Æ27 0Æ23 0Æ36 0Æ30 0Æ36

± ± ± ± ± ± ± ± ±

0Æ70 0Æ45 0Æ37 0Æ34 0Æ24 0Æ20 0Æ35 0Æ39 0Æ82

CV, coefficient of variation. *Plasmid diluted in 1· TE with 30 lg ml)1 of herring sperm DNA. pGEM-T easy vector with Mycoplasma suis 16S ribosomal RNA gene as insert. Number of plasmid copies ⁄ reaction tube.

samples having a low number of target copies (8Æ76 · 105, 2Æ25 · 105 and 8Æ8 · 104 target copies ml)1 of blood) as measured by qPCR, only faint bands were inconsistently detected by cPCR. Subsequent DNA sequencing of the qPCR products of the cPCR negative sample showed a 99Æ4% similarity (GenBank accession number HM232859) to Myc. suis strain Illinois 16S rRNA gene sequence (GenBank accession number AF029394). For blood samples collected from the 40 Brazilian sows, 11 (27Æ5%) were positive by cPCR and 16 (40%) were detected as positive by the addition of Southern blot. All samples that were positive by cPCR were also positive by Southern blot (Guimaraes et al. 2007a). Surprisingly, a total of 32 (80%) of the 40 sow samples were positive by the qPCR. All samples that were positive by cPCR and ⁄ or Southern blot were positive by qPCR. The average Ct number for these additional positive samples (negative by cPCR and Southern blot) was 31Æ5 (range from 28 to 37 Ct), whereas the average Ct number for those samples positive by cPCR and ⁄ or Southern blot was 25Æ3 (range from 22 to 30 Ct). Samples that were negative by cPCR but positive by Southern blot (n = 5) showed an average Ct of 29Æ6 (range from 25 to 30). In general, cPCR and Southern blot failed to detect samples with a Ct >30 or approx. 98% similarity (98Æ1 and 99Æ4%, GenBank accession numbers HQ844556 and HQ844558) to Myc. suis strain 422

Illinois, while the other two showed 97Æ5% similarity (GenBank accession numbers HQ844557 and HQ844559) to Myc. suis isolates from China (GenBank accession number HQ259257). The latter two isolates showed only 96Æ7% similarity to Myc. suis strain Illinois. Blood samples from the peccaries were all negative when tested by both the conventional and qPCR assays. Discussion The assay developed in this study was shown to be highly sensitive and specific for the detection of Myc. suis infected pigs. As compared with cPCR, the sensitivity of our assay was 100-fold greater for both plasmid inserts of target DNA and extracted DNA from whole blood. For this reason, 17 (48Æ57%) of the 35 qPCR positive Indiana domestic pigs and Brazilian sows failed to be detected as positive by cPCR. Our qPCR assay detected twice the number of positive samples when compared with a Myc. suis specific Southern blot assay. As chronically infected animals comprise the majority of Myc. suis cases in the field, the enhanced sensitivity of qPCR is particularly important for gaining a better understanding of the extent and impact of such infections. Furthermore, this technique is extremely beneficial for high-throughput laboratories, such as those serving the swine industry, as the elapsed time for obtaining results is shorter and the technique is less labour intensive than cPCR. The TaqMan MGB probes have the advantages of being more specific and sensitive for the detection of target DNA (Kutyavin et al. 2000). The MGB moiety increases the probe melting temperature (Tm) and allow for the


Log of target copies per ml of blood


14


(a)

12 10 8 6 4 2 0

Days 46

PCV

34%

27%

Slide

–

+

Log of target copies per ml of blood

35

14

53

30%

60

71

80

34%

36%

35%

–

–

–

+

(b)

12 10 8 6 4 2

Days

0 4

16

21

35

60

71

80

PCV

32%

37%

35%

36%

37%

46%

40%

Slide

–

–

–

–

–

–

–

Figure 2 Load of target copies ⁄ mL of blood in the (a) acutely infected pig (pig 1) starting at the first day of qPCR positivity and (b) chronically infected pig (pig 2) (loads are in logarithmic scale). The first four blood samples of pig 1 (days 0, 4, 7, 14) were negative and are not shown in the graph. Peaks of Mycoplasma suis bacteremia are indicated with a triangle. Packed cell volume (PCV) and blood smear evaluation (Modified Wright stain) for each day are indicated below the x axis (‘+’ sign indicates 15–20 organisms per erythrocyte, whereas the ‘)‘sign indicates no observable organisms). Antibiotic treatment for pig 1 started at day 46 for five consecutive days (as indicated in the Material and methods).

design of a shorter and more specific probe when compared with regular TaqMan probes (Kutyavin et al. 2000); MGB probes can even be used to detect single-nucleotide polymorphisms (de Kok et al. 2002; Higgins et al. 2004; Decaro et al. 2006). The MGB probes also bind more efficiently to the target DNA, which increases sensitivity by decreasing background noise (Kutyavin et al. 2000). Accordingly, the MGB detection system employed in this study proved to be specific for Myc. suis based on the assay specificity testing against other porcine mycoplasmas, as well as on in silico analyses using the blast algorithm. The agreement between qPCR and cPCR results and the DNA sequencing of amplification products further corroborate the high specificity of our assay.

Hemotropic mycoplasma quantification can be a valuable tool for the identification of acutely infected pigs. Herein, Myc. suis quantification was based on 100% DNA extraction efficiency. Because of the lack of experimental evidence about hemoplasma DNA extraction efficiency in blood samples and in order to compare our results to other hemoplasma studies, a specific percentage of the DNA extraction efficiency could not be estimated. Therefore, at peaks of bacteremia, the acutely infected animal in this study showed loads of 4Æ86 · 1011 target copies ml)1 of blood. On the blood smear evaluation, this pig had 15–20 organisms ⁄ erythrocyte and additional organisms were seen in the background. Given the known total erythrocyte count of 5 · 106 ll)1 with 70% of erythrocytes having on the average 18 organisms, an approximate number of organisms ml)1 of blood would have been 6Æ3 · 1010 [(5 · 106) (0Æ7) (18) (1000)]. While this calculated value is somewhat lower than that obtained by qPCR, free organisms in the background (not attached to erythrocytes) could easily account for this fold difference. In contrast, a previous study reported an average load of 1Æ50 · 106 (range: 3Æ50 · 104–7Æ90 · 108) organisms ml)1 of blood in pigs with acute disease based on qPCR (Hoelzle et al. 2007). Assuming similar total erythrocyte counts, a calculated estimate for numbers of organisms per erythrocyte is only 3 · 10)4 (7 · 10)6–1Æ5 · 10)1 organisms ⁄ erythrocyte). The reason for this discrepancy is not clear, but may include a lower efficiency of DNA extraction in Hoelzle study (Hoelzle et al. 2007) or an overall lower bacteremia that can lead to similar clinical signs by the German strain of Myc. suis. Although it has been suggested that the German strain of Myc. suis is capable of erythrocytic invasion, this does not explain the discrepant numbers (Groebel et al. 2009). While the presence of intracellular parasites might lower the number of organisms observed on a blood smear, the qPCR results would include the detection of these organisms. As mentioned above, a recent report described that Myc. suis organisms may have cellular invasion capability (Groebel et al. 2009). The observation that motivated the study was that only marginal numbers of organisms were seen on blood smear even when pigs had high loads of 109–1010 organisms ml)1 of blood as detected by qPCR. In the present study and as previously reported (Messick et al. 1999), organisms were consistently seen in high numbers on blood smear evaluations at peaks of bacteremia, even for a few days after the initiation of oxytetracycline treatment. As invasion capability may vary among different mycoplasma and ureaplasma strains (Dusanic et al. 2009; Marques et al. 2010), further studies should be conducted with the North American ⁄ Illinois Myc. suis strain to determine whether or not it has the ability to invade erythrocytes.


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The number of target copies ml)1 of blood as well as absence of overt clinical signs of infection in pig 2, in animals from the Indiana farm and in Brazilian sows suggest that these pigs were chronically infected with a low circulating bacteremia. Further, organisms were not observed on stained peripheral blood smears of pig 2 nor from any of the Indiana pigs. Interestingly, the organism load in the blood of animals from the Indiana farm at 10 days of age was lower (8Æ80 · 104 and 6Æ70 · 104 organisms ml)1 of blood) when compared with older animals (pig 2 and pigs from Indiana farm at 3 and 6 months of age). Additional animals at this age (10 days old) should be evaluated to confirm if this is an actual trend for infection. No previous reports were found in the literature regarding the status of Myc. suis infection of pigs in Indiana, USA based on molecular testing. In this study, the frequency of Myc. suis infection based on qPCR in domestic pigs from Indiana (7Æ5%) was comparable to that of feeder pigs in Germany (13Æ9%) (Ritzmann et al. 2009). In contrast, 80% of the sows from Brazil were positive by qPCR. It is unknown whether Brazilian pigs have a higher infection rate in general or if the age of animals, and possibly increased exposure (via management practises and ⁄ or vector exposure), might account for the high frequency of infection in the sows. In China, 86Æ0% (148 ⁄ 172) of pigs at different ages tested positive for Myc. suis using a cPCR assay (Yuan et al. 2009). It is also important to recognize that the lack of standardized techniques among laboratories as well as the use of techniques with varied sensitivity may greatly influence the frequency of detection in different studies. Negative results for Myc. suis detection in the peccaries evaluated herein does not preclude the possibility of infection with other hemoplasma species. Although peccaries are phylogenetically related to domestic pigs, they are classified within a different family; domestic pigs (S. scrofa) are classified within the Family Suidae, whereas peccaries (T. tajacu and T. pecari) are classified within the Family Tayassuidae. Nevertheless, they are both in the Superfamily Suiodae, Order Artiodactyla (Harris and Li-Ping 2007). As hemoplasmas tend to be host speciesspecific, Myc. suis may not infect these animals. Another consideration for these negative results is that too few peccaries were sampled to detect low numbers of positives within the herd. In a previous study in Texas, five of seven peccaries had organisms attached to the erythrocytes based on blood smear evaluations (Hannon et al. 1985); however, molecular studies were not conducted on these samples and it is unknown whether the observed organisms were Myc. suis or another hemotropic mycoplasma. A recent PCR study, however, evaluated 359 European wild boars (S. scrofa) and found 36 (10Æ03%) Myc. suis positive animals (Hoelzle et al. 2010). Thus, Myc. suis is 424

likely present in wild pigs and other related populations, but additional studies need to be conducted to evaluate its distribution among the Suiodae members. Given that the peccaries evaluated herein had ectoparasites, and hemoplasmas have been described in other animal species from the same Zoos (Guimaraes et al. 2007b; Vieira et al. 2009), further studies are also warranted to determine if peccaries are infected with a different hemoplasma species. In conclusion, the TaqMan qPCR technique developed in this study was highly sensitive (outperforming a Myc. suis cPCR assay) and specific for the detection and quantification of Myc. suis in the blood of domestic pigs. As Myc. suis infection can cause nonspecific clinical signs, such as hyperthermia, inappetance, anorexia and sometimes icterus, the quantification of organisms may be used to indicate if these signs are consistent with the acute form of the disease. In addition, this technique may also be used in future studies that attempt to follow the cultivation of Myc. suis in vitro. Acknowledgements The authors are in debt to all veterinarians and staff of Itaipu Binacional, Universidade Federal do Parana and Zoologico de Curitiba for the peccaries blood collection. The authors also thank the Purdue Animal Care and Use Committee as well as all employees involved in animal handling and care. This study was conducted by Ana M.S. Guimaraes as a partial fulfilment of the requirements for her PhD at Purdue University. Funding for her PhD fellowship was provided by CAPES (Brazilian High Personal Improvement Coordination) – Fulbright combined sponsorship, while these research studies were supported with monies from Hatch funding, project no. 73095. References Applied Biosystems (2003). Creating standard curves with genomic DNA or plasmid DNA templates for use in quantitative PCR. Available from URL: http://www. appliedbiosystems.com/support/tutorials/pdf/quant_pcr. pdf, accessed on November 2007. Brownback, A. (1981) Eperythrozoonosis as a cause of infertility in swine. Vet Med Small Anim Clin 76, 375–378. Decaro, N., Elia, G., Martella, V., Campolo, M., Desario, C., Camero, M., Cirone, F., Lorusso, E. et al. (2006) Characterisation of the canine parvovirus type 2 variants using minor groove binder probe technology. J Virol Methods 133, 92–99. Dusanic, D., Bercic, R.L., Cizelj, I., Salmic, S., Narat, M. and Bencina, D. (2009) Mycoplasma synoviae invades non-phagocytic chicken cells in vitro. Vet Microbiol 138, 114–119.



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