Occurrence of Mycoplasma parvum in German Pigs of Different Age Groups Using a Novel Quantitative Real-Time PCR Assay
Abstract
:1. Introduction
2. Materials and Methods
2.1. Blood Samples and DNA Preparation
2.2. 16S rDNA and Amplicon Sequencing
2.3. Phylogenetic Analysis
2.4. M. suis and Ca. M. haemosuis qPCR
2.5. Primer Design
2.6. Cloning of the qPCR Fragment (qPCR-Positive Control)
2.7. Quantitative Real-Time PCR
2.8. Intra- and Interassay Repeatability
3. Results
3.1. Identification of Mycoplasma parvum Positive Samples
3.2. Development of a M. parvum-Specific Quantitative Real-Time PCR
3.3. Hemotrophic mycoplasma Infection in Fattening Pigs, Sows, and Boars
3.4. Mycoplasma parvum Phylogeny
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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HM Species and Name in the Phylogenetic Tree | Acc. Number (Version) | Host Species | Country of Origin |
---|---|---|---|
Mycoplasma parvum | |||
M. parvum ON703815 * | ON703815.1 | pig | Germany |
M. parvum ON703816 * | ON703816.1 | pig | Germany |
M. parvum ON703817 * | ON703817.1 | pig | Germany |
M. parvum ON703818 * | ON703818.1 | pig | Germany |
M. parvum ON703819 * | ON703819.1 | pig | Germany |
M. parvum ON703820 * | ON703820.1 | pig | Germany |
M. parvum ON703821 * | ON703821.1 | pig | Germany |
M. parvum MK287838 | MK287838.1 | pig | Brazil |
M. suis FN391022 | FN391022.1 | pig | Germany |
Ca. M. haemosuis JX489601 | JX489601.1 | pig | China |
M. wenyonii FN392885 | FN392885.1 | cattle | Germany |
M. ovis MH379798 | MH379798.1 | sheep | Brazil |
M. coccoides AY171918.1 | AY171918.1 | mouse | USA |
M. haemofelis U88563 | U88563.2 | cat | USA |
Ca. M. haemominutum AY150979 | AY150979.1 | cat | South Africa |
Intra-Assay Repeatability | Interassay Repeatability | |
---|---|---|
Plasmid Copy Number | Mean CT (±SD) * | Mean CT (±SD) |
107 | 15.23 (±0.44) | 15.02 (±0.92) |
106 | 19.01 (±0.27) | 18.94 (±0.92) |
105 | 22.78 (±0.11) | 21.92 (±0.94) |
104 | 26.20 (±0.15) | 25.25 (±0.96) |
103 | 29.93 (±0.07) | 29.28 (±0.75) |
102 | 32.81 (±0.73) | 31.89 (±0.99) |
101 | 35.92 (±0.81) | 35.51 (±0.54) |
Fattening Pigs | ||||||
---|---|---|---|---|---|---|
M. parvum1 | M. suis2 | Ca. M. haemosuis 2 | ||||
qPCR positive pigs (total) | 72/200 (36.00%) | 38/200 (19.00%) | 35/200 (17.50%) | |||
bacterial blood-loads (per mL blood) | Average: | 9.34 × 106 | 7.62 × 107 | 1.61 × 105 | ||
Range: | 3.41 × 103–4.82 × 108 | 9.58 × 102–1.14 × 109 | 5.52 × 103–1.55 × 106 | |||
Co-infections | ||||||
M. parvum | + | M. suis | 17/200 (8.50%) | |||
M. parvum | + | Ca. M. haemosuis | 19/200 (9.50%) | |||
M. suis | + | Ca. M. haemosuis | 12/200 (6.00%) | |||
all species (triple infection) | 5/200 (2.50%) | |||||
Sows | ||||||
M. parvum1 | M. suis1 | Ca. M. haemosuis 1 | ||||
qPCR positive pigs (total) | 15/60 (25.00%) | 4/60 (6.67%) | 13/60 (21.67%) | |||
bacterial blood-loads (per mL blood) | Average: | 1.18 × 106 | 3.58 × 104 | 4.83 × 105 | ||
Range: | 5.48 × 103–1.53 × 107 | 5.66 × 103–5.49 × 104 | 2.39 × 103–3.56 × 106 | |||
Co-infections | ||||||
M. parvum | + | M. suis | 0/60 (0.00%) | |||
M. parvum | + | Ca. M. haemosuis | 3/60 (5.00%) | |||
M. suis | + | Ca. M. haemosuis | 1/60 (1.66%) | |||
all species (triple infection) | 0/60 (0.00%) | |||||
Boars | ||||||
M. parvum1 | M. suis3 | Ca. M. haemosuis 3 | ||||
qPCR positive pigs (total) | 8/183 (4.37%) | 0/183 (0.00%) | 0/183 (0.00%) | |||
bacterial blood-loads (per mL blood) | Average: | 1.12 × 105 | / | / | ||
Range: | 6.23 × 103–6.26 × 106 | / | / | |||
Co-infections | ||||||
M. parvum | + | M. suis | 0/183 (0.00%) | |||
M. parvum | + | Ca. M. haemosuis | 0/183 (0.00%) | |||
M. suis | + | Ca. M. haemosuis | 0/183 (0.00%) | |||
all species (triple infection) | 0/183 (0.00%) |
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Ade, J.; Hoelzle, K.; Stadler, J.; Ritzmann, M.; Hoelzle, L.E. Occurrence of Mycoplasma parvum in German Pigs of Different Age Groups Using a Novel Quantitative Real-Time PCR Assay. Pathogens 2022, 11, 1374. https://doi.org/10.3390/pathogens11111374
Ade J, Hoelzle K, Stadler J, Ritzmann M, Hoelzle LE. Occurrence of Mycoplasma parvum in German Pigs of Different Age Groups Using a Novel Quantitative Real-Time PCR Assay. Pathogens. 2022; 11(11):1374. https://doi.org/10.3390/pathogens11111374
Chicago/Turabian StyleAde, Julia, Katharina Hoelzle, Julia Stadler, Mathias Ritzmann, and Ludwig E. Hoelzle. 2022. "Occurrence of Mycoplasma parvum in German Pigs of Different Age Groups Using a Novel Quantitative Real-Time PCR Assay" Pathogens 11, no. 11: 1374. https://doi.org/10.3390/pathogens11111374