Tools for molecular characterization of Mycoplasma hyopneumoniae
Maria Pieters, Douglas Marthaler, Lucas Dos Santos
Outline
• Molecular characterization
• Available tools
• How to use the information?
Molecular characterization
DNA studies shed light on relationships and diversity
Epidemiological investigations – Outbreak investigation – Surveillance in clinical cases
M. hyopneumoniae molecular characterization
• RAPD (Random amplified polymorphic DNA analysis)- Arthiushin and Minion, 1996
• AFLP (Amplified fragment length polymorphism) – Kokotovic et al., 1999
• PFGE (Pulse Field Gel electrophoresis) – Stakenborg et al .,2005
• PCR - RFLP (Randon fragment length polymorphism) – Stakenborg et al .,2006
• Sequencing of single locus – Mayor et al., 2007
• MLST – Multiple-Locus sequence typing – Mayor et al., 2008
• Multiple- Locus of VNTR analysis (MLVA) – Vranckx et al., 2011; Charlebois et al., 2013
Tools available for molecular characterization of M. hyopneumoniae at UMN
• Sequencing P146
• Multi-Locus Variable number tandem repeat Analysis (MLVA)
Sequencing P146
• Mayor et al., 2007 – Serine repeats (repeat 3)
• P146 gene
– UMN VDL – In house
M. hyopneumoniae P146 gene
• Paralog of P97 • Encodes a 148.2-kDa protein • Extensively processed on the surface of M.
hyopneumoniae
Bogema et al., 2012
Fragments of P146
Endoproteolytic processing
Repeat region 3 - Serine repeats
P146 amino acid phylogenetic tree
USA
BRA
BRA FRA CHN
CHN
Blue = System A Red = System B Green = GenBank Black = USA-VDL
98% identity (28 aa changes)
Mycoplasma spp. typing using MLVA
• M. mycoides subsp. mycoides (McAuliffe et al., 2007)
• M. pneumoniae (Degrange et al., 2009)
• M. genitalium (Ma et al., 2008; Cazanave et al., 2012)
• M. capricolum subsp. capripneumoniae (Manso-Silvan et al., 2011)
• M. bovis (Pinho et al., 2012; Spergser et al., 2013; Amram et al., 2013)
• M. hyopneumoniae
MLVA Typing
• Vranckx et al., 2011 – 4 VNTRs
• Dos Santos et al., 2015
– 2 VNTRs : R3 P146 and R1 P97
• Multi-Locus Variable-number Tandem-repeat Analysis (MLVA) typing method based on Variable copy Number of Tandem Repeats (VNTR)
• VNTR loci are selected that are sufficiently discriminatory
• Conserved primers are designed outside the tandem repeat for each VNTR
Use of MLVA to type M. hyopneumoniae
Vranckx et al., 2011
VNTR Loci
• Cilium Adhesin proteins – P97
• 3 VNTR Repeat regions – Repeat Region 1 (18 bp) – AAKPEV – Cilium binding site – At least 8 repeats for functional binding (Minion et al., 2000)
– P146 • 3 VNTR repeat regions
– Repeat Region 3 (3 bp)- Serine – Located in a P85 fragment that is associate with binding to porcine
respiratory cilia
Amplified fragments and standards move through a capillary and are detected using Capillary Electrophoresis Genetic Analyzer
MLVA for M. hyopneumoniae
MLVA for M. hyopneumoniae
Data analysis
• The fluorescence intensities are color-coded and displayed as peaks in the electropherogram
• Data can be analyzed as categorical characters • Population modeling networks can be calculated
using the cluster analysis application
More than one strain can circulate in a herd
Vranckx et al., 2011
MLVA typing – US samples
– To investigate the genetic variability of M. hyopneumoniae among US clinical samples submitted to the UMN-VDL (during a 6 month period)
– To compare the genetic variability of M. hyopneumoniae
between clinical samples from different countries
Materials and methods
• 209 Samples • 17 States • 19 Systems
Materials and methods
• Mexico (n=26) – 2 Systems
• Brazil (n=37) – 19 systems – 8 states
Stability of MLVA
• 12 passages of M. hyopneumoniae – Reference strain
• MLVA - high reproducibility and showed to be very consistent • M. hyopneumoniae - homogeneity in vitro passages
Passage 1 = 14-21 Passage 3 = 14-21 Passage 6 = 14-21 Passage 9 = 14-21 Passage 12 = 14-21
Stability of MLVA
Inoculum Experimentally infected
Vaccinated prior to infection
Maternal immunity prior
to infection
MLVA Type 14-21
M. hyopneumoniae Strain 232
Results • Discriminatory Power
Country D value U.S. 0.979
Mexico 0.880 Brazil 0.858
Combination 0.9816
* D value of 1.0 would indicate that a typing method was able to distinguish each member from all other members of that population
Frequency of distribution of MLVA types at US by manager system
• 209 samples • 19 Systems • 86 MLVA types
Frequency of distribution of MLVA types in Minnesota
Minnesota • 107 samples
54 MLVA types
MLVA type Frequency p97 p146
8-7 7 8 7
15-21 11 15 21
15-25 13 15 25
Frequency of distribution of MLVA types in Mexico
Mexico • 26 samples
12 MLVA types • 2 production systems
MLVA type Frequency p97 p146
10-18 8 10 18
10-19 4 10 19
10-17 3 10 17
Frequency of distribution of MLVA types in Brazil
Brasil • 37 samples
21 MLVA types • 19 systems
MLVA type Frequency p97 p146
12-14 14 12 14
12-15 2 12 15
12-38 2 12 38
Frequency of distribution of MLVA
types in three countries
• The MLVA type was not related to the country of origin
• Some MLVA types were exclusively detected in some countries
– D=0.9816 – 112 MLVA types – 273 samples
Mycoplasma hyopneumoniae variability within a swine production system Lucina Galina-Pantoja, Kalie Pettit, Lucas Dos Santos, Rick Tubbs, Maria Pieters
Material and methods • Three nursery-to-finish sites with endemic mycoplasmal infection • Originated from 8 sow farms which received all breeding stock from
one multiplier • N= 95 samples
(Tracheal aspirates and bronchial swabs)
Objective : to characterize the diversity of the M. hyopneumoniae variants within a production system
Mycoplasma hyopneumoniae variability within a swine production system
• The four variants identified within this study suggest limited variation
Summary • Sequencing the complete P146 gene
provides more discriminatory power than sequencing only the Repeat Region 3.
• MLVA of P97 and P146 identifies subtle changes in virulence factors of M. hyopneumoniae
Future directions
• Understanding the meaning and usefulness of sequencing and typing
• Correlating sequences/types with virulence
• Comparing/contrasting sequencing and MLVA in the same set of samples and correlating with clinical information
(Fano, Sponheim)
Thank you!