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!