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MURDOCH RESEARCH REPOSITORY This is the author’s final version of the work, as accepted for publication following peer review but without the publisher’s layout or pagination. The definitive version is available at http://dx.doi.org/10.1016/j.vetpar.2011.12.010 McInnes, L.M., Dargantes, A.P., Ryan, U.M. and Reid, S.A. (2012) Microsatellite typing and population structuring of Trypanosoma evansi in Mindanao, Philippines. Veterinary Parasitology, 187 (1-2). pp. 129-139. http://researchrepository.murdoch.edu.au/8678/ Copyright: © 2012 Elsevier B.V.. It is posted here for your personal use. No further distribution is permitted.

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Page 1: MURDOCH RESEARCH REPOSITORY · Page 2 of 43 Accepted Manuscript 2 20 ABSTRACT 21 Trypanosoma evansi, a blood-borne protozoan parasite with an extensive geographical range is 22 the

MURDOCH RESEARCH REPOSITORY

This is the author’s final version of the work, as accepted for publication following peer review but without the publisher’s layout or pagination.

The definitive version is available at http://dx.doi.org/10.1016/j.vetpar.2011.12.010

McInnes, L.M., Dargantes, A.P., Ryan, U.M. and Reid, S.A. (2012) Microsatellite typing and population structuring of Trypanosoma

evansi in Mindanao, Philippines. Veterinary Parasitology, 187 (1-2). pp. 129-139.

http://researchrepository.murdoch.edu.au/8678/

Copyright: © 2012 Elsevier B.V..

It is posted here for your personal use. No further distribution is permitted.

Page 2: MURDOCH RESEARCH REPOSITORY · Page 2 of 43 Accepted Manuscript 2 20 ABSTRACT 21 Trypanosoma evansi, a blood-borne protozoan parasite with an extensive geographical range is 22 the

MURDOCH RESEARCH REPOSITORY

This is the author’s final version of the work, as accepted for publication following peer review but without the publisher’s layout or pagination.

The definitive version is available at http://dx.doi.org/10.1016/j.vetpar.2012.01.007

McInnes, L.M., Dargantes, A.P., Ryan, U.M. and Reid, S.A. (2012) Microsatellite typing and population structuring of Trypanosoma

evansi in Mindanao, Philippines. Veterinary Parasitology, 187 (1-2). pp. 129-139.

http://researchrepository.murdoch.edu.au/8678/

Copyright: © 2012 Elsevier B.V..

It is posted here for your personal use. No further distribution is permitted.

Page 3: MURDOCH RESEARCH REPOSITORY · Page 2 of 43 Accepted Manuscript 2 20 ABSTRACT 21 Trypanosoma evansi, a blood-borne protozoan parasite with an extensive geographical range is 22 the

Accepted Manuscript

Title: Microsatellite typing and population structuring ofTrypanosoma evansi in Mindanao, Philippines

Authors: L.M. McInnes, A.P. Dargantes, U.M. Ryan, S.A.Reid

PII: S0304-4017(11)00848-XDOI: doi:10.1016/j.vetpar.2011.12.010Reference: VETPAR 6183

To appear in: Veterinary Parasitology

Received date: 3-9-2011Revised date: 3-12-2011Accepted date: 13-12-2011

Please cite this article as: McInnes, L.M., Dargantes, A.P., Ryan, U.M., Reid, S.A.,Microsatellite typing and population structuring of Trypanosoma evansi in Mindanao,Philippines, Veterinary Parasitology (2010), doi:10.1016/j.vetpar.2011.12.010

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Microsatellite typing and population structuring of Trypanosoma evansi in Mindanao, 1

Philippines

2

3

McInnes, L.M.1*

, Dargantes, A.P.1,2

, Ryan, U.M. 1

and Reid, S.A.3

4

1Division of Health Sciences, School of Veterinary and Biomedical Sciences, Murdoch 5

University, Murdoch, Perth WA 6150 6

2College of Veterinary Medicine, Central Mindanao University, University Town, Musuan, 7

Bukidnon, Philippines 8710 8

3Public Health Division, Secretariat of the Pacific Community, B.P. D5, 98848 Noumea cedex, 9

New Caledonia 10

11

* Author for correspondence: 12

Linda M. McInnes 13

Division of Health Sciences, School of Veterinary and Biomedical Sciences, Murdoch 14

University, Murdoch, Perth WA 6150 15

Phone: +61893602495 16

FAX: +61893104144 17

Email: [email protected] 18

19

*Manuscript

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ABSTRACT 20

Trypanosoma evansi, a blood-borne protozoan parasite with an extensive geographical range is 21

the causative agent of the livestock disease known as surra. A total of 140 out of 179 T. evansi 22

isolates collected between 2006 and 2007 from 44 villages (comprising of 16 reported surra 23

outbreaks) in 3 provinces (Agusan del Sur (ADS), Surigao del Sur (SDS) and Agusan del Norte 24

(ADN)) in Mindanao, Philippines were each successfully genotyped using a suite of 7 25

polymorphic microsatellites. The study identified 16 multi locus genotypes (MLG) within the T. 26

evansi isolates and evidence of the spread of surra outbreaks from one village to another, most 27

likely due to the movement of infected animals. Genotyping provided evidence of population 28

sub-structuring with 3 populations (I, II and III (only 1 isolate)) identified. The most abundant 29

population was II, which was the predominant population in ADS and SDS (p=0.022). In 30

addition, buffalo mortality was statistically higher in outbreak areas associated with isolates from 31

population I (13.6%) than with isolates from population II (6.9%) (p=0.047). The present study 32

has highlighted the utility of microsatellite loci to improve understanding of the epidemiology of 33

T. evansi and in tracking surra outbreaks. 34

35

KEYWORDS 36

Trypanosoma evansi, surra, epidemic, microsatellites, DNA, Mindanao, Philippines. 37

38

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1. Introduction 39

Trypanosoma evansi is endemic in Africa, the Middle East, Central and South America, and 40

many parts of Asia including the Philippines (OIE, 2008). Trypanosoma evansi, although able to 41

infect most classes of mammals, is principally a livestock disease (surra), causing considerable 42

mortality and morbidity in bovines, equines and camelids worldwide. In the Philippines, surra 43

outbreaks have been reported in all 13 regions and resultant mortalities in horses, buffaloes and 44

cattle between 1989 and 1997 have been estimated at a cost of US$1.15 million per annum 45

(Manuel, 1998). There has recently been a large number of severe outbreaks in the Philippines 46

with an outbreak in the North Samar province reported to have caused buffalo mortalities in 47

excess of a thousand over a 6-month period (Cresencio et al., 1994; Manuel, 1998). 48

Surra was first introduced into the Philippines in the early 19th

century with the 49

importation of infected cavalry horses from China by the American forces during the American-50

Spanish war (Manuel, 1998). The initial and successive surra cases which occurred in the 51

Philippines were predominately reported as high mortalities of equines (Manresa, 1935; Randall 52

and Schwartz, 1936; Manuel, 1998). Mindanao, the second largest and southernmost island in 53

the Philippine archipelago appeared to be free of surra until 1989 when the first reported cases of 54

surra (38% equine and bovine mortality) were recorded in the southern Mindanao province of 55

Sarangani (Mercado, R. pers. comm., 2010). Subsequent to the Sarangani outbreak, further surra 56

outbreaks have occurred in a range of Mindanao provinces (Fig. 1). At present, the economic 57

losses due to surra in Mindanao have been estimated at US$1.58 million per year from every 10 58

villages where the disease is moderately to highly prevalent (Dobson et al., 2009). 59

Surra presents as a highly variable clinical disease with the variation in disease 60

manifestation (acute, sub-acute or chronic) associated with differences in host susceptibility 61

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and/or parasite strain virulence (OIE, 2010). An important feature of T. evansi infections is the 62

sporadic nature of surra outbreaks which are often localised. High mortalities are most often 63

attributed to the movement of infected animals into areas previously free from infection or the 64

movement of surra-free animals into endemic areas (Payne et al., 1988). The endemic nature of 65

T. evansi in the absence of high mortality outbreaks has led to speculation that surra may be 66

enzootically stable in some areas such as Indonesia (Payne et al., 1991a; Payne et al., 1991b). 67

Microsatellites, which are highly discriminatory genetic tools have been used to examine 68

T. cruzi phylogeography (Llewellyn et al., 2009a; Llewellyn et al., 2009b), differences in T. cruzi 69

clinical disease (D'Avila et al., 2009), T. brucei gambiense population structuring (Koffi et al., 70

2009; Simo et al., 2010), T. brucei mixed strain infection investigations (Balmer and Caccone, 71

2008), mating in T. congolense (Morrison et al., 2009) and T. evansi population structuring in 72

Kenya (Njiru and Constantine, 2007; Salim et al., 2011). As T. evansi is widely believed to have 73

evolved from T. brucei which was spread beyond the tsetse fly belt (Hoare, 1972). The genetic 74

similarity between T. brucei and T. evansi has led others to propose that T. evansi should be 75

considered a subspecies of T. brucei rather than a species (Lai et al., 2008). This genetic 76

similarity between T. brucei and T. evansi means the T. brucei genome is highly useful for 77

development of T. evansi genetic tools. In the present study, microsatellite markers developed 78

from T. brucei genetic information were used to characterise the isolates and parasite populations 79

from barangays (the Philippines‟ equivalent of a village) without surra outbreaks and those 80

isolates involved in the late 2005 to 2007 T. evansi outbreaks in 16 barangays in Mindanao, 81

Philippines and their possible association with clinical disease, patent parasitaemia and temporal 82

and spatial sub-structuring. 83

84

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2. Materials and methods 85

2.1. Sources of isolates and sample collection 86

A total of 179 T. evansi isolates were collected from infected livestock (mostly buffaloes) 87

from 44 barangays (with or without surra outbreaks) in the provinces of Agusan del Sur (ADS), 88

Surigao del Sur (SDS) and Agusan del Norte (ADN), as shown in Figures 2a and b, in Mindanao, 89

southern Philippines during surra epidemiological surveys in 2006–2007. The field surveys were 90

conducted in a total of 89 barangays from 36 municipalities in 5 provinces in Mindanao 91

involving more than 3,000 village livestock (Dargantes, 2010). From 16 surra outbreak 92

barangays (Table 1), 660 local swamp buffaloes (Bubalis bubalis), 24 cattle, 50 horses and 33 93

goats were randomly bled. Whole blood (6-9 mL) was aseptically collected from the jugular vein 94

of livestock into a labelled ethylenediamine-tetra acetic acid (EDTA) vacutainer vial (Vacuette®, 95

Greiner Bio-One, USA). Blood samples were placed inside a cooler box until examination. The 96

microhaematocrit centrifugation technique (MHCT) and mouse inoculation test (MIT) were used 97

to detect T. evansi in blood samples as described in Dargantes et al. (2009). Mice parasitaemic 98

with T. evansi were bled and the blood was stored in EDTA vials at -20ºC until required. 99

Information on each animal sampled including geographical origin (barangay, municipality and 100

province), MHCT parasite enumeration, card agglutination test for Trypanosoma evansi (CATT), 101

host species, blood packed cell volume (PCV), age and body condition was recorded. Data on the 102

percentage mortality of buffaloes, cattle, horses and goats in conjunction with the outbreaks of 103

surra were obtained from the municipal veterinary personnel or local barangay officials. All field 104

and laboratory activities were conducted with approval from the Murdoch University Animal and 105

Human Ethics Committees (Permit numbers R881/01 and 2007/034, respectively). 106

107

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2.2. DNA extraction 108

Whole genomic DNA was extracted from blood collected from mice inoculated with the 109

blood of a T. evansi-infected original host (M) and from the whole blood from the corresponding 110

original host (OH) using a modified protocol with a MasterPure™ DNA Purification Kit 111

(EPICENTRE® Biotechnologies, Madison, Wisconsin, U.S.A.) as described by McInnes et al. 112

(2011). DNA was stored at -20ºC until use. 113

114

2.3. Microsatellite typing tool development 115

Microsatellites were identified from T. brucei genome sequences sourced from GenBank 116

by screening for repeating DNA motifs utilising the software program Tandem Repeats Finder 117

(Benson, 1999). Primers were designed in flanking regions of selected microsatellites chosen on 118

the basis of perfect repeating motifs, originating from different contigs and where possible from 119

different chromosomes to minimise the chance of linkage. An initial 15 microsatellites were 120

chosen for analysis (11 dinucleotide microsatellites and 4 trinucleotide microsatellites) and 121

primers were designed with the aid of software program Amplify v3.1. Primers were tested on 3 122

different T. evansi isolates (collected in different years from livestock in Central, and East Java 123

and Sumbawa, Indonesia) previously shown to be genetically different (C. Constantine, pers. 124

comm., 2007) and the resultant amplicons electrophoresed on 4% High resolution agarose 125

(Fisher Biotec, Australia) to ascertain the best candidates for further analysis. Promising 126

microsatellites, which appeared to display some degree of heterogeneity amongst the three 127

isolates, were chosen for fluorescent labelling and multiplexing. Of the 15 microsatellites, only 4 128

were deemed useful after evaluation was completed on 20 T. evansi isolates (11 loci were 129

discarded due to monomorphism, unscoreable results and poor PCR amplification). Chosen loci 130

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Tbb1 and Tbb10 were combined in one multiplex (TEM1) and Tbb5 and Tbb9 in a second 131

multiplex (TEM2) with fluorescent labels as indicated in Table 2. 132

All designed primers were tested on the DNA extracted from the blood of a mouse 133

experimentally infected with a T. evansi isolate using the following PCR conditions: 94ºC for 5 134

min followed by 35 cycles of 94ºC for 30 sec, 56ºC for 20 sec and 72ºC for 30 sec, finishing with 135

a final extension of 72ºC for 45 min. All multiplex PCRs were carried out in 25 µL reactions 136

comprising of 1 x PCR buffer, 1.5 mM MgCl2, 0.1 mM of each dNTPs, 0.4 µM of each primer, 137

0.04 units of Tth+ DNA polymerase (Fisher Biotec, Australia) and 1 µL DNA template. All PCR 138

amplicons generated from this T. evansi isolate were purified using a MO BIO UltraCleanTM 15 139

DNA Purification Kit (MO BIO Laboratories Inc. West Carlsbad, California, USA) and 140

sequenced using an ABI PrismTM

Terminator Cycle Sequencing kit (Applied Biosystems, Foster 141

City, California, USA) on an Applied Biosystem 3730 DNA Analyzer to confirm PCR 142

amplification of correct loci. 143

144

2.4. Microsatellite typing of samples 145

DNA extracted from the Philippine T. evansi samples (both M and OH samples) were 146

amplified using the two developed multiplexes (TEM1 and TEM2) following conditions 147

described above as well as with microsatellite primers designed by Biteau et al. (2000) for the 148

following loci: MORF-CA, MT3033-AC/TC, M6C8-CA, MT3033-TA and MEST19-AT/GT. 149

The five loci identified by Biteau et al. (2000) were multiplexed to conserve resources. Loci 150

MORF-CA and MT3033-AC/TC were combined in one multiplex (Bite1) with MORF-CA 151

fluorescently labelled with 6-FAM (Applied Biosystems, Life Technologies, Carlsbad, 152

California) and MT3033-AC/TC with Vic (Applied Biosystems). Loci M6C8-CA, MT3033-TA 153

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and MEST19-AT/GT were combined in a second multiplex (Bite2) with the forward primers for 154

M6C8-CA labelled with NED (Applied Biosystems), MT3033-TA with Vic (Applied 155

Biosystems) and MEST19-AT/GT with 6-FAM (Applied Biosystems). Conditions for the Bite1 156

and Bite2 multiplexes were as follows: 94ºC for 5 min followed by 30 cycles of 94ºC for 30 sec, 157

53ºC for Bite1 and 50ºC for Bite2 for 30 sec and 72ºC for 30 sec, and a final extension of 72ºC 158

for 45 min. The PCR reaction reagents for both Bite1 and Bite2 were the same as previously 159

described for the TEM1 and TEM2 multiplexes. All fluorescently labelled PCR amplicons were 160

analysed on an ABI 3730 DNA Analyzer (Applied Biosystems Inc.) in conjunction with a 161

GeneScan™ 600 LIZ® Size Standard (Applied Biosystems). Allele identification was performed 162

manually using the ABI program Genemapper v3.5 (Applied Biosystems). The term multilocus 163

genotype (MLG) is applied in this paper to isolates with identical genotype established from 164

several genetic loci. 165

166

2.5. Population analysis 167

Population structuring analyses of datasets using the 4 (developed as part of the present 168

study) and 7 loci (the 4 developed loci and 3 loci published by Biteau et al. (2000) were 169

conducted using the software program Structure 2.3.3, which is a Bayesian model-based 170

clustering method for inferring population structure and assign individuals to populations from 171

multilocus genotype data (Pritchard et al., 2000). Simulations using the admixture and correlated 172

frequencies model were run for K=1-7 with a burn in period of 20,000 and 100,000 MCMC 173

repetitions and the average log probability of data clustering (L(K)) from 15 replicates 174

calculated. 175

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The construction of Neighbour-Joining (NJ) phylogenetic trees of microsatellite data 176

from T. evansi isolates were carried out using Powermarker 3.25 (Liu and Muse, 2005), and the 177

trees visualised using MEGA 4.1 (Tamura et al., 2007). The NJ genetic distance trees were 178

constructed with the shared allele model (DAS) (Jin and Chakraborty, 1994) and 1,000 bootstrap 179

replicates for all isolates typed at the 4 loci (TEM1 and TEM2) (159 isolates) and with all 7 loci 180

(140 isolates). 181

Microsatellite allelic frequencies, Nei‟s unbiased measures of genetic distance and 182

inbreeding coefficient (FIS), Wright‟s fixation index (FST) values and departure from Hardy–183

Weinberg equilibrium (heterozygote deficiency or excess) were calculated with the genetic 184

software GENEPOP v.3.4 (http://genepop.curtin.edu.au) originally developed by Raymond and 185

Rousset (1995). In addition, the standardised index of association (IAS), a measure of the 186

association between alleles at different loci, was calculated using the software program LIAN - 187

LInkage ANalysis, Version 3.5 (http://adenine.biz.fh-weihenstephan.de/cgi-bin/lian/lian.cgi.pl) 188

developed by Haubold and Hudson (2000). The null hypothesis of linkage equilibrium for the T. 189

evansi dataset was tested by Monte Carlo simulation (1,000 iterations) and a parametric test 190

(Haubold et al., 1998). The multi locus genotype (MLG) index (G/N), a measure of clonality 191

expressed as a fraction derived from the number of MLGs (G) compared with the number of 192

isolates (N), was calculated. Genic differentiation with calculation of FST values between 193

populations (determined by Structure 2.3.3), provinces, year of sampling and provinces together 194

with years of sampling were calculated using GenAlEx (Peakall and Smouse, 2006). 195

196

2.6. Statistical analysis 197

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The populations identified amongst the isolates were recorded and coded together with 198

other information gathered about each host in a dataset using the Statistical Package for Social 199

Sciences (SPSS) 17.0.1 (SPSS Inc., Chicago, IL, USA). The statistical significance of any 200

differences in values for categorical variables (population, geographical origin, MHCT, CATT 201

and body condition) was determined using Pearson‟s Chi-square (χ2) (p<0.05) and for continuous 202

variables (host animal age, mortality and PCV) using Student‟s T-test (p<0.05). 203

Analysis of the predominant T. evansi population (as determined by Structure 2.3.3), and 204

buffalo mortality required the removal of any sampling locations that could not be assigned a 205

predominant population (i.e. barangays with 2 isolates, each assigned by Structure 2.3.3 to 206

different populations). This reduced the dataset analysed from 44 to 41 barangays. 207

208

2.7. Geographical representation of T. evansi microsatellite information 209

The spatial relationships of the T. evansi isolate genotypes were visualised by plotting 210

populations, epidemics and buffalo mortality figures together with isolate genotypes on a map of 211

Mindanao using a geographical information system (GIS) program Quantum GIS 1.5.0-Tethys 212

(http://qgis.org/). 213

214

3. Results 215

3.1. Microsatellite selection procedure 216

The loci M6C8 and MT3033TA sourced from Biteau et al. (2000) which were initially 217

combined in two multiplexes, were monomorphic in 120 Mindanao T. evansi isolates and these 218

loci were therefore discarded. 219

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DNA sequencing of amplicons of each of the loci (Tbb1, Tbb5, Tbb9, Tbb10) developed 220

in the present study confirmed that the loci amplified from T. evansi isolates were the same loci 221

identified from the T. brucei genome sequence data (data not shown). Therefore a total of 7 loci 222

(4 developed during the present study and 3 sourced from Biteau et al. (2000)) were used for 223

further analysis. 224

225

3.2. Microsatellite typing of original host (OH) and mouse derived (M) DNA extracts 226

Microsatellite typing with the multiplexes TEM1, TEM2, Bite1 and Bite2 were attempted 227

on 262 DNA samples from 195 isolates: 179 T. evansi mouse derived (M) isolates and 99 T. 228

evansi original host derived (OH) samples. Of the 179 T. evansi M isolates, 83 (46%) had a 229

corresponding OH sample, i.e. DNA extractions from the blood of an infected buffalo and the 230

blood of a mouse infected with a sample from the same buffalo. Trypanosoma evansi M samples 231

were more successfully amplified than the OH samples; 7 loci were amplified from 70.9% 232

(127/179) M isolates but only 13.1% (13/99) OH isolates were amplified at all 7 loci. 233

Of the 195 isolates tested, 6 isolates typed had 3-4 alleles at each microsatellite locus 234

typed. Two OH isolates (A7B-387OH and A73-807OH) and 4 M isolates (A7E-788M, A7E-235

859M, A7E-1079M and SN7-151M) demonstrated mixed loci. The complementary M isolates 236

(A7B-387M and A73-807M) to the two mixed OH isolates (A7B-387OH and A73-807OH) did 237

not demonstrate the same mixture of alleles but contained alleles present in the OH isolate 238

mixtures. 239

240

3.3. Population structuring 241

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An unrooted genetic distance tree with NJ 1000 bootstrap was constructed with the 242

shared allele evolutionary model from 140 isolates typed at 7 loci (Fig. 3) and 179 isolates at 4 243

loci (not shown). Population structuring analyses conducted on 4 and 7 loci datasets using the 244

software program Structure 2.3.3 (Pritchard et al., 2000) were congruent (i.e. T. evansi isolates 245

which were present in both datasets, were assigned to the same populations in both analyses). 246

Furthermore, the highest average log probability of data clustering (L(K)) from 15 replicates 247

calculated for the 7 loci dataset was determined for K=3 (-435.867). The three populations 248

identified were designated I, II and III and are represented in the constructed NJ genetic distance 249

tree (Fig. 3). The predominant population in the 7 loci dataset was population II which 250

comprised 67.1% (94/140) of isolates followed by population I with 32.1% (45/140) of isolates 251

and population III with 0.7% (1/140) of isolates. The analysis also identified 16 MLGs (sets of 252

isolates that appeared to be identical when typed with a given set of genetic markers in a 253

basically clonal species) (Tibayrenc and Ayala, 1991) within the three populations (I, II and III). 254

The largest MLG groups were A and C; MLG A comprised 21 identical isolates and MLG C had 255

55 isolates. 256

257

3.4. Population analysis 258

Analysis of the 140 isolates typed at 7 loci revealed complete heterozygosity at all of the 259

7 microsatellite loci analysed (no homozygotes present in any loci in any isolate genotyped) with 260

significant deviations from Hardy-Weinberg equilibrium (p<0.001). The mean inbreeding 261

coefficient (FIS) of the 7 loci was -0.3458 and negative FIS values were also generated for each 262

individual locus. Levels of polymorphism varied between loci, with the total number of alleles 263

per locus ranging from 3 (MORFCA) to 12 (Tbb5). The MLG index (G/N) for 7 loci was 0.14 (6 264

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unique and 14 repeated MLGs). The standardised index of association (IAS) for the complete 265

dataset was 0.6956 with significant linkage disequilibrium amongst all combinations of loci 266

(p<0.001). 267

The expected and observed heterozygosity, number of alleles and FIS values of each locus 268

within populations I, II and III are presented in Table 3. Nei‟s genetic distance, FST and geneflow 269

(NM) of isolates divided by population (I and II; III excluded due being only 1 isolate), province 270

(ADS and SDS), year of collection (2006 and 2007), or combination of province and year of 271

collection (ADS-2006, ADS-2007, SDS-2006 and SDS-2007) were calculated (Table 4). There 272

was little evidence in population structuring according to province or year of collection (0.022 273

and 0.021 respectively). There were significantly different FST values (0.118-0.154) generated for 274

isolates collected from ADS in 2006 compared with those collected from ADS in 2007 or from 275

SDS in either 2006 or 2007. The FST value between populations I and II was 0.117. There was a 276

significant linkage of all loci for isolates divided on the basis of Structure 2.3.3 population, 277

province, year of collection or province and year of collection based on calculated IAS values 278

(p<0.001). 279

280

3.5. Statistical and geographical analysis 281

No significant differences were seen between population I and II isolates (III was omitted 282

due to insufficient data) and province in regard to any categories except for province and buffalo 283

mortalities (p<0.05). Population II was detected in a significantly higher proportion of samples 284

from ADS (58.7%, 95% C.I. 48.9-68.1%) and SDS (81.3% 95% C.I. 67.4-91.1%) (p=0.022) 285

(Fig. 4). The mean buffalo mortality in locations with a predominant population I of T. evansi 286

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isolates was significantly higher (13.6%) compared to locations with a predominant II population 287

of T. evansi isolates (6.9%) (p=0.047). 288

Analysis of MHCT, CATT, buffalo mortality and PCV with T. evansi MLGs could only 289

be conducted for two MLGs; A and C (sufficient numbers for analysis), which belonged to the 290

same population (II). MLG A which comprised 21 isolates was primarily isolated in Surigao del 291

Sur, whilst MLG C which comprised 55 isolates was predominantly isolated in Agusan del Sur 292

(Fig. 6). There were no significant differences on the MHCT and CATT results and PCV values 293

of hosts infected with either MLG A or C. Buffalo mortality in areas with a reported outbreak 294

had significantly higher mortality when MLG A T. evansi isolates were present than in areas 295

where MLG C isolates were present (p<0.05). The mean buffalo mortality involving MLG A 296

isolates was 17.1% and 5.9% for MLG C isolates. There was a more visible association between 297

geographical location of MLG A isolates and areas of recorded buffalo mortality (4 barangays) 298

compared with MLG C and areas of buffalo mortality (1 barangay) (Fig. 5). 299

300

3.6. Correlation between epidemic and T. evansi MLG 301

Samples were collected from livestock within 3 months of first outbreak report in 9 of the 302

17 reported outbreaks of surra. The T. evansi MLG most likely to be the source of the outbreak 303

could not always be inferred because many of the epidemic sites sampled yielded too few 304

isolates to be conclusive. Several isolates of the same MLG were typed in different outbreaks 305

and isolates collected from some outbreaks comprised more than one T. evansi genotype as 306

presented in Table 5. 307

308

4. Discussion 309

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The population structure of T. evansi in Mindanao exhibited evidence of clonal 310

expansion, in particular within epidemics with the observation of 14 MLGs in the dataset as well 311

as significant linkage, negative FIS values and 100% heterozygosity at each locus. High 312

heterozygosity occurs in clonal organisms due to accumulations of random mutation events 313

(Halkett et al., 2005; De Meeus et al., 2006). Due to the absence of the tsetse fly, vector in which 314

T. brucei is known to undergo sexual recombination (Peacock et al., 2011), T. evansi within the 315

Philippines and other non tsetse fly areas is inferred to be asexually reproducing. The complete 316

heterozygosity observed in the T. evansi isolates from the Philippines is incompatible with 317

meiotic segregation (Tibayrenc et al., 1990) and supportive of T. evansi reproduction being 318

asexual in the Philippines. However, heterozygosity may also be due to the typing of mixed 319

strain infections. The presence of 14 T. evansi MLGs in Mindanao isolates, however, negates the 320

notion that the heterozygous microsatellite profiles produced are the result of mixed infections 321

(the combination of two homozygote strains giving rise to two alleles at each locus). The finding 322

of several MLGs amongst the Philippine isolates is in contrast to a recent study conducted by 323

Salim et al. (2011) on T. evansi in camels within Sudan. Salim et al. (2011) noted that the 324

absence of any MLGs in their dataset was unusual for what is believed to be a clonal organism. 325

They argued that their dataset may have been subject to “frequent allelic dropouts, Wahlund 326

effects or both”. 327

Mixed strain infections of T. evansi were evident in 6 isolates (the presence of 4 alleles at 328

each locus), 2 of which originated from OH isolates. Mixed infections have been reported in 329

cattle (T. congolense) (Morrison et al., 2009), humans (T. brucei) (Truc et al., 2002) and in pigs 330

(T. brucei) (Jamonneau et al., 2003). Intra-host variation has been observed in the ribosomal 331

internal transcribed spacer 1 of T. evansi from buffalo and cattle from Thailand, which could be 332

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due to natural mixed strain infections (Khuchareontaworn et al., 2007; Areekit et al., 2008). 333

Mixed trypanosome species and strain infections are also known to occur in vectors such as the 334

tsetse fly (MacLeod et al., 1999). Tabanids are considered opportunistic feeders (Magnarelli and 335

Anderson, 1980; Gouteux et al., 1989; Muzari et al., 2010) and recent studies have shown that 336

mixed host blood meals are frequent in some tabanid species (Muzari et al., 2010). 337

It is important to recognise that in vivo systems such as the MIT may exert „selective 338

pressures‟ (Jamonneau et al., 2003) that may lead to a reduced recognition of naturally occurring 339

mixed infections. Concentration of trypanosomes by methods such as MIT has however, been 340

very important for trypanosome analysis due to the characteristic low parasitaemia that is 341

observed in most naturally T. evansi-infected hosts. Direct amplification of microsatellites 342

without the aid of any concentration methodologies or PCR sensitivity enhancing strategies is 343

frequently difficult. Duffy et al. (2009) were only able to amplify a suite of 8 microsatellites 344

from 31 of 304 (10%) T. vivax-positive DNA samples. Nested PCRs may have increased 345

sensitivity without the need for a potentially selective concentration process such as MIT, 346

however, the reported increases in sensitivity achieved by nested microsatellite PCR design vary. 347

Valadares et al. (2008) reported a 100-1,000-fold increase in the sensitivity with typing T. cruzi 348

from infected tissue samples, whilst Morrison et al. (2007) only reported a 5-fold increase in 349

sensitivity with a nested PCR using dried blood on FTA filter cards. PCR detection on FTA 350

cards can however be problematic as reported by Cox et al ,(Cox et al., 2010) who observed that 351

single punch nested PCR analyses for trypanosomes can lead to considerable underestimations of 352

prevalence. 353

The three populations (I, II and III) identified amongst the T. evansi isolates genotyped 354

from Mindanao may be evidence of the existence of only three T. evansi lineages in the 355

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northeastern Mindanao provinces (ADS and SDS). The population analysis indicates that the 356

outbreaks investigated in ADS and SDS were primarily caused by two T. evansi lineages which 357

have differentiated over time into two populations (I and II). The isolate AS-76 from Mapaga, 358

Prosperidad, ADS was the only representative of T. evansi population III in Mindanao. 359

Identification of only one isolate for this population suggests that this isolate may be a recent 360

introduction to the northeastern Mindanao provinces (ADS and SDS). The genetic similarity of 361

AS-76 with an isolate LZ-1 (typed at 4 loci) from the Philippine island Luzon suggests that AS-362

76 isolate may have originated from Luzon. Mindanao is, however, an island geographically 363

removed from Luzon with restrictions of animal movement due to Mindanao being considered 364

foot and mouth disease (FMD) free without vaccination by the Office Internationale des 365

Epizooties (OIE) since 2001 (OIE, 2001). It is therefore more likely that AS-76 is an isolate from 366

a lineage of T. evansi present in some of the Mindanao provinces not examined in this study. A 367

comprehensive survey to genotype T. evansi isolates from all Mindanao provinces as well as 368

other Philippine provinces would be required to elucidate the origins of the 3 Mindanao T. evansi 369

populations. The present study did not test any isolates from the southern Mindanao provinces 370

which were the first to report surra outbreaks. 371

The provinces of ADS and SDS in Mindanao, although situated adjacent to one another, 372

are separated by a mountain range with few connecting roads. The mountain range, lack of 373

interconnecting roads and the political insurgency in the region may have restricted animal 374

movement between the provinces and thereby reduced the spread of T. evansi strains between 375

provinces. However, comparison of isolates from these two provinces did not support the 376

hypothesis that there is a significant genetic divide in isolates (low FST values of 0.022) with 377

considerable gene flow (11.3) measured between the provinces. This was also evident with T. 378

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evansi population II isolates present in both provinces and significantly more prominent in both 379

ADS and SDS provinces than population I. The transmission of T. evansi MLGs between 380

provinces was exemplified by MLGs A and C with isolates of these MLGs present in both 381

provinces on either side of the mountain range. 382

Significant variations on the FST values of Mindanao T. evansi based on population sub-383

structuring analysis may suggest that the isolates collected from ADS in 2006 were different 384

from the isolates collected from the same province in the following year as well as from isolates 385

collected from SDS in either 2006 or 2007. However, this could be an anomaly attributed to the 386

small sample size (n= 9) of isolates collected from ADS in 2006. 387

The susceptibility to T. evansi infection varies depending on the host species. However, it 388

is not clear whether host-specific strains exist in the areas sampled. It is interesting to note that 389

the outbreak recorded in Ebro, San Francisco identified a T. evansi MLG (K) from 4 horses 390

which was not present in any of the other surra outbreaks in nearby barangays during the same 391

year. This may have been due to a strain more pathogenic to horses than to buffalo or simply due 392

to successful containment of the Ebro outbreak. In the present study there were no villages 393

sampled which contained both horse and buffalo surra strains. This was usually the result of high 394

surra mortality in horses leading to the elimination of most horses from surra epidemic areas 395

prior to sampling. The high susceptibility or possible high pathogenicity of T. evansi to horses 396

was evident in the outbreaks in Ladgadan, Del Rosario, Mapaga and Causwagan, ADS which 397

recorded equine mortalities in excess of 30%. In Banahao, Lianga, SDS, T. evansi isolates were 398

however recovered from 2 horses which were identified as MLG C, the most likely causative 399

agent of the large outbreak in buffalo in Guadalupe and Concordia. As the Guadalupe/Concordia 400

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outbreak occurred immediately before the Banahao outbreak, it may be due to translocation of T. 401

evansi-infected animals. 402

The evidence that some MLGs were localised to specific barangays is probably a 403

reflection of the transmission dynamics of T. evansi that require close proximity between hosts 404

(Barros and Foil, 2007). In Mindanao barangays, the mechanical transmission of T. evansi by 405

tabanids is likely to be enhanced by the common practice of communal tethering of livestock in 406

pastures often adjacent to or in close proximity to irrigation canals, swamps and rivers or by 407

using a common wallowing pool as in the case of buffaloes (Dargantes, A. pers. comm., 2010). 408

Areas with abundant water are likely to have high population of tabanids, as most tabanids 409

require water for breeding and development (Mitzmain, 1913; Service, 1986; McElligott and 410

Lewis, 1996; Butt et al., 2008). 411

Transmission of T. evansi by animal translocation is a logical explanation for the 412

outbreaks reported in Guadalupe and Concordia, Esperanza, ADS. These outbreaks occurred in 413

villages geographically close to one another, around the same time of the year (Jan-Mar 2007) 414

and were both strongly associated with T. evansi MLG C. The MLG finding and temporal and 415

spatial proximity of the outbreaks suggest this was in fact a single connected event rather than 416

two separate outbreaks. The analysis revealed that the MLG responsible for these two outbreaks 417

was the same, which then caused an outbreak in Ladgadan, San Francisco, ADS in the following 418

year. This may be due to the movement of an infected host from Guadalupe or Concordia to 419

Ladgadan and seasonal fluctuations in tabanid abundance culminating in an outbreak the 420

following year in a new location. 421

In the present study, increased buffalo mortality was more associated with T. evansi 422

population I isolates than with population II isolates, and with MLG C isolates than with MLG A 423

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isolates from population II. However, it is important to note that the variation in pathogenicity 424

could be due to the many other potentially influencing factors such as immune status and other 425

contributing health issues of the hosts. Microsatellite typing is able to assist in elucidating this 426

issue but influencing factors would need to be controlled or a more extensive survey over a 427

considerably longer period of time would be required. Data from the application of microsatellite 428

typing of T. evansi isolates from Mindanao shows that outbreaks were caused by the same T. 429

evansi strain (MLG C), and that the T. evansi strains in the northeastern Mindanao provinces 430

represent three lineages. Furthermore, the present study has demonstrated the value of 431

microsatellites for the study of complex infectious agents such as T. evansi. However, larger and 432

more representative datasets are required to provide a more accurate description of the origin of 433

outbreaks, the transmission factors promoting spread of outbreaks and the importance of strain 434

variation in pathogenicity. 435

436

437

Acknowledgements 438

Field surveys and collection of isolates were carried out in coordination with the Mindanao 439

Unified Surra Control Approach (MUSCA) group headed by Dr. R. Mercado and the help from 440

veterinarians and staff of collaborating government agencies in Agusan del Norte, Agusan del 441

Sur and Surigao del Sur, and the members of the CMU Surra Team. The authors are also grateful 442

for the significant assistance of Drs. J.R. Dargantes, P. Calo, M. Samar, J.A. Abella, R. 443

Sumagang, E. Igsoc, M. Pagobo and of D. Miguel and K. Quero. The financial support from the 444

Australian Centre for International Agricultural Research (ACIAR), Central Mindanao 445

University (CMU), International Foundation of Science (IFS), Murdoch University and the 446

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Australian Biosecurity Cooperative Research Centre (AB-CRC) which contributed to the 447

research presented in the article is acknowledged. 448

449

Conflict of interest statement 450

The authors of this article declare that they have no conflicts of interest with regard to the work 451

presented herein. 452

453

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Table 1

Details of the locations of putative Trypanosoma evansi outbreaks in 16 barangays in Mindanao,

Philippines between late 2005 and 2007 and the associated livestock mortality percentages.

Barangay Municipality Province Epidemic

Year

Epidemic

Months

Mortality %

Buffalo Cattle Goat Horse

Parang Cantilan SDS 2005 Dec NR NR NR NR

San Isidro San Francisco ADS 2006 Jan-Mar 17.7 12.8 0.0 0.0

Ebro San Francisco ADS 2006 Jan-Mar NR NR NR NR

Libas Gua San Miguel SDS 2006 Jan-Mar NR NR NR NR

Ladgadan San Francisco ADS 2006 Jul-Sep 5.9 5.4 0.0 40.0

Mamis Barobo SDS 2006 Jun-Aug 17.1 6.0 9.1 0.0

Del Rosario Sibagat ADS 2006 Mar-Apr 14.6 0.0 0.0 35.0

Mapaga Prosperidad ADS 2006 Mar-Apr 8.3 0.0 0.0 35.7

Bayan Marihatag SDS 2006 Oct-Nov 7.9 0.0 100.0 0.0

San Ignacio Trento ADS 2007 Apr-Jun 16.5 0.0 0.0 0.0

Dimasalang San Luis ADS 2007 Dec-Mar 6.0 17.9 0.0 0.0

Concordia Esperanza ADS 2007 Jan-Mar 5.6 8.3 0.0 0.0

Guadalupe Esperanza ADS 2007 Jan-Mar 6.0 0.0 0.0 0.0

Mambalili Bunawan ADS 2007 Jan-Mar 5.1 4.9 0.0 17.4

Banahao Lianga SDS 2007 Mar-May NR NR NR NR

Causwagan Talacogon ADS 2007 Nov-Feb 14.0 0.0 0.0 41.7

NR: Not recorded

SDS: Surigao del Sur

ADS: Agusan del Sur

Table

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Table 2

Primer sequences and modifications, amplicon sizes, and repeat motifs of microsatellite

multiplexes (TEM1 and TEM2) designed on the basis of T. brucei GenBank accession data.

Locus

name

Primer name - sequence 5´-3´

(modifications: PIG tail, fluorescent label)

T. brucei

Amplicon size

bp

T. brucei

Acc. no. T. brucei motif

TEM1

Tbb1 Tbb1F - TTTGAAGAATTTGTACCTCGGTG`3

Tbb1R- gttcttCTTTCAACCGCCTTATCAGC’3 192 AL92960 GT49

Tbb10 Tbb10F- TGTGGCTCCTACTTTCCGTG`3

Tbb10R- gttcttCTCCCTTTTGTCAGTGTGGC`3 262 NW001076899 CA48

TEM2

Tbb5 Tbb5F- CGCAACAACTTTCACATACGC`3

Tbb5R- gttcttTGAGGGTTCTTCGCTTACAC`3 240 AC159443 CA50

Tbb9 Tbb9F- CGTTTGTCGCTGCCTTTGTG`3

Tbb9R- gttcttTGTAGTGCCGATTAGGGTTG`3 239 NW001076895 TG51

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Table 3

Characterisation of the 7 microsatellite loci used in the population analysis of 3 populations (I, II

and III as determined by Structure 2.3.3 analysis) of T. evansi isolates from Mindanao, the

Philippines.

Population Locus Number of

isolates

Number of

Alleles Ho He FIS

I

Tbb1 45 3 1.000 0.511 -0.957

Tbb10 45 3 1.000 0.511 -0.957

Tbb5 45 5 1.000 0.579 -0.726

Tbb9 45 3 1.000 0.540 -0.850

MORFCA 45 2 1.000 0.500 -1.000

MT3033 45 3 1.000 0.566 -0.768

MEST19 45 3 1.000 0.511 -0.957

II

Tbb1 94 8 1.000 0.789 -0.268

Tbb10 94 7 1.000 0.790 -0.267

Tbb5 94 11 1.000 0.871 -0.149

Tbb9 94 4 1.000 0.750 -0.334

MORFCA 94 3 1.000 0.619 -0.615

MT3033 94 9 1.000 0.826 -0.210

MEST19 94 8 1.000 0.788 -0.268

III

Tbb1 1 2 1.000 0.500 -1.000

Tbb10 1 2 1.000 0.500 -1.000

Tbb5 1 2 1.000 0.500 -1.000

Tbb9 1 2 1.000 0.500 -1.000

MORFCA 1 2 1.000 0.500 -1.000

MT3033 1 2 1.000 0.500 -1.000

MEST19 1 2 1.000 0.500 -1.000

Ho: observed heterozygosity, He: expected heterozygosity, FIS: inbreeding coefficient.

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Table 4

Nei’s genetic distance, Wright’s Fixation Index (FST) and gene flow (Nm) for the different

divisions in T. evansi isolates collected in Mindanao, the Philippines and typed with 7

microsatellite loci. Isolates were grouped by province of origin (Agusan del Sur (ADS) or

Surigao del Sur (SDS)) and/or collection year (2006 or 2007) or population structure as

determined by software program Structure 2.3.3 (populations I and II).

Isolates’ population divisions Nei’s genetic distance FST Nm

ADS / SDS 0.122 0.022 11.276

2006 / 2007 0.142 0.021 11.48

ADS-2006 / ADS-2007 0.893 0.118 1.87

SDS-2006 / SDS-2007 0.049 0.011 22.9

ADS-2006 / SDS-2006 1.204 0.154 1.37

ADS-2007 / SDS-2007 0.066 0.012 20.54

ADS-2006 / SDS-2007 0.903 0.124 1.77

ADS-2007 / SDS-2006 0.171 0.032 7.68

I / II (Structure populations) 0.259 0.067 3.476

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Table 5

The 7 microsatellite multi locus genotypes (MLGs) and number of isolates from putative

Trypanosoma evansi outbreaks occurring in 16 barangays in Mindanao, Philippines between late

2005 and 2007.

Barangay Municipality Province Epidemic

Year Months

MLG typed at 7 loci

and number of isolates (n)

Parang Cantilan SDS 2005 Dec MLG I-2

San Isidro San Francisco ADS 2006 Jan-Mar NT

Ebro San Francisco ADS 2006 Jan-Mar MLG K-4

Libas Gua San Miguel ADS 2006 Jan-Mar MLG I-1 & MLG M-1

Ladgadan San Francisco ADS 2006 Jul-Sep MLG E-1

Mamis Barobo SDS 2006 Jun-Aug MLGA-10, MLG M-4 & unique-2

Del Rosario Sibagat ADS 2006 Mar-Apr NT

Mapaga Prosperidad ADS 2006 Mar-Apr MLG J-1 & unique-1

Bayan Marihatag SDS 2006 Oct-Nov MLG E-2

San Ignacio Trento ADS 2007 Apr-Jun MLG G-6, MLG H-5, MLG M-3 &

unique-1

Dimasalang San Luis ADS 2007 Dec-Mar MLG C-3

Concordia Esperanza ADS 2007 Jan-Mar MLG C-12, MLG L-1 & unique-2

Guadalupe Esperanza ADS 2007 Jan-Mar MLG C-20 & MLG L-1

Mambalili Bunawan ADS 2007 Jan-Mar NT

Banahao Lianga SDS 2007 Mar-May MLG C-2

Causwagan Talacogon ADS 2007 Nov-Feb MLG E-2 & MLG L-1

NT: Not typed at all 7 loci

MLG: multi locus genotype

Unique: an isolate with a unique multi locus genotype

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Fig. 1. A map of Mindanao in the Philippines showing the provinces where surra outbreaks have been reported in 1989-2009 (Dargantes, J.R., 1

Mercado, R. and Alforque, J. pers. comm., 2010).2

Figure

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Fig. 2 a and b. The locations of Trypanosoma evansi isolate sampling and surra epidemics (late 3

2005-2007) in Mindanao, Philippines. a. Depicts the Philippines with an inlay box over an area 4

of Mindanao which is enlarged in b. and contains marked T. evansi sampling locations () and 5

epidemic locations (). 6

7

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Fig. 3. Neighbour-joining tree inferred from the Share allele (DAS) genetic distances calculated 8

for the microsatellite data of 140 Trypanosoma evansi isolates from Mindanao, Philippines 9

genotyped at 7 loci. Unique isolates are identified by the individual code (i.e. A7E-912M) and 10

MLGs (groups of identical isolates) by a letter and a number signifying the number of clones of 11

that type (i.e. A21, is a MLG comprising 21 identical isolates). Percentage bootstrap values were 12

generated from 1,000 replicates. Scale bar represents branch lengths. The three populations (I, II 13

and III) inferred by Bayesian model-based analysis with program Structure 2.3.3 are circled and 14

marked accordingly. 15

16

17

18

19

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Fig. 4. Trypanosoma evansi isolates from Mindanao buffaloes classified into three populations I 20

(blue), II (pink) and III (green) based on analysis of population structuring by the software 21

program Structure 2.3.3. The size of the symbol reflects the number of isolates of that type 22

collected from that location. The three provinces from which samples were collected and 23

analysed were Agusan del Sur (ADS) and Surigao del Sur (SDS) and Agusan del Norte (ADN) 24

(no buffalo isolates collected). 25

26

27

28

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Fig. 5. Geographical representation of the occurrences of Trypanosoma evansi MLG A () and 29

MLG C () isolates from buffalo in Mindanao, Philippines in relation to buffalo mortality 30

figures (). The size of the MLG symbols reflects number of isolates and the size of buffalo 31

mortality symbol the buffalo mortality percentage. The three provinces from which samples were 32

collected and analysed were Agusan del Sur (ADS) and Surigao del Sur (SDS) and Agusan del 33

Norte (ADN) (no buffalo isolates collected). Major roads in provinces are marked. Arrows 34

identify the location of MLG isolates which occurred in another province to all the other isolates 35

of the respective MLG. 36

37