Prevalence of Prevalence of Toxoplasma gondiiToxoplasma gondii in two populations in two populations of British Pipistrelle bats (of British Pipistrelle bats (Pipistrellus pipistrellus Pipistrellus pipistrellus and and P. pygmaeusP. pygmaeus))Dodd, N. S.,Dodd, N. S., Lord, J., Brooks, D.R., Jehle, R. and Hide, G. Lord, J., Brooks, D.R., Jehle, R. and Hide, G.University of SalfordUniversity of Salford

OverviewOverview

Introduction and backgroundIntroduction and background

MethodologyMethodology

ResultsResults

ConclusionsConclusions

Overall thesisOverall thesis

Introduction and BackgroundIntroduction and Background

Investigated Investigated ToxoplasmaToxoplasma in 2 wildlife in 2 wildlife species (species (Apodemus sylvaticusApodemus sylvaticus and and pipistrelle bats)pipistrelle bats)

Pipistrelle manuscript to be submitted Pipistrelle manuscript to be submitted

Generated new data in British batsGenerated new data in British bats

Investigated population genetics of bat hostInvestigated population genetics of bat host

Interrelation of pathogen and hostInterrelation of pathogen and host

Introduction and BackgroundIntroduction and Background ToxoplasmaToxoplasma is a protozoan parasite is a protozoan parasite Complex lifecycleComplex lifecycle Phylum Apicomplexa (malaria)Phylum Apicomplexa (malaria) Wide-rangingWide-ranging Can potentially infect all warm blooded animalsCan potentially infect all warm blooded animals To what extent does it infect bats?To what extent does it infect bats? Characteristically found inCharacteristically found in

high prevalenceshigh prevalences Parasite of the cat Parasite of the cat

(any Felidae)(any Felidae)

HumanToxoplasmosisHumanToxoplasmosis

Causes abortion/miscarriage Causes abortion/miscarriage ~2,000 UK women per year contract toxoplasmosis ~2,000 UK women per year contract toxoplasmosis

during pregnancyduring pregnancy Illness is inversely proportional to gestational ageIllness is inversely proportional to gestational age Ocular diseaseOcular disease HydrocephalusHydrocephalus 1/3 humans chronically infected 1/3 humans chronically infected Morbidity and mortality in AIDs Morbidity and mortality in AIDs

patients patients

EpidemiologyEpidemiology Cats are a critical part of the life cycleCats are a critical part of the life cycle The cat is only host in which the oocyst – The cat is only host in which the oocyst –

producing sexual stage of producing sexual stage of ToxoplasmaToxoplasma can can developdevelop

3 routes of transmission3 routes of transmissioni.i. Defecate infective oocysts into the Defecate infective oocysts into the

environment environment

ii.ii. Ingestion of bradyzoites in tissue cysts Ingestion of bradyzoites in tissue cysts

iii.iii. Primary problem is a congenital infection of Primary problem is a congenital infection of foetus via tachyzoites crossing the placentafoetus via tachyzoites crossing the placenta

The relative contribution of infection routes into The relative contribution of infection routes into epidemiology is contentious epidemiology is contentious

RationaleRationale.... The cat is the The cat is the onlyonly definitive host definitive host Parasite is ubiquitous and highly prevalent Parasite is ubiquitous and highly prevalent Not known to what extent it infect batsNot known to what extent it infect bats British bats are insectivorous, should not becomeBritish bats are insectivorous, should not become

infected by carnivory infected by carnivory Rarely come in contactRarely come in contact

with the ground or catswith the ground or cats British bats are protectedBritish bats are protected

and difficult to studyand difficult to study

Bats collected by the South Lancashire Bat Bats collected by the South Lancashire Bat

GroupGroup

Largely pipistrelle bats (1 Largely pipistrelle bats (1 Myotis myotisMyotis myotis))

Sick/euthanased animalsSick/euthanased animals

Dead Dead

Heads sent for rabies testing by the Heads sent for rabies testing by the Veterinary

Laboratories Agency (VLA, Weybridge)

MethodsMethods

MethodsMethods

DNA was extracted using a DNA was extracted using a standard phenol/chloroform standard phenol/chloroform techniquetechnique Electrophoresis was used to Electrophoresis was used to visualise DNAvisualise DNA

T. gondii T. gondii detected by nested detected by nested SAG1 and SAG3 PCRSAG1 and SAG3 PCR Mammalian tubulin PCR to Mammalian tubulin PCR to check amplification capabilitycheck amplification capability

MethodsMethods

Positively amplified SAG1 reactions Positively amplified SAG1 reactions were sequencedwere sequenced

Compared with published Compared with published T. gondii T. gondii SAG1 sequencesSAG1 sequences

MethodsMethods

Restriction Fragment Length Restriction Fragment Length Polymorphism (RFLP) was Polymorphism (RFLP) was used to strain-type used to strain-type Toxoplasma Toxoplasma

11 published microsatellite 11 published microsatellite markers specific to pipistrellus markers specific to pipistrellus were optimisedwere optimised

Bats were genotyped on a Bats were genotyped on a ABI3130 genetic analyser ABI3130 genetic analyser

2000

129.63bp

140.60bp

110 120 130 140 150 160 170 180

1600

1200

800

400

0

Pea

k In

tens

ity

(RF

U)

Base Pairs

Heterozygous genotype 129.63 bp–140.60 bp for the microsatellite P13 locus by fragment analysis. The green peaks correspond to the HEX-labelled amplified fragments.

Allele binning Allele binning performed on performed on TandemTandem

Scoring errors and null Scoring errors and null alleles checked in alleles checked in MicrocheckerMicrochecker

Allele ranges and HW Allele ranges and HW deviations were deviations were analysed using analysed using Genepop Genepop

Population structure Population structure determined using determined using StructureStructure

MethodsMethods

Size of peaks determined by Size of peaks determined by Peak ScannerPeak Scanner

ResultsResults SAG1 gene amplified to compare with other published SAG1 gene amplified to compare with other published

sequences in the NCBI (National Center for sequences in the NCBI (National Center for Biotechnology)Biotechnology)

CLUSTAL W CLUSTAL W sequence sequence alignment. alignment.

GenBank = GenBank = Toxoplasma gondiiToxoplasma gondii strain RMS-2000-strain RMS-2000-ROU major surface ROU major surface antigen (SAG1) antigen (SAG1) gene Accession gene Accession number: number: GQ253086.1GQ253086.1

ResultsResults Genotyped using 9 polymorphic microsatellite lociGenotyped using 9 polymorphic microsatellite loci Analysis carried out in Analysis carried out in StructureStructure What is the population structure of the collection of What is the population structure of the collection of

bats?bats? Is there any relationship between the bats and Is there any relationship between the bats and

ToxoplasmaToxoplasma infection? infection?

ResultsResults Prevalence of 9.9% (Prevalence of 9.9% (±±7.0%; 95%CI) for 7.0%; 95%CI) for P. pipistrellusP. pipistrellus

Prevalence of 16.67% (Prevalence of 16.67% (±±29.8; 95%CI) for 29.8; 95%CI) for P. pygmaeusP. pygmaeus

One One Myotis myotis Myotis myotis was negativewas negative

Majority of bats were highly related Majority of bats were highly related ((n n = 59; 83.1%) = 59; 83.1%)

Remaining group (Remaining group (n = n = 12) had mixed genetic origins12) had mixed genetic origins

No significant difference in the frequency of No significant difference in the frequency of T.gondiiT.gondii infection infection

or geographical distribution between the 2 groupsor geographical distribution between the 2 groups

First report of First report of ToxoplasmaToxoplasma in British bats in British bats

High levels detected using highly sensitive nested PCRHigh levels detected using highly sensitive nested PCR

Other population genetic studies on pipistrelle bats are Other population genetic studies on pipistrelle bats are

scarcescarce

None have used mutilocus genotyping in relation to None have used mutilocus genotyping in relation to

ToxoplasmaToxoplasma infection infection

One large breeding population in the Lancashire areaOne large breeding population in the Lancashire area

Raises questions of transmission route, possible Raises questions of transmission route, possible

congenital congenital

ConclusionConclusion

Thesis title: The Use of PCR to Investigate Thesis title: The Use of PCR to Investigate the Prevalence of the Prevalence of Toxoplasma gondiiToxoplasma gondii in in Wildlife SpeciesWildlife Species

Found high levels of Found high levels of T. gondii T. gondii in mice in an in mice in an area free from catsarea free from cats

Implies the cat less important in the Implies the cat less important in the epidemiology of epidemiology of T. gondii T. gondii that previously that previously thoughtthought

Work in insectivorous bats eliminates Work in insectivorous bats eliminates carnivory as source of infection carnivory as source of infection

This work could imply that congenital This work could imply that congenital transmission could play a significant role transmission could play a significant role