1. Prevalence ofPrevalence of Toxoplasma gondiiToxoplasma gondii in two populationsin two populations
of British Pipistrelle bats (of British Pipistrelle bats (Pipistrellus pipistrellusPipistrellus pipistrellus
andand 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

2. OverviewOverview
 Introduction and backgroundIntroduction and background
 MethodologyMethodology
 ResultsResults
 ConclusionsConclusions
 Overall thesisOverall thesis

3. Introduction and BackgroundIntroduction and Background
 InvestigatedInvestigated ToxoplasmaToxoplasma in 2 wildlifein 2 wildlife
species (species (Apodemus sylvaticusApodemus sylvaticus andand
pipistrelle bats)pipistrelle bats)
 Pipistrelle manuscript to be submittedPipistrelle 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

4. Introduction and BackgroundIntroduction and Background
 ToxoplasmaToxoplasma is a protozoan parasiteis 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 catParasite of the cat
(any Felidae)(any Felidae)

5. HumanToxoplasmosisHumanToxoplasmosis
 Causes abortion/miscarriageCauses 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 infected1/3 humans chronically infected
 Morbidity and mortality in AIDsMorbidity and mortality in AIDs
patientspatients

6. 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 ofproducing sexual stage of ToxoplasmaToxoplasma cancan
developdevelop
 3 routes of transmission3 routes of transmission
i.i. Defecate infective oocysts into theDefecate infective oocysts into the
environmentenvironment
ii.ii. Ingestion of bradyzoites in tissue cystsIngestion of bradyzoites in tissue cysts
iii.iii. Primary problem is a congenital infection ofPrimary problem is a congenital infection of
foetus via tachyzoites crossing the placentafoetus via tachyzoites crossing the placenta
 The relative contribution of infection routes intoThe relative contribution of infection routes into
epidemiology is contentiousepidemiology is contentious

7. RationaleRationale....
 The cat is theThe cat is the onlyonly definitive hostdefinitive host
 Parasite is ubiquitous and highly prevalentParasite 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 carnivoryinfected 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

8.  Bats collected by the South Lancashire BatBats collected by the South Lancashire Bat
GroupGroup
 Largely pipistrelle bats (1Largely pipistrelle bats (1 Myotis myotisMyotis myotis))
 Sick/euthanased animalsSick/euthanased animals
 DeadDead
 Heads sent for rabies testing by theHeads sent for rabies testing by the Veterinary
Laboratories Agency (VLA, Weybridge)
MethodsMethods

9. MethodsMethods
 DNA was extracted using aDNA was extracted using a
standard phenol/chloroformstandard phenol/chloroform
techniquetechnique
 Electrophoresis was used toElectrophoresis was used to
visualise DNAvisualise DNA
 T. gondiiT. gondii detected by nesteddetected by nested
SAG1 and SAG3 PCRSAG1 and SAG3 PCR
 Mammalian tubulin PCR toMammalian tubulin PCR to
check amplification capabilitycheck amplification capability

10. MethodsMethods
 Positively amplified SAG1 reactionsPositively amplified SAG1 reactions
were sequencedwere sequenced
 Compared with publishedCompared with published T. gondiiT. gondii
SAG1 sequencesSAG1 sequences

11. MethodsMethods
 Restriction Fragment LengthRestriction Fragment Length
Polymorphism (RFLP) wasPolymorphism (RFLP) was
used to strain-typeused to strain-type
ToxoplasmaToxoplasma
 11 published microsatellite11 published microsatellite
markers specific tomarkers specific to
pipistrellus were optimisedpipistrellus were optimised
 Bats were genotyped on aBats were genotyped on a
ABI3130 genetic analyserABI3130 genetic analyser

12. 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 binningAllele binning
performed onperformed on TandemTandem
 Scoring errors andScoring errors and
null alleles checked innull alleles checked in
MicrocheckerMicrochecker
 Allele ranges and HWAllele ranges and HW
deviations weredeviations were
analysed usinganalysed using
GenepopGenepop
 Population structurePopulation structure
determined usingdetermined using
StructureStructure
MethodsMethods
 Size of peaks determined bySize of peaks determined by Peak ScannerPeak Scanner

13. ResultsResults
 SAG1 gene amplified to compare with other publishedSAG1 gene amplified to compare with other published
sequences in the NCBI (National Center forsequences in the NCBI (National Center for
Biotechnology)Biotechnology)
 CLUSTAL WCLUSTAL W
sequencesequence
alignment.alignment.
 GenBank =GenBank =
Toxoplasma gondiiToxoplasma gondii
strain RMS-2000-strain RMS-2000-
ROU major surfaceROU major surface
antigen (SAG1)antigen (SAG1)
gene Accessiongene Accession
number:number:
GQ253086.1GQ253086.1

14. ResultsResults
 Genotyped using 9 polymorphic microsatellite lociGenotyped using 9 polymorphic microsatellite loci
 Analysis carried out inAnalysis carried out in StructureStructure
 What is the population structure of the collection ofWhat is the population structure of the collection of
bats?bats?
 Is there any relationship between the bats andIs there any relationship between the bats and
ToxoplasmaToxoplasma infection?infection?

15. ResultsResults
 Prevalence of 9.9% (Prevalence of 9.9% (±±7.0%; 95%CI) for7.0%; 95%CI) for P. pipistrellusP. pipistrellus
 Prevalence of 16.67% (Prevalence of 16.67% (±±29.8; 95%CI) for29.8; 95%CI) for P. pygmaeusP. pygmaeus
 OneOne Myotis myotisMyotis myotis was negativewas negative
 Majority of bats were highly relatedMajority of bats were highly related ((nn = 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 ofNo significant difference in the frequency of T.gondiiT.gondii
infection or geographical distribution between the 2infection or geographical distribution between the 2
groupsgroups

16.  First report ofFirst report of ToxoplasmaToxoplasma in British batsin British bats
 High levels detected using highly sensitive nested PCRHigh levels detected using highly sensitive nested PCR
 Other population genetic studies on pipistrelle bats areOther population genetic studies on pipistrelle bats are
scarcescarce
 None have used mutilocus genotyping in relation toNone have used mutilocus genotyping in relation to
ToxoplasmaToxoplasma infectioninfection
 One large breeding population in the Lancashire areaOne large breeding population in the Lancashire area
 Raises questions of transmission route, possibleRaises questions of transmission route, possible
congenitalcongenital
ConclusionConclusion

17. Thesis title: The Use of PCR to InvestigateThesis title: The Use of PCR to Investigate
the Prevalence ofthe Prevalence of Toxoplasma gondiiToxoplasma gondii inin
Wildlife SpeciesWildlife Species
 Found high levels ofFound high levels of T. gondiiT. gondii in mice in anin mice in an
area free from catsarea free from cats
 Implies the cat less important in theImplies the cat less important in the
epidemiology ofepidemiology of T. gondiiT. gondii that previouslythat previously
thoughtthought
 Work in insectivorous bats eliminatesWork in insectivorous bats eliminates
carnivory as source of infectioncarnivory as source of infection
 This work could imply that congenitalThis work could imply that congenital
transmission could play a significant roletransmission could play a significant role