DNA Barcoding - Parasites and Vectors
Dan Masiga
Molecular Biology and Biotechnology Department
Eastern Africa Barcode Workshop, Oct 18-19
Insects
Eastern Africa Barcode Workshop, Oct 18-19
Vector-transmitted pathogens
Viruses
Rickettsia
Bacteria
Protozoa
helminths
Eastern Africa Barcode Workshop, Oct 18-19
UNICEF/UNDP/World Bank/WHO - TDR
TDR Disease Focus
1. African trypanosomiasis
2. Chagas disease
3. Leishmaniasis
4. Dengue
5. Lymphatic filariasis
6. Malaria
7. Onchocerciasis
8. Schistosomiasis
9. Tuberculosis
10. Leprosy
http://www.who.int/tdr/grants/strategic-emphases/files/matrix.pdf
Eastern Africa Barcode Workshop, Oct 18-19
Distribution
human-infective
trypanosomes
causing
Easternof Africa
Barcode
Workshop,
Octsleeping
18-19sickness Animal-infective trypanosomes
Are found throughout sub-saharan Africa, with the exception of the Southern tip.
Barcoding tsetse flies and trypanosomes
Well
tsetsefed
fly tsetse
feeding
T. brucei in blood smear
Eastern Africa Barcode Workshop, Oct 18-19
Tsetse flies and trypanosomes
There are about 31 species of tsetse flies, the only
members of genus Glossina, are recognized.
About 10 tsetse-transmitted trypanosome species
and sub-species are known; probably >100 other
species.
Beyond Africa, T. evansi and T. vivax are
transmitted mechanically (without parasite
development in the vector)
Eastern Africa Barcode Workshop, Oct 18-19
Eastern Africa Barcode Workshop, Oct 18-19
Barcoding tsetse and trypanosomes
Loci that have been used
Tsetse:
ITS 1
Trypanosomes: Satellite DNA; small-subunit
ribosomal RNA (ssu rRNA) gene
Eastern Africa Barcode Workshop, Oct 18-19
Identification of trypanosomes based on satellite DNA
Satellite DNA is defined as a portion of DNA in eukaryotes
whose density differs from that of the majority of DNA and
that consists of short, repeating sequences of nucleotide
pairs (typically 5-500 bp long).
Trypanosome (sub)
species
Trypanosoma brucei
T. c ongolense Savannah
T. c ongolense Forest
T. Simiae
T. Vi vax
Satellite monomer size
(bp)
177
369
353
527
174
Eastern Africa Barcode Workshop, Oct 18-19
% GC
29
65
35
55
65
Trypanosome phylogeny (and ID) using ssu rDNA (18S rDNA locus)
Eastern Africa Barcode Workshop, Oct 18-19
Current phylogeny is based on 18S rDNA
phylogeny is based on 18S
(taken from Hamilton et al., 20
Eastern Africa Barcode Workshop, Oct 18-19
Can COI be used for barcoding
trypanosomatids?
Eastern Africa Barcode Workshop, Oct 18-19
Inside a trypanosome
mitochondrion
glycosomes
Eastern Africa Barcode Workshop, Oct 18-19
Kinetoplast DNA (mitochondrial DNA)
20-50 identical maxicircles (20-40 kb)
encode 18 protein encoding (12 nonfunctional) genes, 2 rRNAs and a few
guide (g) RNAs.
5000-12000 heterogeneous
minicircles encode (over 900 different)
small guide RNAs.
Maxi- and minicircles are interlocked
in a giant catenate.
Dyskinetoplastic cells lack minicircles,
maxicircles or both, e.g. T. evansi.
Cannot be transmitted by Tsetse.
Prof. Christine Clayton
EM of the periphery of an isolated kDNA network
Network of
minicircles
condensed
Eastern Africa Barcode Workshop, Oct 18-19
network
Some benefits of using COI
A single locus of accessible size.
Resolve classification of Glossina at sub-generic
level (3 or 4 have been proposed: fusca, palpalis,
austeni & morsitans).
A more accurate indication of relationships and
distances, perhaps providing explaining some key
phenotypes, e.g. transmission efficiencies.
Eastern Africa Barcode Workshop, Oct 18-19
Sandflies, vectors of Leishmaniasis
Lutzomyia spp. (New World )
Phlebotomus spp. (Old World)
Lutzomyia longipalpis lapinha
TDR image Lib ID 00061063
Eastern Africa Barcode Workshop, Oct 18-19
Species of Leishmania
L. major
L. aethiopica
L. infantum
L. donovani
L. tropica
L. mexicana complex
L. guyanensis
L. naiffi
L. braziliensis
L. tarentolae
E. scheideri
Insects
Eastern Africa Barcode Workshop, Oct 18-19
Barcoding and epidemiology/disease
control
Eastern Africa Barcode Workshop, Oct 18-19
Global view of endemic regions for leishmaniasis
With such a wide distribution, you can start
small, but must network to advance.
Eastern Africa Barcode Workshop, Oct 18-19
NHGRI Awards $13M for Next Generation Sequencing
Technologies
[October4,2006]
NEWYORK(GenomeWebNews)TheNationalHumanGenomeResearch
Institutehasawardedelevengrantsworthatotalofmorethan$13millionto
developtechnologiesthatwilllowerthecostofDNAsequencing.
Theagencyhasaneartermgoalofloweringthecostsofsequencinga
mammaliansizedgenometo$100,000,andtoeventuallycutthecostofwhole
genomesequencingto$1,000orless.Loweringthecostwillmakegenome
sequencingpossibleaspartofroutinemedicalcare,NHGRIsaid.
Ninegrantsfundresearchersdevelopingtechnologiesthatwillmakethe$1,000
genomepossibleandtwograntsforresearchersworkingonthe$100,000
mammaliangenome:
Eastern Africa Barcode Workshop, Oct 18-19
