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					            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
    Eastern human-infective trypanosomes causing sleeping sickness Animal-infective trypanosomes
Distribution of Africa Barcode Workshop, Oct 18-19
Are found throughout sub-saharan Africa, with the exception of the Southern tip.
            Barcoding tsetse flies and trypanosomes




                                             Well fed tsetse
                                             tsetse fly 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)         Satellite monomer size   % GC
           species                   (bp)
           Trypanosoma brucei        177                      29
           T. congolense Savannah    369                      65
           T. congolense Forest      353                      35
           T. Simiae                 527                      55
           T. Vivax                  174                      65




Eastern Africa Barcode Workshop, Oct 18-19
         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 non-
     functional) 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.                         EM of the periphery of an isolated kDNA network

•    Dyskinetoplastic cells lack minicircles,
     maxicircles or both, e.g. T. evansi.
     Cannot be transmitted by Tsetse.
                                                  Network of
                                                  minicircles



Prof. Christine Clayton                            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
                                             Species of Leishmania
                                             L. major
                                             L. aethiopica
                                             L. infantum
                                             L. donovani
                                             L. tropica
                                             L. mexicana complex
                                             L. guyanensis
                                             L. naiffi
                                             L. braziliensis
Eastern Africa Barcode Workshop, Oct 18-19   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
[October 4, 2006]

NEW YORK (GenomeWeb News) – The National Human Genome
Research Institute has awarded eleven grants worth a total of more than
$13 million to develop technologies that will lower the cost of DNA
sequencing.

The agency has a near-term goal of lowering the costs of sequencing a
mammalian-sized genome to $100,000, and to eventually cut the cost of
whole-genome sequencing to $1,000 or less. Lowering the cost will
make genome sequencing possible as part of routine medical care,
NHGRI said.

Nine grants fund researchers developing technologies that will make the
$1,000 genome possible and two grants for researchers working on the
Eastern Africa Barcode genome:
$100,000 mammalian Workshop, Oct 18-19

				
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