Trypanosome Infections in Wild Tsetse Flies in Three Ecological Zones in Ghana

by PCR Assay

BYTheophilus Combey

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Outline of Presentation• Introduction• Statement of the problem• Justification• Main and specific objectives• Methodology• Results and discussion• Conclusion• Recommendation

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INTRODUCTION• Trypanosomes are extracellular flagellated protozoans

• Human African sleeping sickness(HAT)

• American sleeping sickness (chagas disease).

• Nagana and sura.

Plate 1. Trypanosoma forms in a blood smear

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INTRODUCTION CONT’D

• Human African Trypanosomiasis (HAT) is caused by Trypanosoma brucei rhodesiense or T. brucei gambiense,

• African Animal Trypanosomiasis (AAT) is caused mainly by T. vivax, T. congolense, T. simiae, T. evansi and T. brucei brucei.

• Tsetse flies

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• It is estimated currently 300,000 – 500,000 cases of (HAT)

– 60 million people at risk in 37 countries (WHO, 2008)

– loss of local livestock from nagana amounts to 4.5 billion U.S. dollars annually (Kioy et al., 2004).

Plate 3. Late symtoms of Trypanosomiasis in Human and Cattle 5

Fig 2; Life Cycle of Trypanosome6

Problem statement• Inaccurate identification of morphologically similar

species or strains and varied adaptations of within Glossina spp.

• Specificity are low for Manual identification of trypanosome

• Available data are derived mainly from Central and Eastern Africa.

• In Ghana and some West African countries, species composition is unknown while scanty information exists about the trypanosomiasis status in Ghana especially regarding molecular epidemiology of the pathogen

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Justification • It is envisaged that data generated in this study will contribute to the existing knowledge and enhance understand the epidemiology of trypanosomes in Ghana.

• Provide current data on the various trypanosomes species present in wild populations of tsetse flies in Ghana which is of great concern in the public health campaign in Ghana

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Main Objective• The primary objective of the study is to assess the distribution of trypanosomes and to characterize them in populations of tsetse flies in Ghana

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Specific Objectives• To screen different species of tsetse flies in Ghana and determine level of infectivity with trypanosomes from the different localities.

• To identify different strains of trypanosomes in the wild populations of tsetse flies.

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Methodology

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Collection sites

A B

C

D

Plate 3 photos showing collection of tsetse flies from the field

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A B

C

Plate 4 Species of tsetse flies found in Ghana A) G. morsitans,B) G. palpalis, & C) G. tachinoides

Plate 5. Abdominal differences between male and female tsetse flies. A =female abdominal tip B = male abdominal tip

Hypopegyium

A B

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Molecular analysis• Cytltrimethylammonium bromide (CTAB) extraction protocol

• PCR amplification was carried out using methods described by (Simo et al., 2012)

• Species identification of positive trypanosome

• Sequencing of positive PCR products

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Statistical Analysis• Statistical analysis were performed using Minitab, version 15.1.20.0. student t-test and Fishers exact test

• Sequence results were saved and imported into Mega 5 software

• Subjected to nucleotide Blast analysis to find the most similar sequences on the NCBI database

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RESULTS and DISCUSSION

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Overall samples collected

Feeding pattern

Increase in fecundity rate

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Samples collected among ecological zones

Predominant Glossina spp

Availability of infected hosts

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Overall Infection Level

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Infection among sexes in the various ecological zones

Low infections wild populations

Interventions

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Trypanosomes identified by species specific primers

Three out of the 5 specific primers detected trypanosomes

Availability of species specific primer

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Sequence results

•A total of seven different sequences (903, 763, 324,626, 968, 761, 763 bp in size)

• Compared with sequences available on the NCBI database using nucleotide BLAST analysis at NCBI website.

Score Expect Identities G aps Strand

1009 bits(546) 0.0 546/546(100% ) 0/546(0% ) Plus/Plus

Query 1 CTTCAATAGAGGAAGCAAAAGTCGTAACAAGGTAGCTGTAGGTGAACCTGCAGCTGGATC 60 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 1 CTTCAATAGAGGAAGCAAAAGTCGTAACAAGGTAGCTGTAGGTGAACCTGCAGCTGGATC 60 Query 61 ATTTTCCGATGATaaaaaaaGTATACATACATAtgtgtacgtgtagtgtaggtgtgtgct 120 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 61 ATTTTCCGATGATAAAAAAAGTATACATACATATGTGTACGTGTAGTGTAGGTGTGTGCT 120 Query 121 atcgaaggttgttgttgtgtgctcgtgtgcctgtgtgcCCCTCGCTCATGCGCATCCCCA 180 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 121 ATCGAAGGTTGTTGTTGTGTGCTCGTGTGCCTGTGTGCCCCTCGCTCATGCGCATCCCCA 180 Query 181 TCCCGCACGCCCCAGTGTTTTGTGTGCTGTGCGATGCGGCGGTGTGTGTTGGGATCGCGC 240 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 181 TCCCGCACGCCCCAGTGTTTTGTGTGCTGTGCGATGCGGCGGTGTGTGTTGGGATCGCGC 240 Query 241 ATTGTCGGGCGCTGTGATGTGCCGGCCACGAACCTTGAAAACCTTGAAGCACGTCTCGCG 300 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 241 ATTGTCGGGCGCTGTGATGTGCCGGCCACGAACCTTGAAAACCTTGAAGCACGTCTCGCG 300 Query 301 AAACACACGTGTCCAAGCACGCGTCTCCATGTCGCTGTCTCTCTCTTGTGTTGCGAAGAT 360 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 301 AAACACACGTGTCCAAGCACGCGTCTCCATGTCGCTGTCTCTCTCTTGTGTTGCGAAGAT 360 Query 361 GCTTACTGTCGTGTTTGCTCCGCCAGGCGGCGCCGCGTGACGTATGTGCTCCGCAAAGAC 420 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 361 GCTTACTGTCGTGTTTGCTCCGCCAGGCGGCGCCGCGTGACGTATGTGCTCCGCAAAGAC 420 Query 421 AAGAAGGGTGTGGAGGAGACGGCGTGTTCTTATGCCGCCTGACGCTTTTTGTGTGCGCAC 480 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 421 AAGAAGGGTGTGGAGGAGACGGCGTGTTCTTATGCCGCCTGACGCTTTTTGTGTGCGCAC 480 Query 481 CGGCTCGGGTCActttttcctccctctcttcttttccccctcttcttttccccgccttcc 540 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| Sbjct 481 CGGCTCGGGTCACTTTTTCCTCCCTCTCTTCTTTTCCCCCTCTTCTTTTCCCCGCCTTCC 540 Query 541 cACGTG 546 |||||| Sbjct 541 CACGTG 546

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Sequence result con’td• High similarity match (93%) was observed between the 968bp sequence product with the and T. congolense Killifi type from East Africa

• A high similarity match (98% and 94%) between the 903bp sequence product and two members (T. congolense and Crithidia fasciculata)

• The 763bp had a high similarity match (98% and 94%) for the same two Trypanosomatidae species.

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Sequence results con’td• Trypanosoma congolense riverine-forest type from the GeneBank nucleotide database matched (100%) with the 761bp sequence product

• Two other members of family trypanosomatidae (namely T. congolense riverine/ forest isolate and Crithidia bombi clone) highly matched the 626bp (90%) and 324 bp sequence (98%)

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CONCLUSION • The predominant Glossina species was

Glossina palpalis palpalis.• The Guinea savanna ecological zone had the highest diversity of trypanosome species

• Three trypanosome species were identified with a single mixed infection.

• Trypanosome strain from E. Africa first time matched to species in Ghana

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RECOMMENDATIONS• Studies should be carried out in other ecological zones to ascertain the species of trypanosomes present in these areas

• Future works on trypanosomes should include more species- specific primers in order to identify more different species from tsetse flies

• Novel approaches to curtail these protozoan parasites in the African continent should be developed 28

References • Simarro, P. P., Jannin, J. & Cattand, P. (2008).

Eliminating human African trypanosomiasis: where do we stand and what comes next? PLoS Med, 5: 55 59.

• Kioy, D., Jannin, J. & Mattock, N. (2004). Human African Trypanosomiasis. Nat Rev Microbiol, 2(3): 186-187.

• Mahama, C. I., Mohammed, H. A., Abavana, M., Sidibé, I., Koné, A., & Geerts, S. (2003). Tsetse and Trypanosomoses in Ghana in the Twentieth Century: Revue Élev. Méd. Vét. Pays trop., 56 (1-2): 27-32

• Morlais, I., Grébaut P., Bodo, J. M., Djoha, S., Cuny, G. & Herder, S. (1998). Detection and identification of trypanosomes by polymerase chain reaction in wild tsetse flies in Cameroon. Acta Trop., 70: 109–117

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Acknowledgement • Prof. Mrs Mary Botchey• Dr. Alexander Egyir Yawson• Dr. Johnson Nyarko Boampong• Dr. Rofela Combey• Rev. Kow Wie-Addo (CCE, UCC) • Staff of GAEC BINARI• Mr and Mrs Adinortey

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THANK YOU

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