Development of efficient tools for improved surveillance of vectors of Dengue and

Chikungunya fever

By Eunice A Owino

SupervisorsProf.Christian Pirk (UP) Dr. Catherine Sole (UP)

Dr. Rosemary Sang (ICIPE)Dr. Baldwyn Torto (ICIPE)

Introduction

Arboviruses have become the most important causes of re-emergent epidemic diseases.

Dengue fever causes the highest human morbidity and

mortality worldwide.

In East Africa, unprecedented outbreaks of dengue reported in countries around the coast of the Indian Ocean including in; Kenya, The Seychelles and The Comoros islands.

Outbreaks of Chikungunya have also been reported

Introduction

The main vector for Dengue virus (DENV) and Chikungunya virus (CHKIV) in Kenya is Aedes aegypti in the stegomyia subgenus.

Unlike most mosquitoes Aedes aegypti is a diurnal mosquito.

Problem statement

Climate change is identified as a major cause of re-emergence.

Global warming poses a likelihood of more outbreaks.

Inter-epidemic surveillance is therefore important for preparedness.

More research also important.

However, currently surveillance tool for these vectors in outbreak areas is CO2-baited Centers for Disease Control (CDC) light trap.

Problem statement

Short comings of the CO2

baited CDC light trap

• Non specific

• Targets nocturnal mosquitoes.

• Aedes spp under represented in captures

Problem statement

Short comings of Man landing

Catches

• Ethically unacceptable

• Differences in attraction & efficiency

• Labour intensive.

• Low productivity.

Problem statement

Efficient monitoring methods urgently needed.

Prevent mortality and morbidity.

Justification

Ample evidence; blood seeking mosquitoes locate hosts via odours from the host.

Visual cues e.g light have also been used as effective lures to trap mosquitoes.

It would therefore provide a solution if an effective trap would be developed from the two cues – visual and odour cues.

Developed trap would lead to timely preparedness for outbreaks of;

DENV CHIKV and Yellow fever virus also spread by Aedes spp

Help in research.

Hypotheses

Vectors of Dengue and Chikungunya viruses can effectively be monitored with CDC Light Emitting Diode (LED) traps.

There is a variation in vector response to odour profiles from different humans and to different body regions.

Certain vector species can be monitored effectively with odour baits combined with LED traps.

The Aedes aegypti mosquitoes captured from Kilifi and Busia districts are infected with Dengue and Chikungunya viruses and different genetically.

Main Objective

To develop efficient monitoring tools for Dengue and Chikungunya vectors using visual and odour cues.

Specific objectives

1. To evaluate different coloured LED-traps to attract Aedes spp.

2. To evaluate the attractiveness of different human body odours, isolate and identify electrophysiologically active compounds from the volatiles.

3. To conduct behavioural studies using the identified compounds in laboratory and field assays for their effect on mosquito response.

4. To isolate Dengue and Chikungunya viruses from the Aedes aegypti mosquitoes captured from Kilifi and Busia and to evaluate the degree of convergence or divergence of these mosquito populations.

Materials and Methods

Busia district Kenya Kilifi district Kenya

Non specific

Materials and methods

1

•Compare various LEDs against the Miniature CDC light trap.

•Latin square design to be used.

Materials and methods

• Odours to be trapped in Nylon socks, T- shirts, Nylon stockings and Nylon strips.

• To be tested in combination with the best performing LED.

• Highly attractive odour samples to be extracted with dichloromethane

2.

Materials and methods

2

Authentic standard compounds

Materials and methods

• Blends and concentration of different authentic compounds to be tested in randomized field tests with the best performing LEDs and any other conventional traps.

Virus Isolation

Homogenization/ collection of

supernatant/incubation

Addition maintenance medium/incubate/obse

rve for Cyto Pathic Effect (for 2 wks)

Harvest and Store/Passage/Observe CPE

Harvest/Isolate viral RNA and Identify DENV and

CHIKV by RT-PCR using primers specific for DENV and CHIKV

Testing of mosquito in pools: Samples will be Sorted according to species into pools and each pool subjected to the process below

Population genetics

Amplification of the 648 bp fragment of COI using primer

pairs LCO1490 and HCO2198.

Purification of amplicons ,sequencin

g on an ABI sequence analyzer .

Pairwise nucleotide sequence

divergences calculated using the Kimura 2-Parameter

(K2P) model (Kimura, 1980).

Genetic population structure determined

using ≤13 microsatellite loci

xterised in Ae. aegypti (Slotman et

al. 2007)

Microsatellites amplified in a

multiplex PCR and amplified products on

ABI sequencer

Microsatellite markers genotyped

using the GeneMapper

software 3.7 (Applied Biosystems).

FST; AMOVA (ARLEQUIN, 3.1); Mantel

regression (XLSTAT, 2007); RS, HO and HE, FIS and deviation HWE

estimated for each locus in each population (ARLEQUIN, 3.1) .

Presence of linkage disequilibrium (LD)

between all possible pairs of loci in each population tested (ARLEQUIN, 3.1).

To measure divergence or convergence of Aedes aegypti in the two sites Kilifi and Busia

Data Analysis

Objective 1

Counts subjected to Generalised Linear Models (GLM) with log link and Poisson error and the odds that vectors choose a treatment against the control will be estimated at P=0.05

Objective 2Mosquito captures will be subjected to the same analysis as in objective 1.

Data Analysis

Objective 3

Olfactometer bioassays; Mosquito proportions will be subjected to GLMs with a binomial distribution & analysis of deviance at P=0.05.

Field bioassays; Mosquito captures from traps baited by the various blends and concentrations of the different identified chemicals will be subjected to Generalised Linear Models (GLM) with log link and Poisson error and the odds that vectors choose a treatment against the control (Non baited best perfoming LED trap) will be estimated at P=0.05

Data Analysis

Objective 4.Transmission studies The pooled infection rate (IR) program will be used to estimate infection rates at 95% Confidence Intervals (CI) for species from which Chikungunya and Dengue virus will be identified.

Population genetics studiesDifferentiation among populations of Ae. aegypti will be determined by F-statistics (FST) using BIOSYS-1 (Swofford and Selander 1989). The statistical significance of the FST values will be determined using the GENEPOP and Arlequin (Rousset 1997, Excoffier et al. 2005).

Budget

Items Cost in USD Cost in KSh

Chemicals/materials 24,179.00 2006857.00

Stationery 700.00 58100.00

Field Expenses 15,000.00 1245000.00

Literature search 650.00 53950.00

Conferences/meetings/training 4,000.00 332000.00

Communication 500.00 41500.00

Total 45,029.00 3737407.00

Miscellaneous (10% of Total) 4,502.90 373740.70

Grand Total 49,531..90 4,111,147.70

Time plan

ACTIVITIES 

TIME

2011

2012

2013

2014

Research Proposal write-up, Presentation, University registration, field testing of LED or any other traps & raising of insect colony                              

Volatile collection, isolation, characterization, candidate identification & conduction of laboratory bioassays .                                

Field mosquito survey, molecular studies & field evaluation of candidate compounds                                

Manuscripts preparations, thesis write-up, submission & defense                                

THANK YOU