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Bioassay & Susceptibility Test

Bioassay & Susceptibility Test Help
(WHO, Entomology & Vector Control Learner's Guide)
 
Learning Unit 4

Susceptibility and bioassay tests

Learning objectives

By the end of this Unit you should be able to:

         measure the level of insecticide resistance in a vector population

         determine the residual efficacy of an insecticide deposit on a sprayed surface or material at a specified time after the spraying One of the most important reasons to sample vector populations is to determine their susceptibility to insecticides. When insecticides are used in malaria control, it is important that you monitor changes in the susceptibility level of the target vectors from time to time. The residual efficacy of the insecticide used should also be determined at intervals after its application or use. In this Unit, you will learn the skills required to carry out these activities.

4.1 Susceptibility tests

Physiological resistance to insecticides

 

Susceptibility tests are carried out to determine the proportion of the vector population that is physiologically resistant to a particular insecticide.

 

Physiological resistance to insecticides has been defined as the ‘ability of a population of insects to tolerate doses of an insecticide which would prove lethal to the majority of individuals in a normal population of the same species.

The efficacy of indoor residual spraying (IRS) and insecticide impregnated nets (ITNs) depends, among other things, on the proportion of vectors resting on the sprayed surface and on the susceptibility of the vectors to the insecticide used. It is therefore important to monitor the development and extent of insecticide resistance in a given vector population.

 

Equipment

Susceptibility test kit (including exposure/holding tubes, copper and silver rings, insecticide-impregnated filter papers, oil-impregnated control papers, sucking tubes), thermometer, wooden box with large holes, towels, cotton wool, paper cups with cover nets, rubber bands, markers or wax pencils, mosquito cage.

 

 

Method of determining the susceptibility of adult mosquitoes

The standardized WHO method involves checking the mortality of several female Anopheles of a known species exposed in special tubes to filter papers impregnated with a lethal concentration (known as discriminating dose) of a given insecticide dissolved in mineral oil.

                          

 

 

                 

 Collect as many mosquitoes by means of an aspirator (Fig. 4.1a).

         Transfer 15-25 fed mosquitoes to a special plastic holding tube lined with insecticide free paper (Fig. 4.1b).

         Connect a plastic tube lined with filter paper impregnated with mineral oil (used as control) with the holding tube and transfer 15-25 mosquitoes to the tube through a hole in the slide between the 2 tubes (Fig. 4.1c); also transfer the same number of osquitoes to plastic tube with insecticide-impregnated filter paper. (The filter papers are held in place by special rings). Use separate sucking tubes to transfer mosquitoes to the exposure and control tubes to avoid contamination.

         Close the slide and allow the exposure and control tubes to stand upright for the determined period, which is usually one hour (Fig. 4.1d).

         After the exposure period transfer the mosquitoes back to the holding tube, which should stand upright for 24 hours, with a piece of moist cotton wool on the gauze end and in a wooden box with large holes for ventilation and covered with a damp towel (Fig. 4.1e); you should monitor temperature and humidity in the box.

 

         Count dead mosquitoes killed by the contact with the insecticide and those killed in the control tube at the end of this recovery period.

 

         You will need four replicates of the experiment to calculate % mortality in the exposure and control tubes.

 

         The rates of mortality are:

 

o       Control mortality where C = (number of dead mosquitoes)/(total number of  mosquitoes) in control tube 

 Exposure mortality where E = (number of dead mosquitoes)/(total number of mosquitoes) in tube with insecticide

  • If control mortality is greater than or equal to 5% and less than or equal to 20%, the value for exposure mortality E should be corrected by using the following formula (Abbott’s formula):

 

Where, E is the (uncorrected) exposure mortality expressed in % and C is

the control mortality expressed in %.

 

For instance, if control mortality C is 10% and crude exposure mortality E

is 40%, the corrected exposure mortality is [(40 – 10)/100 – 10)] * 100 =

33% . If control mortality is greater than 20%, the experiment should be

discarded.

 

 

4.2 Bioassay tests

The residual efficacy of an insecticide on a sprayed surface is determined by bioassay tests. This is done by checking mortality of the target mosquito vector exposed to the sprayed surface at intervals of weeks or months after the spraying. This technique can be also used to evaluate the quality of a residual spraying operation. It is also used to dermine residual efficacy of an insecticide on bed nets. This can be used to decide when to re-treat bed nets and also to assess the quality of treatment.

 

 

 

 

Equipment

Bioassay kit (including plastic cones, adhesive sponge tape, bent sucking tube, normal sucking tubes), cardboard paper, small nails, hammer, cotton wool, paper cups with cover nets, rubber bands, markers, mosquito cage, wooden box with large holes, towels The plastic cone and the special (bent) sucking tube are shown in Fig. 4.2.

 

 

 

 

Residual efficacy of insecticide on sprayed surfaces

  • Line the edges of the plastic cone with adhesive sponge tape
  • Using nails or tape, fix the cone to the sprayed surface. (Fix different cones at three heights: low, middle and high) 
  • Nail insecticide-free cardboard paper to the wall and fix plastic cones to the paper (to be used as a control)
  • Transfer 10 mosquitoes (preferably susceptible strains from an insectary) into each cone and put a piece of cotton wool in the opening of the cone (use separate sucking tube to transfer mosquitoes to control cones)
  • After a specified exposure period (usually 30 minutes), carefully take out the mosquitoes from the cones and transfer them to separate (and labeled) paper cups
  • Count the number of mosquitoes dead (or ‘knocked down’) at the end of the exposure period, but do not remove mosquitoes that appear dead, as some of them might later recover
  • Place damp cotton wool on top of the paper cups, put them in the wooden box and cover with damp towel
  • After 24 hours, count the number of mosquitoes dead and calculate percent mortality in both the exposure and control paper cups
  • Repeat the experiment on different walls (in the same house) and in different houses to have a representative sample. In each experiment, use the same number of mosquitoes for exposure and control
  • If control mortality is between 5% and 20%, the exposure mortality should be corrected by using Abbott’s formula described under susceptibility tests. If control mortality is greater than 20%, the experiment should be discarded.

 

 

 

Residual efficacy of insecticide on bed nets

The bioassay procedure for insecticide-treated bed nets is similar to the procedure used for sprayed walls, except that the cone is attached to the fabric with a rubber band, and the mosquitoes are usually exposed only for three minutes.

 

Exercise 4.1

Susceptibility tests: You will be provided with live, fed female mosquitoes in the laboratory. In pairs, prepare holding tubes and introduce 15 fed females to the holding tubes. When all groups have introduced the mosquitoes, half the groups will prepare exposure tubes and the other half control tubes. After all tubes have been prepared, all groups will be asked to transfer the mosquitoes to the exposure and control tubes and to label each tube with the group number indicating whether it is an exposure or a control tube, and the time of the day. After one hour of exposure, you should transfer the mosquitoes back to the holding tubes for 24 hours of observation. After 24 hours, all results will be combined to calculate mortality rates.

 

Exercise 4.2

Bioassay tests: Again, as in Exercise 4.1, you will form pairs and half the groups will install cones on treated bed nets and half on untreated bed nets. Each pair should transfer 10 fed mosquitoes to the cones, and after three minutes the mosquitoes should be taken out and placed in paper cups for 24 hours of observation. Again, the results will be combined for calculation of mortality rates.

 

(Please visit the WHO website for full & detailed document) to complete your referene.

 

 

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