Micromotility Meter

2.6.1. Parasites and incubation conditions

Adult T. colubriformis and T. circumcincta of resistant (T.c. MOX-R and O.c. WAM) and susceptible (T.c. and O.c. McMaster) isolates were obtained from donor sheep. For detailed information collection of the worms see chapter 2.3.4.

The adult T. circumcincta were manually picked with a small sized forceps and placed in groups of 7-8 into the wells of a 24-well plate (Falcon Tissue Plates). The adult T. colubriformis were suspended in RPMI medium (appendix), aspirated with a 200 µl pipette and transferred in the wells (roughly 30 adults per well). Finally the wells contained the worms, RPMI medium and different concentration of the drugs tested at a volume of 1.5 ml.

The worms were incubated for 24 hours in an incubation atmosphere (Heraeus incubator) containing 5% CO2.

For the experiment the worms were placed into single glass tubes (inner diameter 6mm), containing the incubation medium (1.5 ml) and enough fresh medium to reach a certain necessary level in the tubes (meniscus).

2.6.2. Test drugs

Four macrocyclic lactone anthelmintics were evaluated in this study: Ivermectin (Sigma), Moxidectin (Fort Dodge), Ivermectin B1b and Milbemycin A4 (MST).

The dilution of drugs were made in RPMI medium maintaining a steady concentration of 1%

DMSO and giving a final drug concentration of 10^-5 M and 10^-9 M (resistant) and 10^-7 M and 10^-11 M (susceptible). For dead controls the worms were killed by heating in a water bath at 70°C for 20 min. Three replicates (single tubes) for every concentration (five) were prepared for each drug to be tested.

2.6.3. The Micromotility Meter

A Micromotility meter similar to that one described by Folz (FOLZ et al., 1987a), was used in this study. These tubes were monitored in a 37°C warm water bath, then singly placed in the channel of the Micromotility meter and motility was measured for ~ 1 minute.

On the base of the Micromotility meter is a light located, which projects upwards through the tube and its contents. It refracts horizontally from the meniscus to the outside of the tube, where it gets measured by a photo detector. Movement of the worms cause a variation of the reflected light rays and therefore a variation in the signals received. The average deviation of the signals from its mean value is determined by means of an amplifier, a converter and a computer. The numerical representation of this signal is termed the motility index. This motility index gives a

good measure of the motor activity of the helminths tested. Dead helminths generated a reading comparable to those obtained from pure liquid, active helminths caused higher indices then less active worms.

a) photodiode b) pin lamp

c) knob to adjust position of test tubes

d) plastic light pipe

e) aluminium housing for test tube

f) connector to computer g) test tube

h) meniscus

Fig. 2.4.: Cross section of one channel of a Micromotility meter

2.6.4. Standardisation and performance of the assay

For the standardisation of this assay three parameters were thought to be critical. 1) the amount of worms per tube for the different species, 2) the time of incubation of the worms in the drug prior to measuring and 3) the concentration range of the drugs.

Several test assays were carried out in order to optimise these parameters.

Three different numbers of worms per tube were tested for each species without incubation in drugs. Based on the method of Power (POWER, 2005) for T. colubriformis a number of ~30

worms (25-35) was found to be optimal. For T. circumcincta tests were carried out with 2, 5 and 10 worms in RPMI medium. The results obtained for 2 worms per tube were scattered and of no good reproducibility. 5 and 10 worms gave close and repeatable results, so that a number of 7-8 worms was chosen.

The time of incubation was tested by measuring the motility of susceptible worms for only three concentrations of IVM (1-0.01 µM) after 2 and 24 hours. Even if there were no significant differences, the 24 hours incubation showed less scattered results. An incubation time of 24 hours was chosen to allow sufficient uptake of the drug and enough time to affect the motility of the worm.

In order to optimise the concentration range, two different serial dilutions were tested. The 1:3 dilution from 10^-6 M to 10^-8 M (same as used in the LMA) was not able to detect the whole spectrum of the drug. A 1:2 dilution from 10^-5 M - 10^-9 M (resistant isolates) and 10^-7 M – 10^-11 M (susceptible isolates) was chosen.

2.6.5. Data analysis

PicoScope© software from Pico Technology, USA was used to receive the readings from the Micromotility Meter on a computer and to analyze certain parameters. Figure 2.5. shows two typical traces obtained from T. colubriformis following incubation in different media.

The program measures the difference between the peaks of the received electric signals (mV, comparable to the amplitude) at given time points (ms, ca. every 20 ms). We chose a fixed amount of measurements up to 50,000 ms. Approximately the first 10 seconds did not contribute to the calculation to allow adaptation of the test-tube in the Micromotility Meter.

The obtained values were transformed into positive values and an average absolute value calculated automatically by the program. The average absolute values of the three sample triplicates were averaged to gather one value per drug concentration. The positive controls were set as 100%, the dead controls as 0%. The values from the positive controls and all tested

concentrations were corrected against the dead controls and a reduction of motility in percent calculated (using Microsoft Excel© software). The percentages of the four measurements taken were again averaged and used to graph the results in GraphPad Prism©, where an EC50 and the standard error have been calculated.

Further analysis and statistics were performed the same way as described in section 2.4.4.