CRISTOCENTRISMO MORAL Y HERMENÉUTICA
3. Cristocentrismo de las virtudes (L Melina)
Altogether 15 lnhouse LOA plates were prepared for the Larval Development assay of various farm samples. These LOA plates were used for study of detection of anthelmintic resistance on various sheep farms in conjunction with DrenchRite LOA
The lnhouse larval development assay (lnhouse LOA) used in this trial is largely as described by Gill et al (1995) and prepared to be similar to DrenchRite assays although with some variation of anthelmintic analogue used and some minor variation in anthelmintic concentrations.
A detailed protocol is described in Appendix 2.3. In brief, thiabendazole (99.3%
powder, donated by MSD AgVet, New Zealand Ltd. ), levamisole hydrochloride
(Rycozole, oral drench, Youngs Animal Health, New Zealand), ivermectin aglycone
(99.2% powder donated by Virbac laboratories Ltd, New Zealand) were used and
two different sources of ivermectin were used: IVM-1 (Bomectin oral, Bomac laboratories Ltd; ivermectin 0.1% w/v) and IVM-2 (Erase MPC, Schering Plough Animal Health Ltd; 16g/L, ivermectin). The stock solutions of above drugs were prepared in dimethyl sulphoxide (DMSO) and serially diluted 1:2 with DMSO to give 12 concentrations. Similarly, stock solution of levamisole (6 18j..Jg/ml) was prepared in distilled water and it was serially diluted 1 :2 with distilled water to give 12 concentrations. The molar concentrations of these anthelmintics (21.JI of the drug concentrations in 1501JI of the agar) were also determined.
The tests were performed in 96 well microtitre plate (Falcon, Becton Dickinson Labware, USA) by using various anthelmintic drug concentrations. The lnhouse L OA p lates were prepared by adding 21-11 of the various drug concentrations in all wells of various rows except control wells . 21-11 of d istilled water was added for levamisole control wells and 21-JI DMSO was added for thiabendazole, ivermectin a nalogue-1 and 2 and ivermectin aglycone control wells. Each plate contained two rows of wells for thiabendazole, levamisole and IVM-1 . lt also contained one row of wells for each of IVM-2 and ivermectin ag lycone. Then 1 501-JI of 2% agar (Bacto agar, Y-1 000 Sigma) was added in each of 96 wells and allowed to set.
After 1 hou r of p reparation of the LOA plates , 50-70 clean nematode eggs in 201-J I of distilled water was added to each of the 96 wells . The number of nematode eggs per well was determined on the basis of availability of total clean eggs in the sample.
These plates were incubated at 25°C for 7 days. On Day 2 of the incubation, 1 0- 201-JI of nutrient media (one gram of yeast extract (Y- Sig ma) in 90ml of 0.85% saline solution and Earle's Balanced salt solution (E751 0, Sigma) was added to every 9ml of yeast solution) (Hubert and Kerboeuf, 1 984 a nd some modification) was added in each of 96 wells when control wells had >60% egg hatching. The LOA plates were scanned every 2nd day to check for drying of the wells and whenever d rying is noticed , 1 01-JI of distilled water was added and this was recorded.
On Day 7 of the incubation the liquid phase from each well was removed and the number of eggs , L 1 , L2 and L3 at each well of the drug concentrations were counted at 1 OOx under compound microscope after staining with Lugol's iod ine . The strongylid genera/ species were identified on the basis of their measurements and morpholog ical features. All counts were done with in two days of the end of incubation .
P late 2.2 lnhouse LOA plate.
2.2.5 Data a na lysis
The eggs, 1st stage larvae (L 1 ), 2nd stage larvae (L2) and third stage larvae (L3)
from each well were counted and recorded . The number of third stage (L3) larvae
in the test wells were compared to the number that developed in control well for each trichostrongylid genus. This adjusted proportion was fitted to a sigmoid curve
(dose response curve) to calculate LCso values, after log transformation, using
SlideWrite version 5.01 (Advance Graphics Software Inc. , U.S.A.).
The interpretation of both assays were calculated by LCso well number and
compared to the Drenchrite LOA manual comparison table of efficacy (see Appendix 2.x). For Trichostrongylus this manual states that where the LC50 occurs
the efficacy can be interpreted as follows: For benzimidazoles Well 4 . 5 ind icates an
efficacy of 94% , Well 5 an efficacy of 87%; For levamisole Well 5.0 indicates an efficacy of 94% Well 5.5 an efficacy of 83%; For the combination of benzimidazole
used to detect the presence of resistance in ivermectin analogues but not as yet, to q uantify efficacy. For ivermectin analog ues the LCso Well (mean) 5.5 and above, can be interpreted as indicating some level of resistance.
The molar concentration of the LCso was also calculated by using the software programme SlideWrite version 5.01 (Advance Graphics Software l nc, U . S.A.) and expressed as micromole (IJM) and IJg/ml in the agar phase. The goodness of fit (R2 value) of the sigmoid curve is also shown . For those farms where both DrenchRite and lnhouse LOA plates were used the results were compared by using NCSS and PASS 2000 (Hintze
J . ,
2001 , NCSS and PASS Number Cruncher Statistrical System, Kaysville)2.3. Res ults of Drench Rite and l nhouse LOA tests
All together samples from 25 farms were processed that contained 1 0 samples from each fa rm (total 250 samples). Five fa rms had zero faeca l egg counts and these samples could not be assessed for LOA.
Samples from 1 4 farms were assessed with DrenchRite LOA plates and resu lts from 1 2 farms were recorded for data analysis. Results are shown in Table 2.2 and Fig . 2 .2.
S imi larly, Samples from 1 5 farm samples were assessed with lnhouse LOA plates for detection of the level of anthelmintic resistance. Of these , useful results for analysis were achieved for 1 2 farms but for 3 farms LOA plates were discarded due to poor larval g rowth in various control wells. Results are shown in Table 2 . 3 and Fig . 2 . 1 . The details o f data obtained from DrenchRite and l nhouse LOA is g iven in Appendix 2.6, that is included in the compact disk (CD).
The predominant genera (n=20), on LOA plates and larval cultures was
Trichostrongylus spp. (78%), Chabertia and Oesophagostomum spp. (1 7%) and