Research Method Validation

Transcription

1 Evaluation of FECPAK and McMaster methodology for counting nematode eggs in sheep faeces Author: Good, B., Hanrahan, J.P. & Crowley, B.A. - Teagasc Research Centre, Athenry Co. Galway, Irelan Introduction The first experiment was designed to compare results from both methods using faecal material from a well-mixed composite with particular reference to the following issues. Variation within subsamples of FECPAK and McMaster prepared material using a minimum of 4 chambers per sample (sieved aliquot). Variation between sieved aliquots of FECPAK prepared material The second experiment involved individual animal samples to evaluate both methods over a range of FEC values and to examine the following. Variation within subsamples of FECPAK and McMaster prepared material i.e. 4 chambers were filled from the first aliquot of material sieved A second sample from the same individual was processed as above to assess the repeatability of a faecal egg count taken from the same animal on a given day Composite Material To compare FEC obtained from FECPAK methodology to the McMaster methodology (replicate samples taken from one-pooled composite) Experiment was carried out on two occasions using composite samples First composite sample Fresh faecal samples from a group of rams (mixed breeds) were collected, pooled and thoroughly mixed. Replications (n=8) each consisting of independent 3 g faecal sample, were processed according to McMaster methodology. Two chambers were loaded. The volume under each grid =0.15 ml and each egg counted represents 50 eggs per gram of faeces (epg). PG 1

2 From the same composite sample, 5 replications (limited to 5 because of material shortage) each consisting of 10 g faecal sample was prepared using the FECPAK methodology, loading two chambers. The volume under each grid = 0.5 ml. Each egg counted represents 30 epg. To compare FEC between different aliquots of 'slurry' The question as whether any difference was observed between aliquots could only be addressed where a large volume of sample (as in FECPAK) was a product of the method. A total of 4 more aliquots were poured separately through sieve into jug and two chambers loaded per aliquot as described above for two of the replicate samples. As the McMaster method yield an insufficient volume of material for more than one aliquot, this was not examined. Second composite sample The previous experiment was repeated having collected a fresh pool of ram faeces (from the same rams as before) and mixing thoroughly. Sixteen replications each consisting of 3 g faecal material was processed according to the McMaster methodology. For 8 of the replications 4 chambers were filled while for the remaining 8 replications, 6 chambers were loaded. (Chambers loaded after inverting tube with prepared sample five times and aspirating 0.15ml, this process was repeated until required numbers of chambers filled). Eleven replications, each consisting of 10 g faecal material was prepared from the same composite sample using the FECPAK methodology, loading 6 chambers (Chambers were loaded after agitating sample in the jug aspirating 0.5 ml approx., this process was repeated until required numbers of chambers filled). FEC between different aliquots of slurry obtained from FECPAK method A total of 2 more aliquots were poured separately through sieve into jug and six chambers loaded per aliquot as described above for all eleven of the replicate composite samples. Analysis and results Analysis employed SAS procedures. FEC was assigned a log transformed value (loge(epg+50). Initial analysis was carried on all data collected for the two composites. The effect of composite method composite*method PG 2

3 subsample(composite*method) aliquot (composite*subsample) were included in the model. No overall difference in FEC was observed when using FECPAK and McMaster methods (see table 1) although an interaction between compos*method was observed (P>0.001). As may be expected a difference in FEC between the two composites (taken at different times) was observed. No significant difference in FEC was observed between the aliquots prepared using the FECPAK methodology. These data were included in the analysis above. Table 1. Summary results of FEC observed using both methods on 2 separate composite samples The second composite study involved a relatively large set of samples and was used to examine in detail the various sources of variation. The FECPAK and McMaster data were analysed separately. The mean FEC (loge(x+50) scale) was 5.72 (s.e 0.019) for FECPAK method compared with 5.64 (s.e ) for McMaster method applied to the same material. The difference (0.08 s.e ) was not significant. Analysis 2. Summary of ANOVA on sampling variation in two methods - second composite study The variance components due to sampling of faecal material from the composite were and for McMaster and FECPAK, respectively. Thus, the sampling procedure was more variable with the former method. However it is PG 3

4 necessary to add in the Aliquot variance (estimate is ). Thus the overall sampling variance for FECPAK becomes The s.d. for the two procedures then becomes & Clearly the McMaster procedure has more inherent variation. Faecal Material From Individual Animals A comparison of FEC using FECPAK and McMaster methodology over a range of FEC values. On day one, rectal faecal samples were collected from 24 lambs (14 Suffolk, 10 Texel ewe lamb replacements) and placed unmixed into separate cartons identified by their individual tag number. In the lab, approx. 14-to15 g of faeces were removed at random from each carton. All remaining faecal material was placed in fridge. The g subsample was subsequently mixed thoroughly from which 10 g was used to determine FEC using the FECPAK method and 3g used to determine FEC using the Mc Master method (4 chambers were filled for each sample). Once FEC had been determined using both methods a second 14 to15 g of faecal material if available (11 Suffolk, 6 Texel) was taken from the individual cartons that had been placed in the fridge and procedure repeated as before. Due to time constraints there was an interval of 13 days between the first and second FEC determinations. Results Based on a single chamber, the s.d was 1.10 for McMaster and 1.13 for FECPAK. The repeatability, on a within-breed basis was 0.82 for FECPAK and 0.71 for McMaster methods, respectively (d.f. = 22 in each case). There was a significant (P>0.01) difference in FEC between two methods (4.9 (s.e. 0.04) for FECPAK and 5.2 (s.e. 0.04) for McMaster) when all observations were combined. This difference, on a within animal basis is relatively minor. The values obtained from individual sheep are plotted in Figure 1. The within breed correlation between the methods was PG 4

5 Table 3. Summary FEC results observed in two breeds of sheep using both methods on individual samples Using all of the data available the means were 5.3 and 5.5 for FECPAK and McMaster, respectively Figure 1 Scatter diagram of FEC obtained using separate methods (FECPAK and McMaster) for individual animals faecal material Summary of overall results No consistent or important difference in FEC obtained from FECPAK or McMaster methodologies, good correlation observed over a range of values. More inherent sampling variation in McMaster methodologies compared to FECPAK but this may be due to the small sample size (3 g as opposed to 10 g of faecal material examined). No difference in FEC observed between different aliquots of FECPAK 'slurry'. PG 5