Materials and Methods

The parasite egg recovery techniques used in the study were modifications of the original McMaster or Stolle methods. In the modified McMaster technique, 26 mL of sodium nitrate solution (specific gravity, 1.20) was placed in a calibrated vial and feces were added to attain a final volume of 30 mL and thoroughly mixed with the solution. Separate aliquots were withdrawn with a 1-mL syringe and placed under each of the paired slide chambers.^a The numbers of parasite eggs from both chambers counted between the grid lines were added, and the result was multiplied by 25 to yield the EPG of feces.

In the centrifugal flotation technique,¹⁴ a 2-g fecal sample from each horse was placed in a 250-mL flask, diluted to 100 mL with tap water, and homogenized by shaking 40 times. A 10-mL aliquot containing 0.2 g was poured into a 15-mL tube and was packed by use of centrifugation at 430 X g for 10 minutes. The supernatant liquid was removed by quickly inverting and righting the tube over a sink. The sediment was mixed with sucrose solution (specific gravity, 1.20) that was added to form a positive meniscus at the top of the tube. A 22 × 22-mm coverslip sealed the top of the tube, which was centrifuged at 190 X g for 10 minutes. The coverslip was placed on a slide and examined microscopically. The number of parasite eggs counted was multiplied by 5 to yield the number of EPG of feces.

Cultures were made for providing infective larvae to determine the general identity of the parasites that produced the eggs.¹⁵ A 40-g sample of feces from each horse was mixed with an equal volume of vermiculite^b and incubated at approximately 26°C for 10 to 12 days. The larval cultures were processed overnight in Baermann sedimentation funnels (diameter, 25.5 cm) to concentrate infective strongyle stage 3 larvae. From these preparations, the number of LPG of feces and larval species differential counts were determined. The LPG count was obtained by counting the number of larvae in a 0.1-mL aliquot of a 20-mL suspension of larvae cultured from 40 g of feces. The larval number was multiplied by 200, then divided by the number of grams of feces (40) to yield the LPG count. If the sample contained < 40 g of feces, the larval number in the 0.1-mL aliquot was multiplied by 200, then divided by the weight of the sample to yield the LPG count. The larval species differential count was derived via microscopic identification of 100 stage 3 larvae sequentially encountered during the larval number determination. For differential counts of larvae in fecal cultures after horses were treated (in which larval numbers were low), larvae were concentrated in the bottom of the tube from which the larval sample was withdrawn in 0.1 mL of sediment. In this study, all egg counts were performed by one person and all larval cultures were performed by a different person.

Fecal samples from 101 horses were examined. All feces were obtained fresh from each horse and processed on the same day via both techniques. A preliminary test was done with 10 replicate subsamples from 1 horse (experiment I). Fresh feces (approx 250 g) were homogenized in a pan and randomly sampled to provide material for centrifugal flotation or the McMaster technique. In experiment II, samples obtained from 43 horses that had no anthelmintic treatment for at least 9 months were analyzed by use of the McMaster technique, centrifugal flotation, and larval culture. In experiment III, samples were obtained from 57 horses for which owners noted on a questionnaire that the horses were treated with an anthelmintic and provided the approximate date of treatment and the specific anthelmintic that had been given.

In experiment I, ANOVA was used to compare EPG numbers obtained from replicate subsamples by both techniques.¹⁶ In experiment II, an exact paired t test for differences of means of the egg counts determined via the 2 diagnostic methods was performed. For all comparisons, a value of P < 0.05 was considered significant.

Results

Experiment I—Mean strongyle EPG count determined by use of centrifugal flotation was 725 (coefficient of variation, 11.8%). The count by the McMaster technique was 525 (coefficient of variation, 23.8%), which was 72% of the EPG count obtained by centrifugal flotation.

Experiment II—Mean EPG count obtained via centrifugal flotation was 539, and EPG count obtained via the McMaster technique was 437 (ie, 81% of the EPG count obtained by use of centrifugal flotation [P = 0.007]). Larval cultures yielded 231 LPG. This represented 43% of the centrifugal flotation EPG count, typical of the loss during development from egg to infective stage 3 larvae¹⁷ and subsequent use of the Baermann procedure to obtain larvae for identification. All larvae examined were cyathostomins (small strongyles).^18–21

Experiment III—The strongyle EPG numbers were attributed to information of previous anthelmintic treatment of 57 horses by the owners. Except for 2 horses given pyrantel tartrate^c daily, the horses were treated with ivermectin^d or ivermectin-praziquantel combination.^e,f On the basis of posttreatment analyses, 3 groups of data evolved (IIIa, IIIb, and IIIc). In IIIa, no eggs or larvae were detected in samples from 24 horses 28 days after treatment. In samples from 18 horses (IIIb, 58 days after treatment), larvae (mean, 10 larvae/sample) were cultured but eggs were not detected via centrifugal flotation or the McMaster technique. In IIIc, samples were obtained from 15 horses (41 days after treatment). In samples from 10 of these horses, eggs were detected via centrifugal flotation but not via the McMaster technique. In samples from 5 horses, eggs were detected via centrifugal flotation and the McMaster technique. The McMaster EPG count was only 68% of the centrifugal flotation EPG count. Among the 15 samples in which eggs were detected, larval cultures yielded 5 LPG in 2 samples and 0 LPG in 13 samples. When a concentrated sample was withdrawn from the bottom of each conical tube, a mean of 21 larvae were found in 12 of 15 samples.

Overall, in all 3 experiments, 79% of individual EPG counts derived via centrifugal flotation were greater than the EPG counts derived via the McMaster technique, and 21% of the EPG counts determined via the McMaster technique were equal to or greater than those determined via centrifugal flotation.

Discussion

Data regarding ivermectin obtained via clinical administration of the drug with fecal sampling at 2-week intervals via centrifugal flotation for EPG and cultures for LPG and larval identification have established a baseline measurement of efficacy.^13,14 The practical results from those experiments suggest that a combination of egg and larval culture data may be necessary to clearly define the fecal egg count reduction test, especially in cases of suspected drug resistance. Pre- and posttreatment samples from individual or multiple horses are required at a 2-week interval for the fecal egg count reduction test.

In the present study, anthelmintic effectiveness was indicated by the negative results of egg and larval counts from horses that were sampled approximately 28 days after treatment. Such results at this brief posttreatment time were not surprising as this was well within the cyathostomin egg reappearance period for ivermectin. Likewise, the 18 horses that were sampled at 58 days had only larvae detected, which indicated good efficacy. With the ivermectin posttreatment egg reappearance period profile as a basis, the 15 treated horses with positive results of egg and larvae determinations had a much shorter egg reappearance period of 41 days, suggesting development of resistance to the anthelmintic. Results of this experiment indicated the advantage of use of larval cultures for identification of possible resistant cyathostomins when EPG and LPG numbers are low or zero.

Our results indicated that centrifugal flotation was superior to the McMaster technique, especially at low-level fecal egg numbers, which we consider important. The combination of parasite egg numbers obtained from centrifugal flotation and larval culture data may be advantageous for evaluation of anthelmintic efficacy and potentially serve as a better diagnostic baseline for further studies on development of drug resistance.