• 1.

    Belding DL. Chapter 51: the examination of helminths. In: Textbook of clinical parasitology. New York: Appleton-Century-Crofts, 1952;975980.

    • Search Google Scholar
    • Export Citation
  • 2.

    Bello TR, Gordon VL. Rapid concentration of strongyle eggs from equine feces for in vitro studies. Am J Vet Res 1970;31:22852288.

  • 3.

    Coleman SU, Klei TR, French DD, et al.Prevalence of Cryptosporidium sp in equids in Louisiana. Am J Vet Res 1989;50:575577.

  • 4.

    Kazacos KR. Improved method for recovering ascarid and other helminth eggs from soil associated with epizootics and during survey studies. Am J Vet Res 1983;44:896900.

    • Search Google Scholar
    • Export Citation
  • 5.

    Herd RP. Performing equine fecal egg counts. Vet Med 1992;87:240244.

  • 6.

    Alcaino HA, Baker NF. Comparison of two flotation methods for detection of parasite eggs in feces. J Am Vet Med Assoc 1974;164:620622.

    • Search Google Scholar
    • Export Citation
  • 7.

    Craig TM, Diamond PL, Ferwerda NS, et al.Evidence of ivermectin resistance of Parascaris equorum on a Texas horse farm. J Equine Vet Sci 2007;27:6771.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Slocombe JOD, Lake MC. Efficacy of daily pyrantel tartrate (Strongid C) against strongyles in ponies on pasture. J Equine Vet Sci 2007;27:6770.

    • Search Google Scholar
    • Export Citation
  • 9.

    Craven J, Bjorn H, Henriksen SA, et al.Survey of anthelmintic resistance on Danish horse farms, using 5 different methods of calculating faecal egg count reduction. Equine Vet J 1998;30:289293.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Craven J, Bjorn H, Barnes EH, et al.A comparison of in vitro tests and a faecal egg count reduction test in detecting resistance in horse strongyles. Vet Parasitol 1999;85:4959.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Tarigo-Martinie JL, Wyatt AR, Kaplan RM. Prevalence and clinical implications of anthelmintic resistance in cyathostomes of horses. J Am Vet Med Assoc 2001;218:19571960.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Bello TR. Preparing an anthelmintic for equine practice, in Proceedings. 27th Annu Meet Am Assoc Equine Pract 1981;1117.

  • 13.

    Bello TR. Antiparasitic treatment of horses with pyrantel and fenbendazole followed by continual ivermectin treatments, in Proceedings. 35th Annu Meet Am Assoc Equine Pract 1989;419429.

    • Search Google Scholar
    • Export Citation
  • 14.

    Bello TR. Alternative antiparasitic treatment of horses with pyrantel pamoate suspension and ivermectin oral solution compared with horses treated only with ivermectin oral solution. J Equine Vet Sci 1996;16:166170.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Bello TR. Controlled test and clinical evaluation of dienbendazole against naturally acquired gastrointestinal parasites in ponies. Am J Vet Res 1989;50:19761980.

    • Search Google Scholar
    • Export Citation
  • 16.

    Triola MF. Chapter 11: analysis of variation. In: Elementary statistics. 9th ed. Boston: Pearson Education, 2004;602634.

  • 17.

    Bello TR. The control and treatment of internal equine parasites. Somerville, NJ: Monograph, American Hoechst Corp, 1981;119.

  • 18.

    Morgan BB, Hawkins PA. Chapter 11: helminths of the horse. In: Veterinary helminthology. Minneapolis: Burgess Publishing Co, 1949;5455.

    • Search Google Scholar
    • Export Citation
  • 19.

    Soulsby EJL. Chapter 31–33. In: Textbook of veterinary clinical parasitology. Philadelphia: FA Davis Co, 1965;795869.

  • 20.

    Lucker JT. Comparative morphology and development of infective larvae of some horse strongyles, in Proceedings. Helminthol Soc Wash 1936;3:2225.

    • Search Google Scholar
    • Export Citation
  • 21.

    Lucker JT. Description and differentiation of infective larvae of three species of horse strongyles, in Proceedings. Helminthol Soc Wash 1938;5:15.

    • Search Google Scholar
    • Export Citation

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Comparison of two fecal egg recovery techniques and larval culture for cyathostomins in horses

Thomas R. BelloSandhill Equine Center, 1944 N May St, Southern Pines, NC 28387.

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 DVM, PhD
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Tammy M. AllenSandhill Equine Center, 1944 N May St, Southern Pines, NC 28387.

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Abstract

Objective—To compare the McMaster and centrifugal flotation techniques and larval culture for recovery of cyathostomin (small strongyle) eggs from the feces of horses.

Sample Population—Fecal samples from 101 horses.

Procedures—In experiment I, homogenized fresh feces from a single horse were randomly subsampled by use of each technique for 10 replicates. In experiment II, samples from 43 horses that had no anthelmintic treatment were analyzed by use of McMaster, centrifugal flotation, and larval culture techniques. In experiment III, 57 horses were treated with an anthelmintic by owners, and fecal samples were analyzed as for experiment II.

Results—In experiment I, use of the McMaster technique recovered 72% of the eggs obtained by use of centrifugal flotation from paired subsamples. In experiment II, use of the McMaster technique recovered 81% of the eggs obtained by use of centrifugal flotation. Only cyathostomins resulted from individual larval cultures. In experiment III, 24 samples had negative results for all 3 tests, 18 samples had positive results only with larval cultures, and 15 samples had positive results of centrifugal flotation (only 5 of which had positive results via the McMaster technique).

Conclusions and Clinical Relevance—Centrifugal flotation consistently was superior to the McMaster technique, especially at low fecal egg numbers. The combination of centrifugal flotation and larval culture may provide the best accuracy for evaluation of anthelmintic efficacy.

Abstract

Objective—To compare the McMaster and centrifugal flotation techniques and larval culture for recovery of cyathostomin (small strongyle) eggs from the feces of horses.

Sample Population—Fecal samples from 101 horses.

Procedures—In experiment I, homogenized fresh feces from a single horse were randomly subsampled by use of each technique for 10 replicates. In experiment II, samples from 43 horses that had no anthelmintic treatment were analyzed by use of McMaster, centrifugal flotation, and larval culture techniques. In experiment III, 57 horses were treated with an anthelmintic by owners, and fecal samples were analyzed as for experiment II.

Results—In experiment I, use of the McMaster technique recovered 72% of the eggs obtained by use of centrifugal flotation from paired subsamples. In experiment II, use of the McMaster technique recovered 81% of the eggs obtained by use of centrifugal flotation. Only cyathostomins resulted from individual larval cultures. In experiment III, 24 samples had negative results for all 3 tests, 18 samples had positive results only with larval cultures, and 15 samples had positive results of centrifugal flotation (only 5 of which had positive results via the McMaster technique).

Conclusions and Clinical Relevance—Centrifugal flotation consistently was superior to the McMaster technique, especially at low fecal egg numbers. The combination of centrifugal flotation and larval culture may provide the best accuracy for evaluation of anthelmintic efficacy.

In early studies, the zinc sulfate centrifugal flotation technique was used to detect hookworm and whipworm eggs in humans. There have been variations of this technique over time.1 The centrifugal flotation technique has been used for in vitro studies,2 for recovering Cryptosporidium oocysts from foals,3 and for recovering ascarid and other helminth eggs from soil.4

A detailed description of the McMaster technique for counting equine strongyle eggs was provided by Herd.5 Comparisons of the 2 techniques favor centrifugal flotation for recovery of eggs from various hosts.6 In studies7,8 on recovery of Parascaris equorum eggs and pyrantel tartrate efficacy, the McMaster technique was used initially and findings were confirmed by use of centrifugal flotation.

The McMaster technique has been used in combination with larval cultures for detecting development of anthelmintic resistance on Danish horse farms.9,10 The centrifugal flotation technique, supported by larval cultures that were made from the feces of individual horses, has been used for strongyle group identifications and to measure response to long-term administration of anthelmintics.11–13 Because the apparent increased development of specific anthelmintic resistance is becoming widespread,9–11 a combination of techniques may be necessary for efficacious diagnosis of herd or individual infection. Therefore, the purpose of the study reported here was to compare the McMaster and centrifugal flotation techniques for recovery of cyathostomin eggs from the feces of horses, with diagnostic support by use of larval cultures.

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 vermiculiteb 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 tartratec daily, the horses were treated with ivermectind 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.

Abbreviations

EPG

Eggs per gram

LPG

Larvae per gram

a.

PARACOUNT-EPG, Chalex Corp, Wallowa, Ore.

b.

Terra-Lite, WR Grace & Co, Cambridge, Mass.

c.

Strongid C, Pfizer Animal Health, New York, NY.

d.

Zimecterin, Merial Ltd, Duluth, Ga.

e.

Zimecterin Gold, Merial Ltd, Duluth, Ga.

f.

Equimax, Pfizer Animal Health, New York, NY.

References

  • 1.

    Belding DL. Chapter 51: the examination of helminths. In: Textbook of clinical parasitology. New York: Appleton-Century-Crofts, 1952;975980.

    • Search Google Scholar
    • Export Citation
  • 2.

    Bello TR, Gordon VL. Rapid concentration of strongyle eggs from equine feces for in vitro studies. Am J Vet Res 1970;31:22852288.

  • 3.

    Coleman SU, Klei TR, French DD, et al.Prevalence of Cryptosporidium sp in equids in Louisiana. Am J Vet Res 1989;50:575577.

  • 4.

    Kazacos KR. Improved method for recovering ascarid and other helminth eggs from soil associated with epizootics and during survey studies. Am J Vet Res 1983;44:896900.

    • Search Google Scholar
    • Export Citation
  • 5.

    Herd RP. Performing equine fecal egg counts. Vet Med 1992;87:240244.

  • 6.

    Alcaino HA, Baker NF. Comparison of two flotation methods for detection of parasite eggs in feces. J Am Vet Med Assoc 1974;164:620622.

    • Search Google Scholar
    • Export Citation
  • 7.

    Craig TM, Diamond PL, Ferwerda NS, et al.Evidence of ivermectin resistance of Parascaris equorum on a Texas horse farm. J Equine Vet Sci 2007;27:6771.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 8.

    Slocombe JOD, Lake MC. Efficacy of daily pyrantel tartrate (Strongid C) against strongyles in ponies on pasture. J Equine Vet Sci 2007;27:6770.

    • Search Google Scholar
    • Export Citation
  • 9.

    Craven J, Bjorn H, Henriksen SA, et al.Survey of anthelmintic resistance on Danish horse farms, using 5 different methods of calculating faecal egg count reduction. Equine Vet J 1998;30:289293.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10.

    Craven J, Bjorn H, Barnes EH, et al.A comparison of in vitro tests and a faecal egg count reduction test in detecting resistance in horse strongyles. Vet Parasitol 1999;85:4959.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 11.

    Tarigo-Martinie JL, Wyatt AR, Kaplan RM. Prevalence and clinical implications of anthelmintic resistance in cyathostomes of horses. J Am Vet Med Assoc 2001;218:19571960.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12.

    Bello TR. Preparing an anthelmintic for equine practice, in Proceedings. 27th Annu Meet Am Assoc Equine Pract 1981;1117.

  • 13.

    Bello TR. Antiparasitic treatment of horses with pyrantel and fenbendazole followed by continual ivermectin treatments, in Proceedings. 35th Annu Meet Am Assoc Equine Pract 1989;419429.

    • Search Google Scholar
    • Export Citation
  • 14.

    Bello TR. Alternative antiparasitic treatment of horses with pyrantel pamoate suspension and ivermectin oral solution compared with horses treated only with ivermectin oral solution. J Equine Vet Sci 1996;16:166170.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 15.

    Bello TR. Controlled test and clinical evaluation of dienbendazole against naturally acquired gastrointestinal parasites in ponies. Am J Vet Res 1989;50:19761980.

    • Search Google Scholar
    • Export Citation
  • 16.

    Triola MF. Chapter 11: analysis of variation. In: Elementary statistics. 9th ed. Boston: Pearson Education, 2004;602634.

  • 17.

    Bello TR. The control and treatment of internal equine parasites. Somerville, NJ: Monograph, American Hoechst Corp, 1981;119.

  • 18.

    Morgan BB, Hawkins PA. Chapter 11: helminths of the horse. In: Veterinary helminthology. Minneapolis: Burgess Publishing Co, 1949;5455.

    • Search Google Scholar
    • Export Citation
  • 19.

    Soulsby EJL. Chapter 31–33. In: Textbook of veterinary clinical parasitology. Philadelphia: FA Davis Co, 1965;795869.

  • 20.

    Lucker JT. Comparative morphology and development of infective larvae of some horse strongyles, in Proceedings. Helminthol Soc Wash 1936;3:2225.

    • Search Google Scholar
    • Export Citation
  • 21.

    Lucker JT. Description and differentiation of infective larvae of three species of horse strongyles, in Proceedings. Helminthol Soc Wash 1938;5:15.

    • Search Google Scholar
    • Export Citation

Contributor Notes

Presented in part at the 52nd Annual Meeting of the American Association of Veterinary Parasitologists, Washington, DC, July 2007.

The authors thank Dr. J. McInerney for assistance with statistical analysis.

Address correspondence to Dr. Bello.