Counting nematode eggs in equine faecal samples

THE control of gastrointestinal nematodes in horses often relies on routine anthelmintic prophylaxis, which aims to reduce parasite burden in individual animals and to limit contamination of the pasture with nematode eggs. However, treating all the horses in a group is likely to involve the unnecessary treatment of uninfected or lightly infected animals (Comer and others 2001), and to accelerate the development of anthelmintic resistance (Coles 2002). Routine chemoprophylaxis at fixed intervals is also likely to lead to unnecessary drug administration, and is insensitive to climate-driven fluctuations in the risk of infection (Reinemeyer 1999). Individual horses that are egesting large numbers of eggs should be identified and treated, protecting others in the group from high levels of infection, while leaving some parasites in refugia (Coles 2003). A major obstacle to such targeted anthelmintic prophylaxis in horses is the limited availability of suitable faecal egg count tests. The widely used McMaster technique (MAFF 1986) was developed mainly for sheep and is poorly sensitive at low egg densities (Mes and others 2001) that might indicate that treatment is required in horses. It also requires specialist equipment, and the expense of laboratory diagnosis is likely to be a significant disincentive to many horse owners. More sensitive concentration techniques for egg detection generally require good laboratory facilities (Mes 2003), and are often less reliable for the estimation of high egg counts (Levine and others 1960, Rossanigo and Gruner 1991). A new faecal egg count test devised by FECPAK NZ is used with cattle and sheep and has the potential to surmount these difficulties. A larger amount of faeces is examined than by the McMaster technique, so sensitivity should be greater (Hunter and Quenouille 1952). Centrifugation is not required, and the test is available as a complete kit which could be used by a veterinarian or horse owner on the premises. This short communication compares the FECPAK test and McMaster technique to examine the predicted improvements with the FECPAK system. The FECPAK test, like the McMaster technique, is based on the flotation-dilution principle described by Stoll (1930). In the standard FECPAK test for cattle, a minimum of 10 g faeces is mixed thoroughly in a plastic bag with three times the volume of water. A sample of the mixed suspension (45 ml) is then removed, made up to 230 ml with saturated salt solution and poured through a filter of 1 mm aperture to remove coarse debris. An aliquot is removed from the well mixed filtrate and placed in a custom-made acrylic counting chamber with a volume of 1 ml under the grids (compared with 0·3 ml on the McMaster slide). After 30 seconds, nematode eggs that have floated to the top of the chamber are counted under a microscope. The multiplication factor to transform the number of eggs counted to estimated faecal egg density depends on the dilution used: in this case, each egg counted represents 20 eggs per gram of faeces (epg). In common with other tests of this type, excessive dilution reduces sensitivity, whereas insufficient dilution inhibits egg flotation and visibility and also leads to an underestimation of egg density (Dunn and Keymer 1986). The optimum dilution should therefore take into account the consistency of the faecal material and the sensitivity required from the test. To ascertain the best dilution for use in horses, faeces from two horses with a naturally acquired mixed strongyle infection were analysed using the FECPAK test. Initial dilutions of 1 g faeces in 1 ml water (for a high number of expected eggs) to 1 g in 10 ml were used. The expected number of eggs was calculated from the mean number of epg in the three most dilute samples x the mass of faeces examined in the counting chamber. In less diluted suspensions, fewer eggs were counted than expected (Fig 1). A 4 ml sample of water added initially to each gram of faeces represented the most concentrated workable dilution. At this dilution, each egg counted represents 25 epg. The relative sensitivities of each test were compared using faeces from an uninfected horse to which a known number of strongyle eggs had been added to final densities of 50, 100 and 200 epg. Each test was repeated five times at each egg density. Using the FECPAK test, eggs were detected in each case, whereas using the McMaster technique, six false negative results were obtained (three of five at 50 epg and three of five at 100 epg). The greater sensitivity of the FECPAK test is to be expected, because more faeces are examined than by the McMaster technique, and the chances of missing eggs present at low density are consequently reduced. Assuming that the faecal sample is well mixed and that eggs are randomly distributed in suspension, the expected proportion of false negatives at each faecal egg density can be predicted from the Poisson distribution, with the mean being equal to the expected number of eggs per slide (Peters and Leiper 1940). The assumption of Poisson distribution was tested by taking serial counts from well mixed samples of naturally infected horse faeces using both the McMaster and the