23 May 2016
David Rendle looks at targeted worming in equines and considers the factors involved when employing faecal worm egg counts, such as handling and different methods.
David Rendle
Job Title
Figure 3. A standard McMaster slide showing the grids and chambers that can all be counted to increase the accuracy of the result.
Targeted worming is advocated as the gold standard in equine intestinal parasite control as it enables a reduction in anthelmintic use and, in so doing, reduces the selection pressure for anthelmintic resistance.
Fortunately, for the future of equine parasite control, targeted worming plans are being widely adopted (Figure 1) and are cheaper to horse owners than traditional interval dosing strategies1. However, with the promotion and supervision of targeted worming plans comes a responsibility to ensure horses remain adequately protected against parasite-associated disease.
It is, therefore, important to appreciate the limitations of faecal worm egg count analysis and acknowledge sources of potential variation.
The aim of a faecal worm egg count is not to establish the level of parasitism in the individual animal, but to establish whether that animal poses an unacceptable threat to others in the population by excessively contaminating its environment with worm eggs.
As many horses in the population as possible are left untreated to maximise the number of parasites, which are not exposed to a selection pressure or, in parasitology jargon, to maintain refugia.
By only selecting those animals in a population shedding an unacceptably high level of strongyle eggs (typically more than 250 eggs per gram; epg), anthelmintic use can be reduced by around 80%2,3 – markedly reducing the selection pressure for resistance.
A faecal worm egg count only estimates the number of mature female nematodes and provides no indication of the number of male, immature and encysted forms, therefore, providing a poor indication of the total worm burden in an individual horse4.
Most horses will be parasitised by tens of different cyathostomin species, as well as ascarids, tapeworms and (rarely, but potentially increasingly in the UK) large strongyles. Some strongyle species are far more prolific egg producers than others and the pathogenicity of different species is likely to be different; a worm egg count will not be able to determine the species of strongyles present.
Until accurate serological tests are available to quantify the level of endoparasite infection, it will remain impossible to reliably determine the level of infection with immature nematodes antemortem. Therefore, when determining the need for anthelmintics, egg counts should not be interpreted in isolation, but with additional knowledge of previous results, results from cohorts, management on the property (and, hence, risk of exposure) and knowledge of recent anthelmintic administration.
Record-keeping and interpretation are facilitated by the use of online databases that can be updated and viewed by both vet and owner (Figure 2).
A number of studies have demonstrated a minority of horses in a population will be responsible for excreting the majority of cyathostomin eggs. In the UK, 11% of 1,221 Thoroughbreds5 and 15% of 928 leisure horses6 were shown to excrete 80% of the total eggs produced in their respective populations.
This 80:20 rule provides an opportunity to single out the high shedders for treatment. High shedders may continue to excrete higher levels of nematode eggs than others in the group and, therefore, merit long-term closer monitoring, and even different management, compared to low shedders7.
However, individual worm counts may vary over time relative to others in the same group, especially in the face of high levels of challenge2. Just because a horse has a low count – relative to its cohorts – on one or more egg counts, does not indicate it will always remain low.
In addition to the level of exposure, other factors will predispose horses to higher worm egg counts. Age is the most important, with younger horses tending to carry higher numbers of patent nematodes and, hence, excreting a greater number of eggs. It is assumed it takes five years or six years for immunity to parasites to develop to optimal levels.
The horse’s general health and the presence of disease may affect levels of endoparasitism and egg production. Pituitary pars intermedia dysfunction is associated with the production of higher faecal worm egg counts, presumably as a consequence of reduced immunity to parasites8.
The way in which faeces are handled prior to reaching the laboratory can have a marked effect on subsequent counts, as eggs that have hatched will not be counted. Eggs hatch in aerobic conditions at a temperature-dependent rate. Below 10°C, hatching will take more than 10 days, but above 20°C, it may take as little as a day9. If samples are kept in a standard refrigerator (at less than 6°C), egg counts should be stable for up to five days9. The use of ziplock bags, which air can be expelled from, is preferred to the use of larger, unsealed bags or pots that contain a large amount of air.
Eggs are not evenly distributed in horse faeces10 and may be clumped, leading to both overestimation and underestimation of worm egg counts, particularly when the count is low11. There is, therefore, the potential for significant errors if small unrepresentative samples are submitted. As many small pinches from a pile of faeces as possible should be collected, with a minimum of three faecal balls being sampled12.
A number of methods of estimating nematode egg numbers in faeces are available. Those more involved tend to have a lower detection limit and greater repeatability, but will be more time-consuming.
Methods with an inferior detection limit will rely on greater multiplication factors, which will exaggerate any variability and increase potential errors. Methods with lower detection limits (less than or equal to 5epg) are preferred and should always be used when quantifying responses to anthelmintics13.
Regardless of the method used, the amount of faeces examined will be the single biggest factor that determines the accuracy of the result. A minimum of 3g of faeces should be used; increasing to 10g halves the coefficient of variation10.
In human medicine, multiple worm egg counts may be performed from the same sample to increase accuracy. In veterinary species, this is not routine (except in experimental studies) as variation is typically insufficient to impact clinical decision-making and the costs are prohibitive, especially when factored against reducing costs of the anthelmintics. The variation that may occur in multiple replicates from the same horse has been illustrated in a review article14.
The McMaster method remains the most widely used and best accepted method for quantifying nematode eggs in domestic animal species. The method relies on the equal distribution of eggs in a saturated salt solution, which, in turn, relies on the salt solution containing the eggs being thoroughly mixed immediately prior to loading the counting slide.
In a busy practice laboratory, it is very easy for samples to be mixed inadequately or left to stand and separate after mixing – thus, in time, the eggs rise to the top of the saturated solution. If the slide is loaded with solution from the top of the mixture, there will be an overestimation of the faecal worm egg count. If it is loaded from lower down then the number of eggs will be underestimated. Numerous modifications of the original McMaster method have been published.
Although the different variations have not been compared using equine faeces, studies in pigs and sheep conclude the use of more faeces and, hence, a reduced dilution (and subsequent multiplication) factor, results in greater accuracy and sensitivity15,16.
Denwood et al10 looked specifically at sources of variability in worm egg counts in equine faecal samples using a modified McMaster method and concluded the quantity of faeces examined was the greatest source of variance.
Another means of increasing accuracy is to examine a greater volume of fluid from each faecal suspension. Each McMaster slide contains two grids, each within a chamber. By counting more than the standard single grid, the multiplication factor is reduced and accuracy, therefore, increased (Table 1; Figure 3).
In sheep faeces, accuracy is further increased by counting further slides17,18; however, the return in increased accuracy diminishes with the number of counts performed. The extra time required to count more than one slide is difficult to justify, when performing two counts by McMaster rather than one has shown to have little influence on the decision as to whether anthelmintics are required19.
It is important the correct flotation media is used and specific gravity is checked prior to use. A sodium chloride solution with a specific gravity of 1.2 can be prepared by adding around 400g of sodium chloride to 1L of water.
The FECPAK method was designed to be used on farms without the need for complex equipment. A large quantity of faeces (10g) is used with a multiplication factor of 20, which is better than the McMaster method, unless two chambers are being counted as part of the McMaster protocol. In a study of equine faeces, FECPAK had a higher sensitivity and was less likely to underestimate worm egg counts compared to a modified McMaster method20. However, the cost of the system is relatively high.
The Ovatec Plus method provides a quicker, qualitative means of assessing faecal worm egg counts. Regrettably, no data on the method has been published.
Data was presented at a professional meeting21 and it was concluded the absence of eggs provided complete certainty the sample contained fewer than 250epg of nematodes. Therefore, the presence of any eggs indicates treatment with anthelmintics may be necessary. The distributors recommend, where eggs are identified, a quantitative method, such as McMaster, is also performed, which adds time and expense.
Only 3g of faeces is used, which is a cause for concern as it is accepted more reliable results will be obtained with greater volumes, using methods that have been subjected to greater scrutiny.
Ovatec Plus is not appropriate for assessing responses to anthelmintics, but provides a quick means of differentiating very high shedders from very low shedders, if that is all that is required.
With resistance to anthelmintics common in the UK, assessment of anthelmintic efficacy, particularly of fenbendazole and pyrantel, should be routine. Sadly, despite their simplicity and low cost, faecal worm egg count reduction tests are not being performed widely.
Considering the potential for variation outlined in this article, it is important sampling and counting are as accurate as possible. A method with a minimum detection limit of less than or equal to 25epg (preferably less than or equal to 5epg) should be used22. It is also recommended as many horses as possible are included, with a minimum of six required to ensure confidence resistance is present.
A worm egg count is performed before, and 14 days to 17 days after, the administration of an anthelmintic and the percentage reduction in faecal worm egg count is calculated:
Percentage faecal worm egg count reduction (% FWECR) = ([mean day 0 FWEC – mean day 14-17 FWEC]/mean day 0 FWEC) × 100.
A reduction of less than 90% for fenbendazole and pyrantel, or less than 95% for ivermectin or moxidectin, is indicative of resistance.
If the reduction in mean counts following treatment is not uniform across the test group, but is attributed to a small number of individuals, the possibility of misdosing or underdosing should be considered and these horses should be tested again with the same anthelmintic to confirm resistance is present.
Targeted worming has to be central to equine parasite control as interval dosing is unsustainable. However, to minimise the risk of parasite-related disease, it is important to appreciate its limitations. A few simple measures to increase accuracy and improve record keeping can ensure the results are accurate enough to enable appropriate clinical decisions to be made.
