Modified McMasters Fecal Egg Count

Accurate evaluation of nematode burdens cannot be made with subjective assessments of egg loads on fecal examinations. Fecal egg counts, such as the modified McMasters fecal egg count (MMFEC), are more objective for understanding patterns of infection and shedding, success of parasite management, when program changes are needed, and whether changes are helping. Annual and biannual fecal egg count monitoring may be suitable for some artiodactylid species; but higher risk species may need more frequent monitoring. The use of spreadsheet technologies may graph trends and establish in-house reference intervals by species or individual to aid in establishing strategic guidelines for monitoring and treatment. Many procedures exist for determining fecal egg counts, but it is important to use the same procedure each time. The MMFEC, with a sensitivity of 50 eggs/g (epg), is commonly used because of the quantitative data and simplicity of technique. Samples may be collected and refrigerated, not frozen, for up to 7 days to prevent larval hatch out in the fecal matter, typically within 12 to 24 hours at room temperature, rendering the sample nondiagnostic. Laboratory techniques for this procedure may be found at the website for the Southern Consortium for Small Ruminant Parasite Control (SCSRPC; http://www.scsrpc.org).¹⁷ A McMasters slide (Chalex, Wallowa, Ore) is required for this procedure (Fig. 75-1). Trichostrongyle-type eggs (oval; ∼80 to 90 µm) seen in the slide grid of the two chambers are counted on low-power (10×) objective. Notation of other parasites may be made but not counted because of poor correlation with nematode infection. The count (epg) is calculated by multiplying the total number of eggs in both chambers by 50.

Figure 75-1 McMasters slide for FEC enumeration of nematode eggs, which are counted inside the slide grid areas of the two chambers using a low-power (10×) objective. The count (epg) is calculated by multiplying the total number of eggs counted in both chambers by 50.16.

(Courtesy Eddy Krecek.)

Fecal Larval Culture, Larval Development Assay, and Fecal Egg Count Reduction Test

Treatment strategies may be refined when trichostrongyle species and resistance status are identified in populations. Diagnostic options to consider include the in vitro larval development assay (LDA), which includes fecal larval culture (FLC), species identification, and/or FLC in combination with the fecal egg count reduction test (FECRT). A 2-year investigation of exotic artiodactylid nematode populations in four zoological facilities (three in Florida and one in California) using FLC showed individual, species, exhibit, and seasonal variability in nematode species.²⁰ Nematode species vary in their anatomic location of infection, potential for morbidity and mortality, and response to therapy. FLC testing may further characterize these factors to strategize treatment options better and may be done on individual or herd samples. The LDA and/or FECRT are also critical to your program. Similar to bacterial and fungal monitoring, the LDA may identify nematode populations and determine their resistance levels. Resistance, the ability of nematodes to survive anthelmintic drugs typically effective for the same species and dose, is the most critical problem facing our industry.

Nematodes have great genetic diversity, with a high rate of reproductive potential; Haemonchus contortus females may produce 5000 to 10,000 eggs/day. With this population growth, resistance will be an inevitable consequence of drug selection. Nematodes that survive because of evolved resistance transfer their alleles to the next generation.^12,15 Adding to this issue, animal transfers among conservation institutions permits the dispersal of resistant nematodes among multiple populations. Clinically, we see resistance when the normal therapeutic dose is no longer effective (<95% reduction in FECRT). Unfortunately, drug resistance occurs long before it is detected clinically. Because LDA and FECRT are not traditionally part of parasite monitoring programs, clinical resistance is often discovered too late. Zoo veterinarians should determine the resistance issues in their populations and implement testing during quarantine, preshipment, and routine monitoring program. When resistance is recognized in early stages through testing, anthelmintics may still be used but need to be managed appropriately. The LDA (DrenchRite, Microbial Screening Technologies, New South Wales, Australia) as well as the FLC are not suited for clinical use and may only realistically be performed in a parasitology diagnostic laboratory. A single DrenchRite test may detect resistance to three classes in one assay, including benzimidazoles, levamisole, and avermectin-milbemycin anthelmintics from a single herd sample.¹⁸ The FECRT is an in-house means of determining whether resistance is present. For this test, FEC sampling is performed before and typically 10 to 14 days after treatment on individual animals. It is necessary to perform pretreatment FEC so that treatment efficacy may be balanced against the level of infection. An untreated control group should be included, if possible, to detect other factors that could influence FEC variations. The FECRT is calculated by the following:

An FECRT of less than 95% indicates an incomplete therapy response and is likely a concern for resistance. Monitoring frequency is program-dependent. Consider performing FLC and LDA tests using monthly or bimonthly sampling for the first year to identify areas of concern and then an annual or biannual monitoring program may be adequate to monitor any significant change in population trends. FECRT should be completed after every treatment is performed. Feces should be submitted fresh or refrigerated but not frozen. Check with your laboratory partners to get specifics on sampling and shipping needs for FLC and LDA testing.

Pasture Larval Count

Exhibit populations, forage populations, seasonal changes, and rain accumulation may all influence which nematode populations are present in exhibits. A 2-year investigation of nematode populations in a Florida zoological facility using the pasture larval count (PLC) assay showed exhibit, exhibit region, species, and seasonal variability. This information has proven helpful for developing animal collection and exhibit management, fecal removal schedules, and savannah forage maintenance, including irrigation strategies. PLC is not an in-house test and also requires a partnership with a university parasite laboratory. Sampling and monitoring frequency are program-dependent and may not be critical to your strategic program. If testing is indicated, consider performing monthly or bimonthly sampling for the first year to identify areas of concern. Follow-up annual or biannual testing may be indicated to monitor any significant changes in population trends. Check with your laboratory partners to obtain specifics of sampling and shipping needs.

Drug Treatment

The current drug resistance crisis shows that total reliance on chemical control for parasites is no longer a viable strategy.^12,15 Intelligent use of anthelmintics is necessary because drugs are a valuable limited resource to be used conservatively, not on a rotational basis. Treatment decisions based on the biology and life stages of parasites, dynamics of resistance selection, biology of the host-parasite relationship, and needs of individual patients are critical. This approach, termed smart drenching in the domestic industry, uses the information about parasite, animal, and drugs to maximize effectiveness of treatments while decreasing the development of resistance.²⁷ Drug strategies should be based on current resistance patterns, with consideration of using only one drug class until resistance develops, synergistic use of classes (different modes of action) to enhance efficacy, and restricted use of one class of drugs only for crisis management. Studies in oral dosing in domestic ruminants have shown that the duration of drug availability is dependent on the flow rate of the rumen. With the benzimidazole and avermectin classes, fasting animals 24 hours prior to treatment decreases rumen motility and increases drug availability and efficacy through increased nematode contact. Dosing accuracy to minimize resistance in exotic species may be a challenge with unknown pharmacokinetic data. Additionally, domestic ruminant studies have shown significant differences in dosing in cattle versus small ruminant species. For oral and parenteral anthelmintics, goats metabolize drugs more rapidly with rule of thumb dosages, resulting in dosages that are 1.5 to 2 times higher than those of sheep or cattle. Levamisole, however, has a narrow margin of therapeutic safety and should be used at no more than 1.5 times the dosage. Anthelmintic drugs are typically most effective orally, but moxidectin in goats has shown a superior pharmacokinetic profile with subcutaneous injection, resulting in slower resistance. The bioavailability of pour-ons in domestic nonbovid species is poor and the pharmacokinetics of absorption is highly variable because of differences in follicular density and skin lipid characteristic.¹³ Studies have supported selective target treatment in domestics, with most parasite dispersal in only 20% to 30 % of animals, allowing treatment of animals only if clinically indicated. A standardized scoring system correlating conjunctival color with the level of anemia (FAMACHA; http://www.ars.usda.gov) was developed for the control of H. contortus and has resulted in a significant drop in individual and herd treatments, resulting in delayed resistance. This program requires anemia to be present before treatment is warranted. Animals are scored using the FAMACHA system histogram typically before parasite season and then every 2 to 3 weeks thereafter.¹⁶ Developing a system such as this in our industry would require a large population data set, correlation of anemia with conjunctival color, and standardization among species, which presents challenges.