Internal nematode parasites, specifically the abomasal worm, Haemonchus spp., are a significant health concern in ruminants, domestic and non-domestic, resulting in morbidity and mortality. Parasite control programs in zoological institutions have historically relied on an empirical, rotational drug program. Veterinarians are being challenged with orally, parenterally, and topically medicating a variety of hoofstock species in the face of estimated body weights, marginal compliance of oral medications, and unknown pharmacokinetic data resulting in sub-therapeutic dosing and drug resistance. Now, drugs alone cannot be relied on to control parasites. The exotic hoofstock industry must take heed and look to the future for alternatives. Successful parasite control can be accomplished if holistic control programs are used with resistance prevention in mind integrating diagnostic tools with strategic parasite control focusing on both animal and environment. Programs such as this are being used in the small ruminant industry and can serve as models. Components of these programs include: objective fecal parasite monitoring systems; fecal larval cultures; and/ or in vitro larval development assays looking at drug sensitivity patterns and resistance issues; fecal egg count reduction rate testing; pasture larval counts to identify "hot zones" for strategic environmental control; and non-chemical alternatives to reduce drug selection pressure and resistance issues.

Parasite Monitoring Strategies

Modified McMasters Fecal Egg Count (MMFEC)

Accurate evaluation of worm burdens cannot be determined with subjective assessments of egg loads on fecal exams on a 1+ to 4+ grading scale. Fecal egg counts, such as the MMFEC, are more objective for understanding patterns of infection and shedding, success of parasite management, when programs changes are needed, and if 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. Utilization of spreadsheet technologies can graph trends and establish in-house reference intervals by species or individual to aid in strategic guidelines for monitoring and treatment. Many procedures exist for determining fecal egg counts but the important thing is to use the same procedure each time. The MMFEC, with a sensitivity of 50 eggs per gram (epg), is commonly used because of the quantitative data and simplicity of technique. Laboratory techniques for this procedure can be found at the website for the Southern Consortium for Small Ruminant Parasite Control (SCSRPC) (http://www.scsrpc.org). Trichostrongyle- type eggs (oval shaped, ~ 80-90 microns) are counted in the slide grid. Notation of other parasites can be made but not counted due to poor correlation with worm infection. In domestic small ruminants, counts of greater than 1000 epg are cause for concern.

Fecal Larval Culture (FLC), Larval Development Assay (LDA) & Fecal Egg Count Reduction Test (FECRT)

Treatment strategies can be refined when trichostrongyle species and resistance status are identified in your populations. Diagnostic options to consider include: in vitro larval development assay (LDA) which includes fecal larval culture (FLC) species identification and/or FLC in combination with fecal egg count reduction test (FECRT).

A 2 year investigation of exotic artiodactylid worm populations in four zoological facilities (three in Florida and one in California) using FLC showed individual, species, exhibit, and seasonal variability in worm species. Worm species vary in their anatomic location of infection, potential for morbidity and mortality, and response to therapy. FLC testing can further characterize these factors to better strategize treatment options and can 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 can identify worm populations and determine their resistance levels, the most critical problem facing the parasite control industry. Clinically, we see resistance at the time when normal therapeutic dose is no longer effective (< 95% reduction in FECRT). Because LDA and FECRT are not traditionally part of our parasite monitoring programs, clinical resistance is often discovered too late. The LDA (DrenchRite®, Microbial Screening Technologies, New South Wales, Australia) as well as the FLC are not suited for in-clinic use and can only realistically be performed in a parasitology diagnostic lab. A single DrenchRite ® test can detect resistance to three classes in one assay including benzimidazole, levamisole, and avermectin/ milbemycin anthelmintics from a single herd sample. The FECRT is an in-house means of determining whether resistance is present. For this test, FEC sampling is performed before and typically 10-14 days after treatment on individual animals. It is necessary to perform pre-treatment FEC so that treatment efficacy can be balanced by level of infection.

The FECRT is calculated as: Pre- treatment FEC - post- treatment FEC/ Pre-treatment FEC x100%. FECRT of less than 95% indicates incomplete therapy response and likely a concern for resistance. FECRT should be completed after every treatment is performed. Check with your laboratory partners to get specifics on sampling and shipping needs for FLC and LDA testing.

Pasture Larval Counts (PLC)

Exhibit populations, forage populations, seasonal changes, and rain accumulation can all influence what worm populations are present in exhibits. A two year investigation of worm populations in a Florida zoological facility using PLC showed exhibit, exhibit region, species, and seasonal variability. This information has proven helpful for developing animal collection and exhibit management, fecal removal schedules, savannah forage maintenance including irrigation strategies. PLC is not an in-house test and requires a partnership with a university parasite laboratory as well. Sampling and monitoring frequency is program dependant and may not be critical to your strategic program. Check with your laboratory partners to get specifics on sampling and shipping needs.

Parasite Control Strategies

Drug Treatment Strategies

The drug resistance crisis shows us that total reliance on chemical control for parasites is no longer a viable strategy. Intelligent use of anthelmintics is necessary where drugs are a valuable, limited resource to use 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 knowledge about parasite, animal, and drugs to maximize effectiveness of treatments while decreasing development of resistance. Drug strategies should be based on current resistant patterns with consideration to use 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 show that 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, increases drug availability and efficacy through increased worm contact. Dosing accuracy to minimize resistance in exotic species can be a challenge with unknown pharmacokinetic data with domestic ruminant studies showing 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 that are 1.5-2 times higher than sheep or cattle. Levamisole, however, has a narrow margin of therapeutic safety and should be used 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 non-bovid species is poor and pharmacokinetics of absorption is highly variable due to differences in follicular density and lipid character of skin. Studies have supported target selective treatment in domestics with the majority of parasite dispersal in only 20 - 30 % of animals allowing treatment of animals only if clinically indicated. A standardized scoring system correlating conjunctival color with level of anemia (FAMACHA, http://www.ars.usda.gov) was developed for control of Haemonchus contortus and has resulted in a significant drop in individual and herd treatments resulting in delayed resistance. This program does require anemia before treatment is warranted. Animals are scored using the FAMACHA system histogram typically before parasite season and then every 2-3 weeks thereafter. Developing a system such as this in exotics industry would require a large population data set, correlation of anemia with conjunctival color, and standardization among species.

Animal Management Strategies

Utilization of mixed species exhibits combining primary grazers with higher risk browsing species has shown to reduce worm burdens on susceptible species by reducing grass length and larval exposure while increasing pass-through species and refugia. The term refugia refers to the population that is not under selection by drug treatment and includes untreated animals as well as the eggs and larvae present on the pasture. This "refuge" of susceptible genes dilutes the frequency of resistant alleles. In the domestic animal industry, increasing refugia is recognized as the key animal management strategy to manage drug resistance allowing non-clinical animals to harbor susceptible worms.

Environmental Control Strategies

Good environmental management can play a role in minimizing disease and reducing drug use as well. Factors such as temperatures > 50 F, more than two inches of rain a month, exposure time to exhibit > 8-12 hours, grazing behaviors (< 3 inches from the ground), immune or nutritionally challenged status, and stocking rates greater than 5 animals/acre can negatively influence worm burdens. Environmental management of pastures and exhibits have been challenged in zoological exhibits by other factors such as animal visibility for guest experience, staff time for fecal removal, and limited acreage for animal environment, but compromises can balance these issues. Limiting population densities and decreasing stocking rates on exhibit can be managed through proper collection planning and herd rotations to limit exhibit exposure time. Rotating species on exhibit as well as multi- species populations will increase the refugia population. Providing diet and enrichment items such as elevated browse and grass higher than three inches can minimize larval exposure and stereotypic grazing behaviors reported in browsing species such as giraffe. Parasite transmission is greatly reduced when animals are browsing or grazing high away from the larvae which usually migrate a couple inches up the grass blade. Tillage of exhibit prior to replanting between grazing seasons is another way to reduce larval contamination on exhibits allowing most of the larvae to die buried under soil resulting in reduction of PLC and concentration of infective larvae. Measures such as exhibit forage burning can be considered to kill larvae. Trees, although great for foraging to reduce grazing behaviors, provide shade and humidity causing animals to congregate thus concentrating feces and allowing increased ingestion of infective larvae. Barns and watering areas can also contribute to animal concentration and exhibit plans should take this into account. Fecal removal strategies can be labor intensive and exhibit geography-dependant but can be cost-effective measures with vacuum systems and labor reinforcement. Water control by eliminating water leaks and pooling, controlling irrigation schedules, and removing animal exposure to moist areas can limit larval development and infection rates as well.

Non-Anthelmintic Control and Treatment Strategies

Alternatives to anthelmintics are needed to address resistance concerns in the face of no new products on the horizon. Copper oxide wire particles (COWP), condensed tannin plants, and nematophagous fungi show promise for both animal and environmental control in studies in domestic species. Limited pilot studies and clinical trials in zoological collections also show promise for non-chemical methods. Some alternative control measures have significant research data to show they work, some have limited data that merit further studies, but many of them lack data, especially in exotic artiodactylids. As you trial these methods, monitor animals closely for morbidity through clinical exam and FEC and be aware of potential toxicity issues. We must stay abreast of emerging technology in the domestic field and consider validating these methods in exotic species.

Copper Oxide Wire Particle Therapy (COWP)

The success of COWP in domestic ruminants has been well- documented showing good efficacy against Haemonchus spp. COWP as well as trace mineral boluses with copper reduce FEC by 60-90% for 21-28 days. COWP are retained in the folds of the abomasums with the low pH causing release of high concentrations of soluble copper ions creating an environment that causes death and/or expulsion of worms. The exact mechanism of action is unknown. Worm cuticle damage has been shown in studies likely disrupting metabolic function of the worm through absorption of toxic levels of copper from the blood or abomasal fluids. Copper may also stimulate a local immune response. FEC reductions with COWP are likely reflective of worm kill versus decreased fecundity in the worm population. A COWP study in four species of exotic artiodactylids showed species variability with efficacy of FECRT > 90% by 7 days post- treatment for 3 species and by 21 days for all species. Dosing was based on the manufacturer's recommendation of 12.5g for cattle less than 227kg; but, lower dosages of 0.1- 0.3 mg/kg have been used with similar efficacy in domestic small ruminants. Copper does not appear to affect intestinal worms so FCL is critical before using COWP in your program. Boluses can be made and administered using the commercially available product (Copasure ®, Butler Animal Health Supply, Dublin, OH, 43017-7545, USA) which can be repackaged into doses suitable for smaller species using gelatin capsules. Copper tends to accumulate in the liver, and chronic elevations in copper levels could predispose to hepatic disease and anemia. Until further studies can be completed, limit COWP administration to no more than once every 6-12 months to limit risk of toxicity.

Condensed Tannins (CT)

Tannins are polyphenolic plant compounds that bind proteins and other molecules. There are two main types of tannins: hydrolysable which may have toxic effects on animals, and bioactive condensed tannins found in legumes and other plants. Effects of CT vary depending on type of tannin, forage, CT concentration, and the animal. Domestic studies have shown efficacy with CT concentrations as low as 20-45 g CT/kg DM, 2- 4.5% DM), while high forage CT concentrations (> 55 g CT/kg DM, 5.5%) may have negative effects such as reduced intake and digestibility (Table 1). Positive effects include an increase in by-pass protein, reduction in FEC, parasite numbers, and egg hatchability. Research has shown CT- containing bioactive plants, like sericea (Lespedeza cuneata), are useful in controlling internal parasite infection in sheep and goats. Studies show that a diet fed with 75% pelleted serecia over a 2-4 week period and forage/ hay products over a 7-8 week period results in FEC reductions of 50%. FEC show increases after sericea feeding was stopped indicating an effect on worm fecundity. Pelleting of ground sericea hay increases ease of storage, transport, and feeding with one study showing improved efficacy in goats compared to sericea hay. Pelleted sericea may facilitate the broader use of this forage in our industry with investigations currently underway in exotic artiodactylid collections showing promise. The mechanism of action is not yet known; but, researchers believe that the CT may affect parasites directly (cuticle absorption causing worm dysfunction) and/or indirectly (improving protein nutrition, amino acid absorption and immune system stimulation). CT also appear to reduce the hatching of eggs and larvae development by binding to the larvae, feed nutrients, or preventing bacterial growth in the feces (larva feed on bacteria) and limiting the feed available for larval growth.

Table 1. Condensed Tannin (CT) Content in Different Forage Species Used For Trichostrongyle Control (in press, Zoo. Wild. Anim. Med. Vol. 7)

Nematophagous fungus

Another alternative treatment is a naturally occurring nematode-trapping fungus, Duddingtonia flagrans, which acts as a biological control agent by parasitizing developing worm larvae in feces. The fungus is ubiquitous, found worldwide, and normally present in the feces at low levels. Spores of this fungus can be incorporated into various diet items and upon ingestion they pass unchanged through the digestive tract and concentrate in the feces. After feces are deposited onto the pasture, the spores germinate forming hyphae that are able to trap and kill the developing larval stages. It is primarily used as a preventive with no therapeutic benefits. It is active against free-living larvae with no effect on adult stages in vivo, the eventual effect being reduction in pasture larval numbers and thus reduced reinfection. Studies in domestic ruminants show positive benefits and a study is currently underway in exotic ruminant species. The fungal spores must be fed daily for two weeks to achieve the full benefit, but every other day feeding has shown acceptable activity as well. Spores are fed at a dose of 250-500,000 spores/ kg body weight with larval reduction occurring 7-14 days after treatment starts. The spores are being produced in Australia (International Animal Health Products, Huntington, NSW, 2148, AU) as well as in experimental settings in the US.

Other Treatment Strategies

Other alternative methods have been reported in the domestic small ruminant industry with no clinical trials in exotic species. Parasite vaccines remain an elusive goal and it will likely need further investigations before effective vaccines become commercially available. Protein supplementation in small ruminants may be beneficial as increasing nutrition and amino acid availability could result in immune system stimulation, but application in zoo species already receiving high quality diet with appropriate protein may limit efficacy and increasing amounts further may be a poor nutritional strategy long term. Historically, tobacco and nicotine sulfate have been recommended in for control of parasites, but dosing has not been well established with limited quantitative effects being reported. Nicotine sulfate is a nerve paralyzing toxin with a narrow margin of safety to achieve worm effect (worm release and excretion) without ill effects to host. Diatomaceous earth is fossilized unicellular marine or fresh water algae and is used as a food ingredient and in swimming pool filters. Caution should be used in giving the non-food grade product to animals due to heavy metal contamination. Scientific studies in domestic goat and sheep species combining it with mineral supplements showed minimal effects unless at a very high level in the diet (5% of the diet). It is postulated that this product may cause fecal pellets to dry out faster which could reduce larval development; however, investigations have been inconclusive. Commercially available herbal anthelmintics contain various mixtures of dried plants or plant products (Artemisia absinthium (wormwood), Allium sativum (garlic), Juglans nigra (black walnut), Cucurbita pepo (field pumpkin), Artemisia vulgaris (mugwort), Foeniculum vulgare (fennel), Hyssopus officinalis (hyssop), and Thymus vulgaris (thyme) and limited investigations show no measurable health benefits and have failed to control worm burdens in small ruminant species. Use caution with commercial products as some herbs can be toxic.

References

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