Anthelminthic Drug Resistant. Inside This Issue. BEYOND numbers

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1 S P R I N G I s s u e # 2 Anthelminthic Drug Resistant Nematodes in Hses: A Case f Fecal Egg Counts Laura Andrews, DVM, Diplomate ACVP Inside This Issue Anthelminthic Drug Resistant Nematodes in Hse: A Case f Fecal Egg Counts...1 New Test: Fecal Egg Count...7 BEYOND numbers Anthelminthic drug resistance in parasitic nematodes has been repted f all currently available classes of drugs in all livestock species. It is an established and serious problem in sheep and goats, with high prevalence of multi-drug resistant (MDR) strains of Haemonchus conttus, the most imptant pathogenic ovine and caprine nematode, and increasing repts of resistance to me than one drug class in Teladsagia (Ostertagia) circumcincta and Trichostrogylus colubrifmis. Resistance is an emerging problem in cattle, with repts of resistance in Cooperaria spp to all three maj classes of commonly used anthelminthics and resistance to at least two classes of drugs in Haemonchus conttus, Teladsagia (Ostertagia) spp. and Trichostrongylus spp. In hses and other equids, there are numerous published repts of resistance in cyathostomins (small strongyles), now judged to be the most pathogenic parasitic nematode in adult hses, to two of the three maj classes of anthelminthics. Resistance to single doses of benzimidazoles (fenbendazole, oxibendazole, oxfendazole) approaches 100% wldwide. Resistance to tetrahydropyrimidines (pyrantel pamoate and tartrate) is close to 50% in the USA. Early indications of developing cyathostomin resistance to the third maj class of anthelminthics, macrocyclic lactones (ivermectin, moxidectin), also have been repted. Resistance is not limited to cyathostomins. Insusceptibility to pyrantel salts, ivermectin and moxidectin has been repted in Parascaris equum (roundwms), the most pathogenic nematode in foals and hses under two years old. The expanding drug resistant populations of cyathostomins and other nematode parasites limit the number of useful anthelminthic drugs at a time when very few new drugs are being developed. The urgent need to slow down the accumulation of drug resistance gene alleles, limit parasite egg production and resulting pasture contamination and preserve the efficacy of remaining anthelminthic drugs by using them less frequently and me selectively has prompted a revision of thinking about the goals of parasite control in hses. Parasite eradication is no longer considered a realistic and desirable goal. It has been shown that (Continued on page 2) RFPT041202

2 maintaining reasonably large numbers of parasites in refugia slows the development of drug resistance in parasites. The imptance of parasites in refugia (parasites in a population that have not been exposed to a particular anthelminthic drug drug class and are susceptible to it) is discussed in me detail later in this article. Individual hses vary widely in their inherent resistance to parasitic nematodes and egg shedding potential; therefe, evidence-based parasite control strategies should be tailed to the needs of individual hses. Central to newer strategies are the use of two labaty tests: fecal egg counts (FECs) and fecal egg reduction tests (FECRTs). Fecal egg counts identify hses with genetically linked parasite resistance (hses that consistently shed few no nematode eggs in the feces and require less frequent dewming) and hses that are highly susceptible to parasite infestation (hses that consistently shed high numbers of eggs and require me frequent and targeted treatment). Fecal egg reduction tests (FECRTs) help identify anthelminthic drug resistance in the parasitic population on a given farm, guide selection of appropriate anthelminthic drugs and optimize dosing intervals f resident hses. To understand why parasite resistance to anthelminthic drugs has become a problem and to avoid repeating past mistakes when developing parasite control plans, it is imptant to understand how dosing regimens used in the past contributed to and accelerated drug resistance. Histical Background The 6-8 week fixed interval dosing schedule recommended f benzamidazole treatment of hses in the 1960s was iginally designed to eradicate large strongyles, particularly Strongylus vulgaris, a nematode with a relatively long minimum prepatent period of six months and a protracted migraty larval stage outside the GI tract, including migration through the cranial mesenteric artery, leading to verminous colic and death in a significant number of infested hses. The frequent administration of benzimidazole drugs greatly reduced clinical disease and death caused by S. vulgaris, which was considered the most pathogenic gastrointestinal nematode in hses at that time. After introduction in 1983 of the macrocyclic lactone ivermectin, a drug that is highly effective against both adult and migrating larval stages of Strongylus spp., large strongyles became increasingly less common; however, the sht, fixed interval dosing schedule that nearly eliminated Strongylus vulgaris faved the proliferation of cyathostomins (small strongyles). By the early to middle 1980s, cyathostomins accounted f nearly 100% of the nematode eggs detected in fecal samples of grazing hses. Cyathostomins have several biological characteristics faving mutations that lead to drug resistance: high intraspecies genetic diversity, very high fecundity, a large population size and a relatively sht, direct life cycle of 5-6 weeks under favable conditions. Cyathostomins also have a high rate of gene dissemination (gene flow) due to movement of the host from location to location f purchase and sale, breeding, training, showing, racing, etc. As different classes of anthelminthics with differing mechanisms of action became commercially available, the sht interval, rapid rotation treatment regimens recommended f parasite eradication led to selection f cyathostomins that were genetically resistant first to benzamidazoles then to tetrahydropyrimidines. The role of mutations in parasites Because mutations causing drug resistance confer a significant survival advantage to affected nematodes (and have no known deleterious effects), these mutations are well conserved by the parasite from generation to generation and considered to be permanent. Recovery of susceptibility to a given drug class does not appear to occur in a resistant population of nematodes. Once drug resistance is found in a resident parasite population, drugs of that class should no longer be used as single agents in hses exposed to that population. In addition to excessively frequent administration of anthelminthic drugs, the likelihood of parasite resistance is increased by dewming during periods when pasture infectivity is low, using po quality, inadequately sted expired drugs and administering doses that are too low f the body weight of the hse. Treatment of hses with daily low dose pyrantal tartrate fmulations is suspected of hastening parasite resistance to pyrantel salts in the USA, one of the few places in the wld where continuous daily dosing is available and popular. While 2 (Continued on page 3)

3 dewming at rigid, fixed intervals that igne the biology of the wm increases the risk of mutations that cause drug resistance, strategic dewming timed to crespond to peak numbers of infectious larvae on pasture reduces that risk. Life cycle of cyathostomins (small strongyles) and influence of climate In cyathostomins, eggs passed in the feces hatch when temperatures range from o F. The first stage larvae (L1) molt sequentially into second stage larvae ( L2), then mobile, infective third stage larvae (L3) that leave the fecal droppings, following moisture trails to crawl up blades of grass where they are consumed by the hse. The rate of larval development is directly proptional to ambient temperature: lower temperatures slow development, while higher temperatures speed it up. Freezing temperatures usually kill eggs, but not infective third stage larvae. Although eggs hatch quickly at temperatures above 85 o F, the resulting L1 die quickly. In warm, but not hot weather, larvae can develop rapidly, becoming infective in as few as three days. The infective L3 are protected by a chitinous sheath that prevents desiccation. Because the sheath lacks an al opening, the L3 larvae survive on sted energy. In hot, dry weather, the energy stes are quickly depleted, and the larvae die. Third stage larvae survive well in cool freezing conditions, and L3 present on pastures in the fall can survive until the following spring early summer. Hses become infested by ingesting L3 while grazing infected pastures drinking contaminated water. In cold climate zones of Nth America, hses are at highest risk of parasitism in March/April through September/October. In hot climate zones, September/October through March/April is the most dangerous time f transmission. Early mning and evening, when dew is on pasture grass and hses are grazing, are the main transmission periods. The number of infective larvae increases after rain; the increase can be dramatic when rain follows a period of hot, dry weather. After ingestion, the L3 migrate to and encyst in the mucosa and submucosa of the cecum and ventral colon. While encysted, the L3 can molt to a stage 4 persist in a state of arrested development (hypobiosis) f a period of time that can be sht as long as 2-3 years. The number of encysted larvae at any given time generally far exceeds the number of adults in the lumen of the bowel. While the larvae are encysted, they are protected from most anthelminthic drugs by the fibrous cyst wall; of currently available drugs, only fenbendazole at a dose of 10 mg/kg f 5 consecutive days and moxidectin penetrate the parasite cyst at concentrations high enough to kill significant numbers of encysted larvae. The emergence of encysted larvae, migration to the lumen of the large bowel and maturation to the sexually mature adult wm are largely dependent on the number of adult wms already present in the bowel lumen; high numbers of adults in the lumen suppress larval emergence. Near-simultaneous emergence of massive numbers of encysted stage 4 larvae causes a syndrome known as larval cyathostomiasis in which emerging larvae cause locally intense hemrhage, edema and inflammation of the mucosa and submucosa of the cecum and colon, weight loss, diarrhea, colic, and sometimes intestinal infarction, with a mtality rate up to 50%. Cyathostomiasis occurs most often in the winter and during hot, dry periods when older adult wms die off. The syndrome also can occur after dewming of hses harbing high numbers of encysted larvae. Imptance of the parasites in refugia In addition to the climate regulated variation in the number of infectious larvae on pasture, the most imptant parasite characteristic to exploit f optimal parasite control is the parasite refugia. The refugia is defined as the subset of parasites in a given population that is not exposed to a specific anthelminthic drug class. In cyathostomins, the refugia comprises the free-living (L1-L3) larvae and, f most drugs, the encysted larvae in the wall of the cecum and colon. It also includes susceptible parasites in resistant hses (low shedders) and hses that have not been exposed to drugs of the same class. The refugia provide a source of non-resistant (susceptible) gene alleles to dilute the resistant (non-susceptible) alleles in the parasite population. This dilution decreases the rate at which resistance develops. Anthelminthic treatment (Continued on page 4) Spring

4 of hses on a given farm should be avoided when the refugia is small. Treatment at that time increases selection pressure f resistance in the resident parasite population. In nthern climates of Nth America with cold winters, the refugia is smallest in the winter when the rate of larval development is slow ( ceases completely). In southern climates with hot summers, the refugia is smallest in the summer when heat decreases the survival of larvae on pasture. Fecal egg counts The most imptant host characteristic to consider when designing a dewming program is host resistance, which is largely genetically determined and inherent. Host resistance has been shown to be remarkably constant over the lifetime of an individual hse. Humal and cell mediated immunity also contribute to host resistance. While acquired host resistance is imptant in controlling Parascaris equum, acquired immunity to Cyathostomins develops slowly and incompletely. Host resistance is determined by the fecal egg count (FEC), a procedure in which the numbers and types of parasite eggs per gram of feces (EPG) are determined by the McMasters other similar quantitative technique that uses specialized slides with two calibrated counting chambers. In an average herd of hses, 80% me of all parasites will be harbed by 20-25% of the hses, the high egg shedders ( high contaminats). The high egg shedders (hses with over 500 EPG on a fecal egg count) are responsible f most of the pasture contamination. Approximately 50% of the animals in the herd will harb low numbers of parasites and shed no low numbers of parasite eggs (less than 200 EPG). The remaining hses will be intermediate between the two groups ( EPG). Fecal egg counts are used to decide how often dewming should be done in an individual hse. Parasite resistant hses should be dewmed as infrequently as possible. Because the cyathostomin life cycle includes an encysted stage of sexually immature larvae, a low fecal egg count at a single time point does not necessarily mean that the hse being tested has a low parasite burden. Fecal egg reduction tests Fecal egg reduction tests (FECRTs) are used to determine if resistant parasites are present in the population on a given farm. In the FECRT, the fecal egg counts are first determined f a variety of hses on the premises that are at least three years old. The initial fecal egg count should be done at least 6-8 weeks after the last treatment with anthelminthic drugs of the benzimidazole and pyrimidine classes (fenbendazole and pyrantel) and weeks after treatment with a macrocyclic lactone drug (ivermectin, moxidectin). Hses are then treated with a specific anthelminthic drug, and the FEC is repeated days later. Only hses classified as high moderate shedders should be included in the group means in fmal calculations. FEC reduction of less than 95% is considered evidence of resistance to ivermectin and moxidectin, while FEC reduction of less than 90% is considered evidence of resistance to fenbendazole pyrantel pamoate. If parasites are resistant to a particular drug class, drugs of that class should not be used again as single agents in that population. The drugs can still be used concurrently with drugs of a different class to treat non-strongyle parasites. Some drugs also can be used at higher doses in a repeat dose regimen. Despite widespread cyathostomin resistance to single doses of fenbendazole, the drug maintains effectiveness in most populations when given at two times the usual single dose f five consecutive days. Use of FECs and FECRTs to design parasite control programs Veterinary parasitologists have recommended that adult hses classified as low shedders be dewmed on a minimum maintenance schedule, two times a year, at the beginning and end of the period of highest exposure to infective parasites on pasture. The twice a year treatment with a larvicidal drug effective against large strongyles, parasites with a minimum prepatent period of six months, maintains near-eradication of that parasite. Moderate shedders ( EPG) should receive an additional anthelminthic treatment during the time of highest risk (a total of three treatments). High shedders (over 500 EPG) should receive at least two additional treatments (at least four total), two of them about 8-12 weeks apart during the period 4 (Continued on page 5)

5 of highest parasite exposure. The following table (modified from Reinemeyer, 2009) is an example of a dosing schedule that should effectively control large and small strongyles in adult hses. It incpates the shedding status of each hse and capitalizes on the life cycle of cyathostomes in nthern and southern climate zones of Nth America. The schedule includes praziquantel treatment f Anoplocephala (tapewms) and boticidal treatments at appropriate times and intervals. The drugs used also control Strongyloides westeri (threadwms) and Oxyuris equi (pinwms). Table 1. Example of a Selective Anthelminthic Program f Control of Strongyles in Adult Hses in Nth America (table adapted from Reinemeyer, C. R. (2009) Egg Shedding Status (Eggs per gram) Months (Maj Transmission Season)* Months (Min Transmission Season) /0 LOW (<200 EPG) (FEC) (FEC) MODERATE ( EPG) BZD/PYR BZD/PYR (FECR) (FEC) (FEC) (FEC) (FECR) BZD/PYR IV MX HIGH (>500 EPG) IV BZD/PYR (FECR) (FEC) (FEC) (FEC) (FECR) Table Legend * Month 0 = March/April f nthern climate zones and September/October in southern climate zones. IV = ivermectin; MX = moxidectin; BZD = a benzamidazole such as fenbendazole; PYR = pyrantel pamoate; P = praziquantal. BZD/PYR: A benzamidazole drug pyrantel pamoate can be used if effective; both drugs can be used concurrently if either drug is effective. The selection of drugs shown in the table assumes that FECRTs have been perfmed previously to identify resistance Boticidal treatment is indicated in Month 2 in Southern temperate climate zones (ST) and Month 8 in Nthern temperate zones (NT). Parasitologists have recommended that newly arrived hses that are staying f longer that six weeks on a farm be treated f encysted larvae using either moxidectin five daily doses of fenbendazol at 10 mg/kg/day. If fenbendazol is used, a single dose of ivermectin moxidectin should be given after the fifth dose. F hses staying less than six weeks, a single dose of ivermectin should be sufficient. New hses should be quarantined f 96 hours befe they are turned out to pasture. (Continued on page 6) Spring

6 Treatment of periparturient mares with a drug effective against encysted larvae has been recommended to minimize foal exposure to parasitic nematodes. Treatment with Moxidectin shtly befe foaling reduces egg shedding f about 12 weeks. An optimal treatment strategy f foals is difficult to design because fecal egg counts do not accurately reflect wm burden in young hses, and emerging parasite resistance to pyrantel salts, ivermectin and moxidectin in some populations of Parascaris equum may limit the choice of drugs necessitate use of me than one drug concurrently. Parascaris equum is the most imptant GI parasite in foals and juvenile hses followed by cyathostomins. As with cyathostomins, the larval stages of Strongylus vulgaris are highly pathogenic in young hses. Although large strongyles are far less prevalent today compared to past years, it is imptant to design parasite control programs to minimize their numbers. Complicating parasite control in foals and juvenile hses is the shter egg reappearance period (ERP) in young hses compared to adults. The egg reappearance period ERP is the interval between drug treatment and return of fecal egg counts to levels that are 80% of the fecal egg count reduction (FECR) f that drug. In non-resistant parasite populations of adult hses, the ERP is 4 weeks f fenbendazole, oxibendazole and pyrantel salts, 6-8 weeks f ivermectin and 12 weeks f moxidectin. The ERPs in juvenile hse are 25-40% shter. To ensure effectiveness of anthelminthic control programs in foals and avoid treating too often too infrequently, it is imptant to determine the FECR f each drug used and closely monit ERPs. Most parasitologists recommend that treatment of foals begin when the foal is two months old and continue at intervals two months apart ( at intervals determined by FECR and ERP). Young hses should receive ivermectin moxidectin at intervals of six months to control Strongylus vulgaris. Larvicidal treatment to minimize the burden of encysted cyathostomins (moxidectin fenbenazole given at 10 mg/kg f 5 consecutive days) is recommended during the late summer/early fall late winter/early spring f foals and juvenile hses that are between six months and two years old. Nonchemical wm control The inevitability of parasite drug resistance to existing drugs and the expanding populations of resistant parasites emphasize that anthelminthic drugs cannot be used as the sole means of parasite control. Nonchemical means of reducing parasite exposure should be employed as often as possible. Pasture management plays an imptant role in reducing parasite transmission. The pastures used f grazing should be large enough f the number of hses, generally at least 1-3 acres per hse in non-arid, temperate climate zones. Overgrazing greatly increases exposure to infective parasites and decreases fage quality. Pasture rotation can be used on larger premises. Pasture turnout after dew is dry in the mning and befe dew settles on grass in the evening reduces parasite exposure at times when infective larvae are most numerous on grass blades. Frequent removal of manure, while impractical in large pastures, can be attempted in smaller pastures with low numbers of hses. Frequent manure removal from barns and paddocks is a necessary management practice. In nthern climate zones of the USA, pastures that are not grazed by hses f three months starting with the onset of warmer weather in the spring become relatively parasite-free. Pastures should not be harrowed when hses are on them and should be free of hses f at least four weeks after harrowing. Harrowing can be done during hot dry weather in southern climate zones and at the end of the grazing season in cold climates. Harrowing in the spring and fall should be avoided because moist weather prolongs parasite survival. Hse manure should only be spread on hse pastures after it has been adequately composted at temperatures high enough to kill parasites (at least 90 o F f two weeks). Concurrent sequential rotational grazing of hses with unrelated livestock species (small ruminants, cattle, camellids) reduces the number of contaminating parasites from all species. Finally, keeping pastures mowed to reduce weeds decreases large rough areas where hses preferentially defecate and parasite contamination is high. 6 (Continued on page 7)

7 Marshfield Labs Now Offers Fecal Egg Count Test code: VFEGG Specimen: grams fresh feces in a sterile container. Minimum: 5 grams. Stage: Room temperature. Refrigerate if kept f me than 6 hours. Cause of Rejection: Specimens submitted in fmalin volume <5 gm. Availability: Set up daily with results available in 1-2 days. Method: Manual Egg Count using McMaster Slide Chamber. References: 1. Kaplan, R. N. (2004) Drug Resistance in nematodes of veterinary imptance: A status rept. Trends in Parasitology, 20 (10): Fleming, S.A., et al (2006) ACVIM Consensus Statement: Anthelminthic resistance of gastrointestinal parasites in small ruminants. J. Vet. Intern. Med., 20: Kaplan, R. N., et al (2004) Prevalence of anthelminthic resistant cyathostomes on hse farms. J. Am. Vet. Med. Assoc., 22 (55): Traversa, D. (2009) Field survey on the efficacy of four anthelminthic drugs against hse cyathostomin infection in Europe. AAEP Proceedings, 55: Kaplan, R. N. (2002) Anthelminthic resistance in nematodes of hses. Vet. Research, 33: Reinemeyer C. R. (2009) Controlling strongyle parasites of hses: A mandate f change. AAEP Proceedings, 55: Shulaw, W. (2008) Fecal egg counts: What do they tell us? Http: //sheep.osu.edu/2008/06/19/ fecal egg counts: what do they tell us? (iginally published in Sheep Team Newsletter, Sept. 2004). 8. Nielsen, M. K. and Kaplan, R. M. (2008) Evidence based equine parasitology: It ain t the 60s anyme. Proceedings des 36 emes Journees Annuelles de l Association Veterinaire Equine Francaise. Parasitologie. 36: Cobb, R. and Boeckh, A. (2009)Moxidectin: A review of chemistry, pharmacokinetics and use in hses. Parasites and Vects, 2 (suppl 2): S Reinemeyer, C. R. (2009) Diagnosis and control of anthelminthic resistant Parascaris equum. Parasites and Vects, 2 (suppl 2): S Ihler, C. F. (2010) Anthelminthic resistance: An overview of the situation in Ndic countries. Acta Veterinaria Scandinavica, 52 (suppl 1): S Steinbach, T. et al (2006) Small strongyle infection: Consequences of larvacidal treatment of hses with fenbendazole and moxidectin. Veterinary Parasitology, 139: Spring