ASSESSMENT OF CHRONIC WASTING DISEASE, MENINGEAL WORM (PARELAPHOSTRONGYLUS TENUIS), AND FLUKE ( MAGNA) IN LARGE UNGULATES AT THE SULLYS HILL NATIONAL GAME PRESERVE

James J. Maskey Jr. and Dr. Rick A. Sweitzer University ofNortll Dakota Grand Forks, ND 58201

February 2004 TABLE OF CONTENTS

I' Page 'I IN"TRODUCTION ...... 1 OBJECTIVES ...... 2 ~ DISEASES EXAMINED ...... 2 Chronic Wasting Disease ...... 2 Parelaphostrongylus tenuis ...... 4 Fascioloides rnagna ...... 9 METHODS ...... 12 White-Tailed Sample Collection ...... 12 Chronic Wasting Disease ...... 13 Examination of Heads for P. tenuis ...... 13 Examination of Fecal Samples ...... 13 Examination of for F. rnagna ...... 13 Terrestrial Gastropods ...... 13 Aquatic Gastropods ...... 16 RESULTS ...... 17 Chronic Wasting Disease ...... 17 P. tenuis ...... 17 l F. rnagna ...... 17 I Terrestrial Gastropods ...... 17 Aquatic Gastropods ...... 18 ~ DISCUSSION...... 19 I Chronic Wasting Disease ...... 19 ~ P. tenuis ...... 19 F. rnagna ...... 24 MANAGEMENT RECOMMENDATIONS ...... 24 General ...... 24 Chronic Wasting Disease ...... 25 P. tenuis ...... 26 F. rnagna ...... 27 LITERATURE CITED ...... 27

111 INTRODUCTION Sullys Hill was established as a national park in 1904. In 1931 it was transferred to the control of the Department of Agriculture in 1931 to be administered as the Sullys Hill National Game Preserve (NGP). The original purpose of Sullys Hill NGP was to preserve (Bos bison), (Cervus elaphus) and white-tailed deer (Odocoileus virginianus) populations, but the mission has since changed to the promotion of wildlife­ oriented education and recreation while still maintaining the identity of a big-game preserve. The preserve consists of 2 units. The big game unit is a 700 acre fenced enclosure contained in Unit I that is inhabited by bison, elk and white-tailed deer. Originally, this area was surrounded by a 7 foot high fence installed in 1916. In 1999, the height of the fence was increased to 12 feet. The bison herd was established in 1918 with the introduction of 2 bulls, 1 cow and 3 female calves from the Portland City Park in Portland, Oregon. Since that time the herd has been periodically supplemented with bulls in order to reduce inbreeding. Bulls have been introduced from Wind Cave National Park, Fort Niobrara National Park, and the National Bison Range. Elk were also introduced to Sullys Hilll NGP in 1917 or 1918 when 15 from Yellowstone National Park were brought to the preserve. Bull elk have also been added to the population over the years as a means of reducing inbreeding. Elk have been introduced from Fort Niobrara and Theodore Roosevelt National Park. Disease testing at Sullys Hill NGP has been conducted in the past, but it has been sporadic. All slaughtered elk and bison were tested for brucellosis in 1983. Results were negative, and the herd was declared brucellosis free. Testing for this disease has not been conducted since. The Fenced Management Plan (Veikly 1984) called for the examination of the lungs of all slaughtered animals for signs associated with bovine tuberculosis, and this disease has apparently not been reported in Sullys Hill NGP. Recently, concerns have developed over the potential of disease to effect the large mammal population at Sullys Hill NGP. In part, this has been the result of the emergence of chronic wasting disease (CWD) in North America, and recently, targeted surveillance for CWD has been conducted (Will Meeks 2003, personal commuication). In addition, since 2001, 7 elk have died of natural causes at Sullys Hill NGP (Cami Dixon 2004, personal communication), and this has led to concerns that other diseases caused by

1 parasites such as brain worm (Parelapkostrongylus tenuis) or (Fascioloides magna) may be negatively impacting the health of large mammals at Sullys Hill NGP. The purpose of this project was to assess the status of disease in the large mammal population at Sullys Hill NGP, and provide recommendations to mitigate the negative effects of diseases or parasites present at Sullys Hill and to prevent the introduction of new pathogens.

OBJECTIVES 1) Determine if Chronic Wasting Disease was present in whit-tailed deer at Sullys Hill National Game Preserve 2) Estimate the prevalence of P. tenuis infection in white-tailed deer 3) Examine the relationship between habitat and intermediate hosts of P. tenuis 4) Determine if the haylot or manure pile areas serve as foci for disease transmission by providing concentrated sources of larval contamination 5) Investigate whether F. magna was present in white-tailed deer 6) Examine the potential for F. magna to be transmitted based on the availability of intermediate hosts 7) Provide management recommendations to help managers address disease issues

DISEASES EXAMINED Chronic Wasting Disease Chronic wasting disease (CWD) belongs to a group of diseases known as transmissible spongiform encephalopathies (TSEs). It is believed that TSEs are caused by proteniaceous agents called prions (Williams et al. 2001). Prions are abnormal proteins that are resistant to protease. Upon entering a susceptible host, the abnormal proteins (PrP res) promote the conversion of normal proteins (PrPc) found in the lymph tissue and central nervous system into PrPres (Williams et al. 2001). Because CWD is fatal to its hosts, it occurrence in a Cervid population has major management implications.

2 Range CWD was first described in 1967 in a captive herd at a Colorado research facility (Williams and Young 1980). It was later discovered in a captive herd of mule deer in Wyoming (Williams and Youg 1980). Infection in captive elk at both facilities was later discovered. CWD was first reported from a free-ranging population in Colorado where infection in wild elk was detected in 1981 (Spraker et al. 1997). CWD has since been found in captive facilities Montana, Colorado, Kansas, Nebraska, Oklahoma, Wisconsin, Minnesota, South Dakota, Alberta, Saskatchewan, and Manitoba. Infection in free-ranging herds has been detected in Colorado, Wyoming, Nebraska, New Mexico, South Dakota, Wisconsin, Illinois, and Saskatchewan (Ver Steeg 2003, Spraker et al. 1997). White-tailed deer, mule deer (Odocoileus hemionus), and elk are the only known to be susceptible to CWD infection (Williams et al. 2001). Transmission and Prevalence The mode of CWD transmission is not precisely known, though it does not appear to be transmitted as a food-borne disease via bone meal or other animal products as is the case with bovine spongiform encephalopathy (a.k.a mad cow disease). The most important mode of transmission appears to be lateral transmission (Miller et al. 2000, Miller et al. 1998, Williams and Young 1992). Because prions persist for long periods in the environment, contamination from an infected animal may potentially infect new animals for a lengthy period. It is thought that lateral transmission is necessary to maintain CWD in free-ranging populations of Cervids (Miller et al. 2000). Maternal transmission is not considered as important in the maintenance of CWD in a population, though it may occur (Williams et al. 2001). Prevalence of CWD infection in endemic areas ranges from 1-8% (Miller et al. 2000). In captive herds prevalence may be much higher. In one herd, over 90% of mule deer present in the herd for 2 years or longer were • infected with CWD (Williams and Young 1980) . Clinical Signs/ Pathogenesis • The clinical signs associated with CWD are slow to develop. The minimum incubation period for the disease is though to be about 1 Yz years. The maximum incubation period is not known, although often the animals in a population showing clinical signs are those that would be considered prime age (Williams et al. 2001 ) .

• 3 • Disease is caused by lesions that result from accumulation of PrPres proteins in the brain. The most common site of protein accumulation is at the parasympathetic vagal nucleus in the medulla, although as prions accumulate, lesions spread to other areas of the brain (Williams and Young 1993). Sign associated with disease are loss of condition, repetitive patterns of movement, reduced feeding, lowered posture of head and ears, drooling, in-coordination, head tremors, and wide stance. Death from CWD is inevitable, and while most animals will survive a few months after the onset of disease, death may occur anywhere from a day to around a year after clinical signs develop (Williams et al 2001). Parelaphostrongylus tenuis P. tenuis (Family: Protostrongylidae, Subfamily: Elaphostrongylinae), the meningeal worm is a parasite that occurs in the dura mater, subdural space, and venous sinuses of the cranium of its definitive hosts (Anderson and Prestwood 1981). It was first described by Dougherty (1945). The normal definitive host for P. tenuis is the white-tailed deer. However, several other ungulate species are also susceptible to infection by this parasite. Life Cycle Deer become infected when they ingest a gastropod infected with third-stage larvae of P. tenuis. Once in the alimentary canal, the larvae exit the gastropod, penetrate the gastrointestinal wall, and cross the peritoneal cavity (Anderson 1965, Anderson 1963). Larvae then follow the spinal nerves to the vertebral canal (Anderson and Strelive 1968). Larvae enter the dorsal horns of the gray matter, develop into subadult worms, and enter the subdural space by 40 days post-infection. Worms continue to mature and migrate anteriorly into the cranium, staying in the subdural space or entering the venous sinuses. Worms mate and eggs are deposited in the veins and traverto the lungs where they hatch into first-stage larvae. These larvae traverse the bronchial tree, are swallowed by the host and are passed out with the feces. The pre-patent period is typically 82-92 days but can be as long as 115 days or more (Samuel et al. 1992, Anderson and Prestwood 1981 ).

4 Range P. tenuis has been reported throughout eastern North America in 31 states and six Canadian provinces (Figure 1). The worm occurs as far west as eastern Saskatchewan and central North Dakota and eastern Oklahoma, and as far north as the limits of the range of white-tailed deer (Wasel and Samuel 2003, Platt 1989, Anderson and Prestwood 1981 , Carpenter et al. 1972,). P. tenuis is limited from northerly expansion beyond where white-tailed deer occur by the lack of a suitable definitive host because transmission cannot be sustained in caribou and populations (Lankester 2000). While P. tenuis is distributed continuously throughout the northern part of the range of white-tailed deer in eastern North America, it is absent from areas of the southeastern United States and has not been reported west of the Great Plains, despite the abundance of white-tailed deer in both of these regions (Comer et al. 1991 , Foreyt and Compton 1991, Kocan et al. 1982, Prestwood et al. 1974, Samuel and Holmes 1974, Bindernagel and Anderson 1972, Prestwood and Smith 1969). Larval sensitivity to environmental conditions or the lack of available intermediate hosts may limit the range of P. tenuis in the Southeast and prevent the expansion of this parasite into western North America (Comer et al. 1991, Shostak and Samuel 1984, Kocan et al. 1982, Samuel and Holmes 1974, Prestwood and Smith 1969). Prevalence Prevalence of infection in white-tailed deer is often greater than 50% (Anderson and Prestwood 1981 ). Annual differences in prevalence have been observed (Peterson et al.1996, Schmitt et al. 1989, Gilbert 1973). These differences are associated with annual differences in environmental conditions such as temperature and rainfall that affect the survival of first-stage larvae and availability of intermediate hosts (Schmitt et al. 1989, Gilbert 1973). The relationship between deer density and prevalence is unclear. Behrend and Witter (1968) and Karns (1967) found that the prevalence of infection was higher in areas of higher deer density, while Gilbert (1973) found prevalence in Maine was highest in an area oflow deer density, and Bogaczyk et al. (1993) found no relation of deer density to prevalence. This suggests that deer density is only one factor that influences P. tenuis prevalence, and prevalence is the result of a combination of deer density and the environmental factors that influence larval survival and gastropod availability.

5 Several studies also examined the relationship of prevalence and host age (Carpenter et al. 1972, Beaudoin et al. 1970, Karns 1967, Anderson 1963). Fawns are often infected early (Gilbert 1973, Behrend and Witter 1968, Karns 1967, Anderson 1963, Degiusti 1963) and it has been suggested that prevalence among fawns can serve as a barometer for the current transmission conditions (Slomke et al. 1995, Bogaczyk et al. 1993, Gamer and Porter 1991). Prevalence of P. tenuis infection increases with white­ tailed deer age, leading some parasitologists to suggest that prevalence is the result of the cumulative probability of infection over multiple seasons of exposure (Bogaczyk et al. 1993, Gamer and Porter 1991, Beaudoin et al. 1970, Karns 1967). McCoy and Nudds (2000) and Slomke et al. (1995) dismiss this hypothesis, however, suggesting that P. tenuis are long-lived and that most deer probably become infected in their first or second season of exposure and remain infected for a long period of time, acquiring few additional worms because of resistance to additional infection. Prevalence has been reported to be higher in does (Gilbert 1973, Prestwood and Smith 1969, Behrend and Witter 1968, Karns 1967, Degiusti 1963). Gilbert (1973) suggested that females may have greater contact with intermediate hosts in the type of cover used when fawning. Other studies have found no differences in prevalence between sexes (Behrend and Witter 1968, Anderson 1963), and Slomke et al. (1995) suggested that this was not the likely mechanism for higher prevalence in females because most females probably become infected before they ever bear fawns. Intensity Intensity of infection is usually low. It typically ranges from one to nine worms (Anderson and Prestwood 1981). Bogazcyk et al. (1993) and Slomke et al. (1995) proposed that resistance to P. tenuis infection may develop, preventing the accumulation of adult worms as deer are repeatedly exposed to P. tenuis. Peterson et al. (1996) and Slomke et al. (1995) reported a lower shedding intensity in older deer and attributed this to the development of an immune response that reduces eggs and larvae, or lowered reproductive output of older worms. Larval shedding intensity also varies seasonally and is greatest from late winter into spring, possibly as a result of winter stress lowering the immune response of white-tailed deer (Peterson et al. 1996).

6 Pathogenesis ofP. tenuis infection There is no evidence that P. tenuis is a significant mortality factor in white-tailed deer, as deer typically tolerate infection very well. The most serious implication of P. tenuis infection in white-tailed deer is lung damage caused by eggs and larvae that may make deer more susceptible to other infections. Prestwood (1970) suggested that animals with mild infections may be more susceptible to accidents and predation. Reichard et al. (2004), however, found that the prevalence of infection of vehicle-killed deer was not greater than that of live deer. P. tenuis causes fatal neurological disease in its accidental hosts, which include several species of wild and domestic ungulates. Neurological disease in accidental hosts is the result of the following factors. Worms are unusually active in the host's neural tissue and tend to coil upon themselves, causing tissue destruction. Also worms fail to leave the neural parenchyma as they develop, and these large worms damage tissue during this prolonged migration. Worms also invade and damage the ependymal canal (Anderson and Prestwood 1981 ). Signs of neurological disease include loss of fear, blindness, holding head to one side, walking aimlessly or in circles, weakness in hindquarters, and paralysis (Olson and Woolf 1978, Carpenter et al. 1973, Anderson 1965). P. tenuis is usually unable to complete its life cycle in its accidental hosts. P. tenuis infection has been experimentally induced or observed naturally in big horn (Ovis canadensis) (Pybus et al. 1996), domestic sheep (Ovis aries) (Anderson and Strelive 1966), pronghorn antelope (Antilocapra americana) (Anderson and Prestwood 1981), (Lama glama)(Baumgartner et al. 1985), domestic (Capra hircus) (Anderson and Strelive 1972), eland (Taurotragus oryx) (Anderson and Prestwood 1981), sable (Hippotragus niger) (Nichols et al. 1986), guanaco (Lama guanacoe) (Brown et al. 1978), bongo (Tragelaphus eurycerus) (Nichols et al. 1986), cow (Yamini et al. 1997), moose (Alces alces) (Anderson 1965), elk (Cervus elaphus)( Samuel et al. 1992, Olsen and Woolf 1978), caribou (Rangifer tarandus) (Dauphine 1975, Anderson and Strelive 1968), mule deer (Odocoileus hemionus hemionus) (Tyler et al. 1980), black-tailed deer (Odocoileus hemionus columbianus) (Nettles et al. 1977), and fallow deer ( dama) (Pybus et al. 1992).

7 , fallow deer, mule deer, black-tailed deer, elk, caribou, and moose are especially susceptible to disease (Raskevitz et al. 1991, Baumgartner et al. 1985, Anderson and Prestwood 1981, Tyler et al. 1980, Olsen and Woolf 1978, Nettles et al. 1977, Carpenter et al. 1973, Anderson and Strelive 1968, Anderson 1965). Infections in wild populations have been observed in elk, caribou and moose, and P. tenuis has been implicated in the decrease or elimination of several populations of these animals in eastern North America (Raskevitz et al. 1991 , Anderson and Prestwood 1981, Gilbert 1974, Carpenter et al. 1973, Behrend and Witter 1968). Elk introductions in areas where P. tenuis is endemic have had varying degrees of success. P. tenuis has not resulted in the elimination of any elk populations, but can be a limiting factor (Raskevitz et al. 1991 , Carpenter et al. 1973). P. tenuis infection has been observed in the Michigan elk herd, but Michigan's elk population does not appear to be limited by P. tenuis (Fay and Stuht 1973, Michigan Department of Natural Resources 2001). P. tenuis infection causes greater fatality in calves than in adults, and the parasite is capable of completing its life cycle in elk, though only small numbers oflarvae are shed (Samuel et al. 1992, Woolf et al. 1977, Anderson et al. 1966). Intermediate hosts ofP. tenuis P. tenuis requires terrestrial gastropods as intermediate hosts. First-stage larvae are found in the mucous coat of deer feces. They are readily washed off, and are subject to drying, solar radiation, and freezing (Shostak and Samuel 1984, Samuel and Holmes 1974, Lankester and Anderson 1968). Large numbers of these larvae can be present in the feces of the definitive host (Peterson et al. 1996). The intermediate host becomes infected while feeding on infected feces. The first-stage larvae actively penetrate the intermediate host or are sometimes ingested by the snail or slug (Platt and Samuel 1984). Within the gastropods, larvae develop to third-stage within 3-4 weeks at summer temperatures (Lankester and Anderson 1968). However, larval development slows or ceases in estivating gastropods (Platt and Samuel 1984, Lankester and Anderson 1968). Several species of pulmonate gastropods are capable of serving as intermediate hosts for P. tenuis. They are alternata, Arion circumspectus, Cochlicopa lubrica, Discus cronkhitei, Discus palatus, Deroceras laeve, D. reticulatum, Haplotrema concavum, Helicina orbicula, thyroidus, Pallifera dorsalis, Phlomycus

8 carolinianus, Polygyrajacksoni, Stenotremafraternum, S. stenotrema, Striatura exigua, Strilobops labrynthica, ova/is, Triodopsis albolabris, T divesta, T multilineata, T notata, T tridentata, Vallonia collisella, Ventridons intertextus, and ' arboreus (Peterson et al. 1996, Beach 1992, Raskevitz et al. 1991 , Rowley et al. 1987, Maze and Johnstone 1986, Kearney and Gilbert 1978, Lankester and Anderson 1968, Anderson 1963). Some species may be more important intermediate hosts than others. On Navy Island, Ontario, Lankester and Anderson (1968) found that the annual species Deroceras laeve was an important intermediate host for P. tenuis because of its abundance and wide distribution. They also found the snail and Z. arboreus were important intermediate hosts. The overall prevalence of infection in gastropods is usually low (0.08-9.0%) (Lankester and Peterson 1996, Pitt and Jordan 1994, Maze and Johnstone 1986, Kearney and Gilbert 1978, Gleich et al. 1977). Despite this low prevalence, it is believed that transmission to deer through accidental ingestion can occur because of the large consumption of food by deer, and because infection can result from a small number of larvae (Lankester and Peterson 1996, Anderson and Prestwood 1981 , Prestwood and Nettles 1977). White-tailed deer become infected with P. tenuis by ingesting gastropods while feeding on vegetation (Anderson 1963). Infected gastropods become available to white­ tailed deer at night or during cool, moist conditions when gastropods emerge from the leaflitter and crawl on vegetation (Anderson 1972). Infected gastropods are commonly found in low, damp forested areas. Fewer infected gastropods are found in grassy areas, but intermediate hosts found in this type of habitat were infected at higher intensities (Lankester and Anderson 1968). They suggested that grassy areas may also be important foci for infection because of their importance as feeding areas for white-tailed deer. McCoy and Nudds (2000) found no evidence that P. tenuis manipulates the behavior of intermediate hosts to make them more susceptible to ingestion by deer, and Lankester and Peterson (1996) calculated that deer could ingest enough gastropods by chance to result in the high prevalence of P. tenuis infection typically observed in white-tailed deer.

9 However, deer may also purposely ingest snails as a source of calcium or protein, although this possibility has not been investigated (Lankester 2000). Fascioloides magna Fascioloides magna is a large trematode that occur in pairs or groups within fibrous capsules in the liver parenchyma of definitive hosts such as white-tailed deer and elk. In addition to its normal definitive hosts, F. magna may infect several other species of ungulates such as bison (Pybus 2000). Life cycle Fluke eggs make their way to the small intestine via the bile collection system and are shed in the feces of the host (Pybus 2000). Eggs may embryonate in moist feces but will only hatch if deposited in aerated water (Campbell 1961). Embryonation takes about 35 days (Swales 1935). Temperatures less than 20 C may retard development and at temperatures greater than 34C eggs will not hatch (Campbell 1961). Eggs hatch into miracidia which swim actively but die within 1-2 days if they are unable to locate a suitable intermediate host ( aquatic snails). If miracidia do encounter a host, they penetrate the tissue of that aquatic snail, undergo development and three generations of asexual reproduction, and leave the snail as cercariae. This process takes from 40-58 days within the snail, and as a result, over 1000 cercaria may be produced from one miracidium. These cercariae encyst on emergent vegetation. Definitive hosts become infected by eating vegetation that contains fluke cysts. Larval F. magna exit the small intestine and move to the host's liver where they migrate through the liver tissue until they become encapsulated (Foreyt et al. 1977, Foreyt and Todd 1976). Range In North America, F. magna is enzootic in the Great Lakes Region, Gulf coast, lower Mississippi region, southern Atlantic seaboard, northern Pacific coast, Rocky Mountain trench, and northern Quebec and Labrador (Pybus 2000). F. magna may occur in wild cervids in North Dakota (Dr. Omer Larsen pers. comm.), but research conducted concurrently with this project has failed to recover liver fluke from moose livers collected during the 2002 and 2003 moose hunting seasons in North Dakota (Maskey 2003, unpublished).

10 Prevalence Prevalence of F. magna infection is influenced by several factors. Prevalence is often greater in areas where hosts congregate for long periods of time or are present at high densities. In an area of the lower Bow Valley in Alberta where elk are present year round, 86% of elk were infected with F. magna (Pybus 2000), and in a walled game park in 100% of elk were infected (Balbo et al. 1987). Prevalence of F. magna infection in ungulate populations can also be influenced by habitat conditions via the influence of habitat on the availability of suitable aquatic gastropod intermediate hosts. For example, prevalence of F. magna infection is often greater in swampy or riparian areas as opposed to upland habitats (Trainer 1969, Mulvey et al. 1991). Prevalence generally increases with age to a point where it tends to level off in older age classes (Flock and Stenton 1969, Foreyt et al 1977, Addison et al. 1988, Lankester and Luttich 1988, Mulvey and Aho 1993). Young of the year animals are usually not infected (Pybus 2000). Prevalence is similar in male and female animals (Pybus 2000). Prevalence in white­ tailed deer is usually no greater than 65-70%. Foreyt (1981) suggested that this may be due to some animals being immune to infection or to ecological separation that is the result of a proportion of the population feeding sparsely on emergent vegetation. Intensity Like many parasites F. magna tends to have an aggregated distribution within the population, with most hosts harboring few flukes and a few hosts bearing heavy infections (Pybus 2000, Mulvey and Aho 1993, Addison et al. 1988, Huot and Beaulieu 1985). Mean intensity in white-tailed deer is often less than 10 flukes, though intensities as high as 20-125 flukes have been observed. In elk as many as 500-560 flukes have been recovered from individual animals (Pybus 2000).

11 Pathogenesis of F. magna infection White-tailed deer and elk are normal hosts for F. magna. Infection in normal hosts can result in significant liver damage as a result of tissue destruction by migrating larvae and encapsulated adults, but unless heavily infected, normal hosts usually survive in relatively good condition, exhibiting few clinical signs (Huot 1989, Torbit et al. 1985, Reimers et al. 1982, Pursglove et al.1977). In dead-end hosts, such as bison, juvenile flukes migrate much more extensively before becoming encapsulated, causing extensive destruction to the liver tissue (Pybus 2000). Extensive fibrosis as a result of the migratory tracts and capsules containing adult flukes can result in damage to 50-90% of the liver and death of the host (Pybus 2000, Aho and Hendrickson 1989, Lankester 1974, Karns 1972). In dead-end hosts, the capsules enveloping adult flukes are much thicker walled than those found in normal hosts and prevent the escape of eggs and the completion of the fluke life-cycle (Pybus 2001). Until recently, F. magna infection had not been viewed as a limiting factor for ungulate populations. However, a long-term decline in moose nwnbers in northwestern Minnesota has been linked to liver fluke infections (Murray et al. 2001). Intermediate Hosts of F. magna Aquatic snails of the genus Lymnaea are considered suitable hosts for F. magna. Snails are typically most abundant in shallow (1-35cm deep) areas of warmer water that have a slightly alkaline pH and light canopy cover (Pybus 2000). Lymnaea species are present in North Dakota (Cvancara 1983)

METHODS White-Tailed Deer Sample Collection In December, 2002, 18 adult white-tailed deer (6 male, 12 female) were culled from the enclosed area of the Sullys Hill NGP by U.S. Fish and Wildlife Service personnel. Heads from 17 (5 male, 12 female) of these deer the liver and a fecal sample from 14 of these deer were collected for disease testing. An additional 30 white-tailed deer fecal pellet groups were collected from the haylot area throughout the winter of 2002-03. All samples were frozen until examination.

12 Chronic Wasting Disease The brainstem and/or retropharyngeal lymphnodes were removed from each head and preserved in 10% formalin. These samples were submitted to the Wyoming State Veterinary Laboratory to be tested for chronic wasting disease. Examination of Heads for P. tenuis Deer heads were examined by first cutting frozen heads sagitally using a butcher's band saw. After allowing heads to thaw 12-16 hours, the cavernous, intercavemous, transverse, and sagittal blood sinuses; surface of the brain; and inner surface of dura mater of each head was examined for adult P. tenuis (Prestwood and Smith 1969). Adult worms recovered were identified using the criteria of Dougherty (1945). Examination of Fecal Samples Each fecal sample collected was divided approximately in half. Half of each fecal sample was screened for P. tenuis larvae using the modified Baermann technique (Georgi 1969). Larval contamination intensity was calculated for each sample as the number of dorsal-spined larvae divided by mass of the fecal sample in grams. In addition, the prevalence of infection by dorsal-spined larvae was determined by dividing the number of samples containing dorsal-spined larvae by the total number of samples collected. The remaining half of each deer fecal sample was screened for the presence of F. magna eggs by the gravity sedimentation technique (Ash and Orihel 1991 ). Prevalence of infection by F. magna was calculated by dividing the number of samples containing F. magna eggs by the total number of samples collected Examination of Livers for F. magna Livers were cut into 2 cm wide slices. These slices were examined for the presence of adult or juvenile F. magna and the signs associated with F. magna infection, e.g. fibrous tissue, migratory tracts, detritus (Lankester 1974). Terrestrial Gastropods Gastropod collection was designed to determine the role of habitat in providing a source of intermediate hosts for P. tenuis and to examine whether the haylot or manure pile areas are serving as foci for transmission by providing a concentrated source of larval contamination. Gastropods were collected using transects consisting often 30 x 30 cm cardboard squares placed Sm apart. Cardboard squares were wetted and covered with

13 clear plastic sheeting to reduce drying. Rocks or sticks were then placed on squares to prevent them from being displaced by wind (Lankester and Peterson 1996). After 48 hours, each cardboard square was checked. All gastropods adhering to cardboard squares or the plastic sheeting were collected and counted. Gastropods from each transect were placed in a plastic container containing wet paper towel. Gastropods were transported to the lab for identification and examination. Sites were re-sampled every two weeks from May - September. Gastropods were identified to the species level using the criteria of Burch (1962). The number of intermediate host species and their relative abundance was determined. In order to examine intermediate host abundance related to habitat type, one transect was placed in each of 5 replicates of woodland and savannah habitat types (Figure 1) (Boag 1982). Abundance of gastropods between habitat types was compared using at-test. Gastropod collection around the haylot area was designed to determine whether this area was serving as a focus for P. tenuis transmission via increased fecal deposition as a result of concentrated deer movements to and from the food source or via larvae being washed off of the haylot area downhill to where they would be able to infect gastropods. Three transects spaced 20 meters apart were placed on the right and left sides of the haylot area, and on the downhill side of the haylot area, an additional four transects were placed at 20 meter intervals until the slope leveled (Figure 2). To determine whether the manure pile serves as a foci for P. tenuis transmission, four transects were placed at 20 meter intervals with the first transect running along the edge of the down hill side of the manure pile and the last transect established where the slope leveled. Three additional transects were placed at 20 meter intervals starting on the opposite side of the manure pile (Figure 2). Collected snails were placed individually in 50 ml test tubes and digested in a solution of 1.0% pepsin and 1.0% HCl for 6-8 hours (Jacques 2001 ). Digests were then placed in a 6 cm petri dish and examined under a dissecting scope for the presence of . Any nematodes recovered were placed in hot ethanol. Nematodes were identified as P. tenuis based on size, morphology, and characteristic C or J shape that 3 rd stage P. tenuis larvae assume after being heat relaxed. Prevalence of P. tenuis infection was calculated for each species of gastropod and for haylot, manure pile, woodland, and

14 savannah transects. The prevalence of infected gastropods collected from the hay lot, manure pile, woodland, and savannah transects was compared using a Fisher' s exact test.

Legend

- - - Roads --- OJerlook Fence i Fence ~=~ l Waterhole ::==~ I Prairie Dog Town ;:::::====; I Savannah == HayPen

.a

135 270 540 810 1,080 - - Meters Figure 1. Approximate locations of terrestrial gastropod sampling transects in Woodland (W) and Savannah (S) habitats and of aquatic gastropod collection sites (P) at Sullys Hill National Game Preserve in summer 2003.

15 oownhill \ \ \ Manure Pile Transects W+EN s Legend

- Hay Pen D Prairie Dog Town Haylot Transects LJ Savannah - Haylot - Manure Pile - Snail Transects 0 37.5 75 150 225 300 Meters • 0

Figure 2. Approximate location and arrangement of terrestrial gastropod sampling transects located around the manure pile and haylot areas at Sullys Hill National Game Preserve in 2003.

Aquatic Gastropods The three areas of open water accessible to elk, deer and bison within the enclosure at Sullys Hill NGP were surveyed for the presence of Lymnaea snails capable of serving as intermediate hosts for F. magna (Figure 1). Once in June and once in July these areas were searched for Lymnaea snails. Snails were collected by dragging a dip net along the bottom of each site in 10 I-meter sweeps spaced approximately 10 meters apart roughly 5 meters from shore. Any vegetation or substrate collected in the dip net was picked through by hand and rinsed through a No. 10 sieve (2mm pore size). Any Lymnaea snails that were recovered were counted and transported to the lab for examination.

16 Snails were examined for F. magna infection by individually placing them into small glass dishes filled with water. The snails were allowed to remain in the dishes over night. The contents of the dishes were then examined for F. magna cercaria. Prevalence • of F. magna infection was calculated for each wetland.

RESULTS Chronic Wasting Disease None of the deer submitted for testing were infected with CWD. P. tenuis Adult P. tenuis were recovered from 58.8% of the deer heads examined (N=l 7; 95% C.I. 32.9-81.6%) examined. Mean intensity was 1.2 worms (95% C.I. 1.0-1.3 worms). Larval P. tenuis were recovered from 78.6% (N=14; 95% C.I. 49.2-95.4%) of fecal samples obtained from culled white-tailed deer. Mean larval shedding intensity for these deer was 1.91 larvae/gram of feces (95% C.I. 1.18-2.55 larvae/gram). Prevalence of P. tenuis larvae in fecal samples collected from the haylot was 30% (N=30; 95% C.I. 14.7-49.4%). Larval shedding intensity for these samples was 2.89 larvae/gram (95% C.I. 1.00- 5.22 larvae/gram). Adult P. tenuis were recovered from 3 deer that were not passing larvae in their feces. Four deer from which no adult worms were recovered were passing P. tenuis larvae in there feces. The estimate of P. tenuis prevalence based on a combination of the recovery of adult and/or larval P. tenuis was 83.3% (95% C.I. 58.6- 96.4%). F.magna No F. magna were recovered from the 14 deer livers that were collected. F. magna eggs were not recovered from any of the 14 fecal samples collected from culled deer or from the 30 fecal samples collected from the haylot. Terrestrial Gastropods Ten species of terrestrial gastropods identified and detected as being present at Sullys Hill NGP based on cardboard transect sampling. Seven of these are known hosts for P. tenuis (Table 1). Gastropods were more abundant in the wooded habitat than in the savannah habitat. No gastropods were collected from savannah transects, while 208 gastropods were collected from woodland transects (P=0.00).

17 Table 1. Summary of data on terrestrial gastropods collected and examined for larvae of meningeal worm (Parelaphostrongylus tenuis) at Sullys Hill National Game Preserve during research in 2003. * Indicates gastropod species that have been identified as known intermediate hosts for the transmission of meningeal worm among Cervids .

• seecies No. collected No. examined No. infected Prevalence{%} Discus cronkhitei* 86 86 3 3.5% Deroceras laeve * 54 49 4 8.2% Succinea ova/is* 65 64 0 0.0% Zonitoides arboreus* 54 54 0 0.0% Cochlicopa lubrica* 30 30 0 0.0% Va/Ionia col/isel/a * 14 14 0 0.0% Vitrina limpida 21 18 0 0.0% Refine/la electrina 2 2 0 0.0% Stenotrema stenotrema * 0 0.0% Euconulus fu/vus 0 0.0% Total 328 319 7 2.2%

Overall, 7 of 319 (2.2%, 95% C.I. 0.9-4.5%) gastropods examined were infected with P. tenuis larvae. P. tenuis larvae were recovered from 4 of 49 (8.2%, 95% C.I. 2.3- 19.6%) Deroceras laeve and 3 of 86 (3.5%, 95% C.I. 0.7-9.9%) Discus chronketei examined (Table 1). None of the 208 gastropods collected from woodland transects were infected. One of 44 (%; 95 % C.I.0.05%-12.03%) gastropods from the haylot area was infected. Six of 67 (9.0%; 95%C.I. 3.35-18.48%) gastropods collected from manure pile area were infected with P. tenuis. Five of 63 gastropods (7 .9%; 95% C.I. 2.62-17 .56 %) from the transects downhill of the manure pile were infected, while 1 of 4 snails (25%; 95% C. I. 0.63-80.59%) collected from the transects uphill of the manure pile was infected. Prevalence of infection in gastropods collected from the manure pile transects did not differ from prevalence of infection in gastropods collected from the hay lot (P=0.241). Aquatic Gastropods A total of 35 aquatic snails of the genus Lymnaea were collected. Ten snails were collected from Pond 1 and 25 snails were collected from Pond 2. Lymnaea snails were not recovered from Pond 3. None of the snails collected were shedding F. magna cercana.

18 • DISCUSSION Chronic Wasting Disease The results of testing done on white-tailed deer from Sullys Hill NGP indicate that CWD is not present in the population. CWD has a long incubation period, the disease does not usually develop until the infected animal is at least 1.5 years old (Williams 2001). Because all deer tested were at least 1.5 years old the likelihood of detecting of CWD should it be present at Sullys Hill NGP deer herd was high. In addition, there is no evidence that CWD is present in North Dakota. CWD has not been reported in captive herds in North Dakota, and no infected animals have been discovered during surveillance of hunter-killed white-tai led and mule deer in 2002 and 2003 (Jacquie Ermer 2002, 2003, personal communication). P. tenuis Prevalence and Intensity The prevalence estimate based on the recovery of adult P. tenuis was greater than other estimates for this area of North Dakota (Wasel and Samuel 2003). However, this estimate and the one based on the recovery of dorsal-spined larvae from deer feces are comparable to those obtained from eastern areas of the range of P. tenuis (Slomke et al. 1995, Bogacyk 1993, Schmitt et al. 1989). The underestimation of P. tenuis prevalence by the recovery of adult worms from the heads of white-tailed deer is likely the result of migration of worms out of the cranium, difficulty of finding worms when there was blood in the brain case (Anderson 1963), or worms being destroyed by the saw blade. As a result, the estimate of infection intensity (1.2 worms) must be considered minimal. The prevalence estimate based on recovery of dorsal-spined larvae from fecal samples may underestimate the true prevalence of infection because this method fails to detect prepatent, single worm, or unisexual infections (Slomke et al. 1995, Bogaczyk et al. 1993, Upshall et al. 1987), or because of seasonal variation in larval shedding. Shedding intensities from the samples collected at Sullys Hill were at the low range of those previously reported (Peterson et al. 1996, Slomke et al. 1995, Pitt and Jordan 1994). Studies have reported that shedding intensity is lowest in the winter, and that infected animals may temporarily cease shedding larvae during this time (Peterson et al. 1996,

19 Slomke et al. 1995). However, prevalence estimates based on fecal samples are a more convenient means of assessing prevalence and more truly reflect the transmission potential of P. tenuis in an area (Slomke et al. 1995). The low shedding intensity in winter may also explain the lower prevalence estimate based on fecal samples collected from the haylot versus that based on the fecal samples from the culled deer. The average mass of the fecal samples collected from the hay lot was less than the average mass of those collected from culled deer. The samples collected from the haylot may not have been large enough to detect infection at the low shedding intensities observed. The combined estimate of P. tenuis prevalence was higher than prevalence estimates based on the recovery of adult worm or first-stage larvae alone. The combined estimate of prevalence is still within the range for other areas where transmission conditions were good (Reichard et al 2004, Slomke et al 1995, Gilbert 1973). In addition, the few studies that have utilized a combined estimate of prevalence have found similar discrepancies between estimates obtained by either the recovery of adult worms from heads or recovery of first-stage larvae from feces versus estimates combining both methods. Reichard et al (2004) estimated prevalence at 90% in Michigan, while Slomke et al. (1995) found an infection prevalence of 84% in Minnesota when combining estimates based on the examination of both heads and feces. Several researchers have proposed that the prevalence of infection of any age class of deer is the result of the cumulative probability of infection over multiple seasons of exposure (Bogaczyk et al. 1993, Garner and Porter 1991, Beaudoin et al. 1970, Karns 1967). McCoy and Nudds (2000) and Slomke et al. (1995) dismiss this hypothesis suggesting that P. tenuis are long-lived and that most deer probably become infected in their first or second season of exposure and remain infected for a long period of time, acquiring few additional worms because of resistance to additional infection. This latter scenario may account for the high prevalence of P. tenuis infection observed in this study, as all but one of the 17 deer examined for which ages were obtained was 2.5 years old or older.

20 Intermediate Hosts Two species of gastropods, D. laeve and D. cronkhitei, appear to be the most important intermediate hosts for P. tenuis at Sullys Hill NGP. Discus laeve was infected at the highest prevalence (8.2%) of the gastropod species collected. Other studies have also found D. laeve to be an important intermediate host (Lankester and Peterson 1996, Kearney and Gilbert 1978, Lankester and Anderson 1968). Lankester and Anderson ( 1968) noted that D. laeve is an annual species with juvenile slugs hatching in late June or early July, maturing, over-wintering, and reproducing before dying the following June or July. This led these authors to suggest that this species would be most important as an intermediate host in spring and early summer because adult D. laeve would have had the longest duration of exposure to P. tenuis. This pattern appears to be occurring at Sullys Hill NGP; all of the infected D. laeve were collected in June, and adult D. laeve became scarce in collections by early July. Discus cronkhitei has been found to be an important intermediate host in several previous studies due to its high relative abundance in the northern part of the white-tailed deer's range and infection prevalence (Boppel 1998, Platt 1989, Maze and Johnstone 1986, Lankester and Peterson 1996, Kearney and Gilbert 1978, Gleich et al 1977). Additionally, this species is available to function in transmission throughout the growing season (Lankester and Peterson 1996). The absence of gastropods in the open savannah habitat of SHNGP was not surprising because previous studies have found that gastropods are rare in meadow habitats especially where the height of vegetation is too short to provide sufficient cover to retain moisture or provide refuge for snails and slugs during hot, dry weather conditions. (Raskevitz et al. 1991 , Kearney and Gilbert 1978, Lankester and Anderson 1968). Gastropods have typically been found to be most abundant in deciduous woodland habitats where shade, leaf litter and woody cover protect snails and slugs from dessication and provide opportunity for estivation during winter (Raskevitz et al. 1991, Kearney and Gilbert 1978, Lankester and Anderson 1968). Factors Contributing to P. tenuis Prevalence The results of this study suggest that there are two main factors contributing to the prevalence of P. tenuis infection observed at Sullys Hill NGP. The first of these is the predominance of woodland habitat ideal for intermediate hosts. Seven species of known

21 intermediate hosts of P. tenuis were recovered, which was comparable to the numbers of intermediate host species recovered in wooded areas of eastern North America where P. tenuis prevalences are comparable to or higher than the prevalence estimated at Sullys Hill NGP. In Michigan's Upper Peninsula where P. tenuis prevalence has been estimated to be between 44-90% (Reichard et al. 2004, Boppel 1998, Schmitt et al. 1989), Boppel (1998) reported 8 species of intermediate hosts. In Pennsylvania, Maze and Johnstone recovered 9 species of intermediate hosts in forested areas where P. tenuis prevalence in white-tailed deer was estimated at 54%. Conversely, concurrent research conducted at the Lonetree Wildlife Management Area (WMA) in central North Dakota, detected only 2 species of intermediate host (D. laeve and S. Ovalis). Natural woodlands are sparse at the Lonetree WMA, and P. tenuis prevalence in deer in this area of North Dakota appears to be very low. The results of this study also suggest that the manure pile and the haylot areas may be serving as foci for infection. Others have suggested that foci for P. tenuis transmission may exist where there is high overlap between white-tailed deer habitat use and intermediate hosts (Lankester and Peterson 1996, Maze and Johnstone 1986, Lankester and Anderson 1968). Lankester and Peterson (1996) reported that prevalence of P. tenuis infection in gastropods was 4 times greater in a white-tailed deer wintering area than it was on deer summer range, and concluded that deer wintering areas may serve as foci for transmission because high deer densities in these areas for several-month periods concentrate larval contamination. At Sullys Hill NGP the haylot and manure pile are in close proximity to each other. Thus, infection foci in these two areas may be the result of heavy larval contamination from concentrated deer movements to and from the haylot. In addition to snails and slugs acquiring infection by feeding on deer feces, it has also been suggested that larvae may wash out of deer feces and remain in the environment to infect intermediate hosts (Lankester and Anderson (1968). Data from the gastropod transects around the manure pile and haylot suggest larvae may be washing from feces in the pile and in the feeding area and infecting snails in the wooded area down slope from these locations. White-tailed deer density at Sullys Hill NGP may also contribute to the high prevalence of P. tenuis infection in deer observed in this study. However, previous

22 studies have found mixed results when comparing prevalence of infection to the number of deer in an area. Behrend and Witter (1968) and Karns (1967) found that the prevalence of infection was higher in areas of higher deer density. Gilbert (1973) noted that P. tenuis prevalence in Maine was highest in an area of low deer density, and concluded that this was due to more favorable transmission conditions in the form of greater precipitation in the low deer density area. Bogaczyk et al. (1993) found no relation of deer density to prevalence and also attributed the results to varying environmental conditions across the area. The area around Sullys Hill NGP (North Dakota hunting unit 2L) has among the highest reported deer densities in the state, yet P. tenuis infection is rare in this area (Wasel and Samuel 2003). This suggests that deer density is only one factor that influences P. tenuis prevalence, and that prevalence is the combined result of environmental factors that influence larval survival and gastropod availability. Therefore, although the density of deer at Sullys Hill NGP is relatively high, the high P. tenuis prevalence in the area may be the result of favorable habitat and environmental conditions for gastropods and P. tenuis larvae, and not deer density per se. P. tenuis Transmission to Elk In order for P. tenuis to constitute a significant mortality factor for elk at Sullys Hill NGP, elk must come into contact with infected intermediate hosts, and elk that are infected with P. tenuis must develop disease. The relatively low number of elk succumbing to P. tenuis infection at Sullys Hill NGP in the past several years versus the high proportion of white-tailed deer that are infected suggests that elk are either less prone to acquiring P. tenuis or that most infected elk develop subclinical infections. The fact that 4 of the 5 elk exhibiting signs consistent with P. tenuis infection were 1.5 years old or less suggests that possibly only younger animals are normally susceptible to P. tenuis infection. Older animals that become exposed may be resistant to the establishment of adult worms or the development of clinical disease, and as a result their exposure would go undetected. Samuel et al. (1992) found that elk exposed to low doses of P. tenuis larvae exhibited no clinical sign or were able to recover from infection. Raskevitz et al. (1991), however, found no evidence that elk were exhibiting subclinical infection, and concluded that the main reason for the persistence of elk in their study area

23 was that elk predominantly used open meadows that were poor habitats for the gastropod intermediate hosts. F.magna F. magna was not detected in white-tailed deer at Sullys Hill NGP. The chance of F. magna naturally spreading to Sullys Hill NGP is minimum because evidence suggests that F. magna is absent or rare in North Dakota. It is thought that the ability of this parasite to disperse naturally from areas where it is enzootic is limited because infected animals shed eggs at a moderate rate, and the short life span of miracidia limits their ability to find snails (Pybus 2000). Therefore, an influx of a high density of infected definitive hosts would be needed to naturally establish F. magna. Nevertheless, F. magna has shown the potential to spread via the translocation of infected animals (Hood et al. 1997), and if F. magna infected elk or deer are introduced to Sullys Hill NGP it is possible that this parasite could become established. Another factor favoring the establishment of F. magna at Sullys Hill NGP via the translocation of infected elk or deer is that Lymnaea snails are present at Sullys Hill NGP and the limited number of open water sources available in the refuge may concentrate animal activity around these water sources and favor F. magna transmission.

MANAGEMENT RECOMMENDATIONS General A handling facility as outlined by the Fenced Animal Management Plan (Veikly 1984) should be constructed. This would allow for serum-based testing on live animals, tonsil biopsies for CWD, and facilitate the tagging and marking of animals. All white-tailed deer, bison, and elk at Sullys Hill NGP should be marked. This can be done inconspicuously using strap tags, ear tattoos, or pit tags that will not detract from the aesthetic experience of the public and will allow better record keeping on individual animals and on overall herd composition. Individual identification will also alleviate concerns of the public and other agencies on the spread of disease from captive herds by providing assurances that all animals at Sullys Hill NGP are accounted for. Marking of animals should be combined with a contingency plan to respond to the escape of any large ungulate from the enclosed area of Sullys Hill NGP. In the event that a large

24 ungulate should escape from the enclosed Sullys Hill NGP, a marking program will allow definitive identification of the escaped animal. This plan should also include procedures for notifying state, tribal, and local agencies of an escape, as well as procedures for recovery of the escaped animal. For example, it may be preferable to destroy any escaped animal because capturing and reintroducing the animal to Sullys Hill NGP may present a risk of disease being introduced to the enclosure. Chronic Wasting Disease The presence of CWD would have serious implications for large mammals at Sullys Hill NGP. As a result of concerns over the spread of CWD, regulations that prohibit the interstate movements of live Cervids are already in place. This eliminates the potential for CWD to be introduced to Sullys Hill NGP from endemic areas in other states. Elk may still be introduced to Sullys Hill NGP from elsewhere in North Dakota, however it has been recommended that the source of animals for translocation be from an area that is documented disease free (U.S. Fish and Wildlife Service 2002). This requires that a sufficient amount of surveillance be conducted in an area to conclude with 99% confidence that CWD prevalence is

25 P. tenuis Concerns over the potential of P. tenuis to impact the health of the elk herd at Sullys Hill NGP should be further investigated. The cavernous, intercavernous, transverse, and sagittal blood sinuses; surface of the brain; and inner surface of dura mater and spinal cord of all elk dying of natural causes should be examined for the presence of P. tenuis. This will allow for determining the number of elk mortalities that are due to P. tenuis infection as well as whether only younger animals are at risk. Elk can be treated with with a pour-on application of ivermectin as a preventative against P. tenuis infection (Foreyt 2001). However, treatment is required every 3 weeks in order to prevent infection (Foreyt 2001 ), and unless it is determined that P. tenuis is a real detriment to the elk herd, it is not advisable to treat elk to prevent infection because of the cost and effort that this measure requires. However, there are other measures that can be taken that may help reduce the rate of P. tenuis transmission to both elk and deer. First, some white-tailed deer fawns should be culled each year. Previous studies have shown that fawns shed P. tenuis larvae at a greater intensity than adult deer (Maskey 2002, Peterson et al. 1996, Slomke et al. 1995), and culling some fawns may reduce the amount of larval deposition. If some deer fawns were removed from the herd in early winter, they could be eliminated as a source of further infection before they had been infected long enough to begin shedding larvae. Culling of these fawns could be incorporated into the existing ungulate management strategies, along with other recommendations that result from the ongoing carrying capacity project at Sullys Hill NGP. Secondly, the manure pile should be relocated to a location where it is not near wooded habitat that would contain intermediate hosts or concentrations of deer and elk activity. This could be accomplished by placing the pile in an open area away from the heavily used trails leading to and from the haylot. Alternatively, a silt fence could be installed on the down slope side of the pile to prevent larvae from washing off the pile into the woods. Finally, the hay feeding area should be relocated to the opposite side of the enclosure. This will place the feeding area at a greater distance from wooded habitat, and will reduce the chance that larvae will wash off the hay pile down slope into wooded habitat. This may also shift at least some of the concentrated deer and elk activity from wooded habitats to more open areas that are currently devoid of intermediate hosts.

26 F.magna Cervids from wild or captive populations where liver fluke is present should not be introduced to Sullys Hill NGP. All known endemic areas of F. magna infection are outside North Dakota, so regulations designed to limit the spread of CWD into the state will also serve to prevent the introduction of F. magna into the state. However, the northeastern edge of North Dakota is in proximity to an area of Minnesota where F. magna is present in wild Cervid populations and has been linked to declines in moose numbers (Murray et al. 2001). It is possible that F. magna may be present in deer and elk in the northeastern part of North Dakota. As a precautionary measure, it may not be advisable to translocate elk or white-tailed deer to Sullys Hill NGP from herds in northeastern North Dakota.

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