ALCES VOL. 47, 2011 MASKEY - LIVER FLUKE IN NORTH DAKOTA MOOSE 1 GIANT LIVER FLUKE IN NORTH DAKOTA MOOSE James J. Maskey, Jr.1 Department of Biology, 10 Cornell Street, Stop 9019, University of North Dakota, Grand Forks, ND 58202, USA ABSTRACT: The giant liver fluke (Fascioloides magna) is a parasite of white-tailed deer (Odocoileus virginianus) and wapiti (Cervus elaphus) that can cause extensive and conspicuous liver damage in moose (Alces alces), a dead-end host. The implication of F. magna as a factor in the long-term decline of moose in northwestern Minnesota has raised concern that a concurrent decline in moose in northeastern North Dakota may also be linked to this parasite. I reviewed data collected from moose hunter check stations in1977-1984 and necropsy reports of non-harvested animals examined in 1983-1992 to estimate past prevalence of F. magna in moose in North Dakota. I also collected livers from harvested moose in 2002 and 2003 to investigate the current occurrence of this parasite. I also surveyed 78 wetlands at 12 sites in 2003-2006 to examine the potential for F. magna transmission based on the occurrence of aquatic snail intermediate hosts. Flukes or signs consistent with fluke infection were observed in 19.6% of harvested moose (n = 158) in 1977-1984, and in 18.8% of moose necropsied (n = 32) in 1983-1992. Fascioloides magna was not recovered from any of the 78 moose livers collected in 2002 and 2003. However, lymnaeid snails were found at 10 of 12 sites in the aquatic gastropod surveys indicating that the intermediate hosts for this parasite occur widely throughout the range of moose in North Dakota. While this represents the first known report of F. magna in North Dakota, this parasite occurs at relatively low prevalence, and there is no evidence that it has been an important factor in recent moose declines, nor that it noticeably impairs the health of moose in North Dakota. Transmission may be limited by the transient availability of wetlands capable of supporting the life cycle of F. magna. ALCES VOL. 47: 1-7 (2011) Key words: Alces alces, Fascioloides magna, intermediate hosts, lymnaeid snails, moose, parasite, population decline, prevalence. White-tailed deer (Odocoileus virgin- ianus) are the normal host for 2 parasites that may cause fatal disease in moose (Alces alces). The best-known of these is the men- ingeal worm (Parelaphostrongylus tenuis), a nematode long implicated as a limiting factor of moose populations (Lankester 2001, 2010). The other is the giant liver fluke (Fascioloides magna), a large trematode that occurs in pairs or groups within fibrous capsules in the liver parenchyma of its normal hosts, white-tailed deer and wapiti (Cervus elaphus) (Pybus 2001). Fascioloides magna has an indirect life cycle, requiring aquatic snails in the fam- ily Lymnaeidae (hereafter lymnaeid snails) to serve as intermediate hosts (Pybus 2001). In dead-end hosts such as moose, juvenile flukes migrate much more extensively than in normal hosts before becoming encapsulated, and as a result, cause considerable destruction of liver tissue. Extensive fibrosis of the migratory tracts and capsules containing adult flukes can damage 50-90% of the liver, and sometimes be suspected of causing death of the host (Pybus 2001). Recently, F. magna was implicated in the long-term decline of moose in northwest- ern Minnesota (Fig. 1) where 89% of moose examined in 1995-2000 were infected with F. magna (Murray et al. 2006). The North Dakota Game and Fish Depart- ment (NDGF) conducts annual winter aerial surveys of moose populations in 3 survey areas (Turtle Mountains, Drift Prairie, and Pembina Hills; Fig. 1). Survey data collected 1Present address: Department of Biology, University of Mary, 7500 University Drive, Bismarck, ND 58504 LIVER FLUKE IN NORTH DAKOTA MOOSE - MASKEY ALCES VOL. 47, 2011 2 over the past decade indicate that while moose popula- tions appear to be stable to increasing in the Turtle Mountains and Drift Prairie areas, moose have declined considerably in the Pembina Hills (Johnson 2002, 2007; Fig. 2). During this same period, white-tailed deer in the state have increased considerably, suggesting in- creased transmission poten- tial of deer parasites (namely F. magna and P. tenuis) to moose (Jensen 2007, Smith et al. 2007). Additionally, because the Pembina Hills area is adjacent to the de- clining moose population in northwestern Minnesota, concern existed that the North Dakota decline also may be related to F. magna infection. This study addressed this concern by 1) examining historical data to estimate past prevalence of this parasite in the moose in North Dakota, 2) investigating the current occurrence of F. magna infection in moose, and 3) determining whether suitable intermediate hosts for this parasite occur in the state. METHODS To estimate the historical prevalence of F. magna in North Dakota moose, I reviewed 2 data sets collected previously by NDGF. The first data set consisted of hunter check-station records for 158 moose harvested in 1977-1984. During these first 8 years of the moose sea- son, hunters were encouraged to bring entire carcasses to check stations where the animals were weighed and the viscera examined to as- sess reproductive status and parasitic infection. The second data set was historical necropsy reports of non-hunting related deaths. These included full necropsies on 32 such moose conducted by the NDGF wildlife veterinarian in 1983-1992 as part of targeted surveillance for wildlife diseases. I reviewed check station data sheets and necropsy reports for evidence of liver fluke infection based on the recovery of flukes from liver tissue or comments in reports that sug- gested fluke infection including unspecified cysts or capsules in the liver, fibrous areas, mi- gratory tracts, detritus, necrosis, congestion or “bad” or “questionable” livers. Additionally, examination of the necropsy reports from tar- geted surveillance allowed me to compare the relative frequency of F. magna infection with that of other pathogens. A Clopper-Pearson bi- nomial confidence interval was calculated for the historical estimate of F. magna prevalence obtained from the check station and necropsy data (Rosza et al. 2000). In addition, I estimated the current occur- rence of F. magna infection in moose by exam- ining 78 moose livers collected from hunters during the 2002 and 2003 moose seasons. Livers were sectioned into approximately 2-cm wide slices and examined for the presence of adult or juvenile F. magna and signs associated Fig. 1. Moose aerial survey units in North Dakota (Turtle Mountains, Drift Prairie, and Pembina Hills) and study areas of Murray et al. (2006) in adjacent northeastern Minnesota (Agassiz National Wildlife Refuge, Red Lake Wildlife Management Area, Thief Lake Wildlife Management Area, Beltrami Island State Forest). ALCES VOL. 47, 2011 MASKEY - LIVER FLUKE IN NORTH DAKOTA MOOSE 3 with F. magna infection such as fibrous tissue, migratory tracts, detritus, liver necrosis, and congestion (Lankester 1974). I also investigated the occurrence of intermediate hosts for F. magna by sampling permanent and semi-permanent wetlands, small lakes, and streams for lymnaeid snails during 4 summer periods (2003-2006). I sampled for the presence of lymnaeid gas- tropods in 78 wetlands that included small lakes and streams at 12 sites (11 in northeastern North Dakota, 1 in northwestern Minnesota; Fig. 3). Each site was sampled by a series of 10 1-m sweeps with a dip net approximately every 10 m within 1-2 m of shore. Af- ter each sweep the contents of the net were examined for aquatic gastropods; lymnaeid snails observed floating on the surface were collected opportunistically. Snails were placed in 70% ethanol or frozen, and were subsequently identified to species using the criteria of Clarke (1973) and Cvancara (1983). RESULTS Based on the review of check station records and necropsy reports, the past preva- lence of F. magna infection in North Dakota moose during the period 1977-1992 was 19.5% (95% C.I., 14.1-25.8%, n = 190). There was evidence of F. magna infection in 31 of 158 (19.6%) harvested moose (Table 1). Liver flukes were recovered from 18 (11.4%) of these moose, while signs suggesting possible F. magna infection were observed in the re- mainder (8.2%; 6 with unspecified cysts, 5 with bad livers, 2 with fibrous tracts). Only the northeastern area of North Dakota (Unit M1C, Fig. 3) was open to moose hunting from 1977-1982, thus 138 of 158 harvest samples originated from this area. In M1C, liver flukes were recovered from 16 (11.6%) moose, and signs consistent with F. magna infection were observed in the remainder (9.4%). Hunting for moose was initiated in Units M4-M10 in 1983; liver flukes were recovered from 2 of 20 moose harvested in Units M4-M10 in 1983 and 1984 (Table 1). Six of the 32 (18.8%) non-hunting related fatalities (1983-1992) exhibited pathology 0 50 100 150 200 250 300 Drift Prairie Turtle Mountains Pembina Hills Fig. 2. Number of moose observed within 3 survey units in North Dakota by the North Dakota Game and Fish Department during winter 1980-2006. Winter surveys were not completed in the Drift Prairie area prior to 1987. Data are from Johnson (2002, 2007). Fig. 3. North Dakota moose hunting units (M1-10) and sites sampled for lymnaeid snails (● = lymnaeids present; o = absent). Numerals represent number of wetlands from which snails were recovered/ number of wetlands sampled at each site. LIVER FLUKE IN NORTH DAKOTA MOOSE - MASKEY ALCES VOL. 47, 2011 4 suggesting F. magna infection (2 with liver congestion, 1 with liver unspecified infection, 1 with fibrosis of the liver, and 1 with fibrous capsules; Table 1). Only a single moose was believed to have died as a result of F. magna based on the amount of liver damage caused by the fluke infection. Flukes or signs of infection were not seen in any of 78 moose livers collected in 2002 and 2003. The moose hunting unit of origin was known for 56 of the samples; however, unit of origin was not available for 22 samples (Table 1). A total of 418 lymnaeid snails represent- ing 3 species (Lymnaea caperata, L. palustris, and L. stagnalis) were recovered from 10 of the 12 sites, and 55 of 78 wetlands sampled (Table 2, Fig. 3). The 2 sites where snails were not found were represented by only a single wetland sampling area. All 3 species collected are known hosts for F. magna (Swales 1935, Foreyt and Todd 1978, Laursen and Strom- berg 1993); Lymnaea palustris was the most common occurring at 9 sites, and L. stagnalis and L. caperata were found at 5 and 4 sites, respectively (Table 2). DISCUSSION To my knowledge, this study represents the first report of F. magna in moose in North Dakota. However, because the historical data were collected by previous investigators, they were subject to a degree of interpretation. First, I assumed that flukes collected by past inves- tigators were actually F. magna, as this fluke has been recovered from moose in adjacent northeastern Minnesota (Karns 1972, Murray et al. 2006), and the only other large liver fluke in North America, Fasciola hepatica, has not been reported in North Dakota (Pybus 2001). Second, I interpreted all signs suggestive of F. magna infection as actually being caused by this parasite; however, certain described signs may have been due to injury (Lankester and Samuel 1998), bacterial infection (Leighton 2001), Echinococcus granulosis, or Taenia hydatigena cysts (Jones and Pybus 2001). As a result, the true prevalence of F. magna in moose in eastern North Dakota may have been lower than the 19.5% estimated from check station records and necropsy reports. Unfortunately, data from white-tailed deer were not available to corroborate the presence Harvested 1977-1984 Unharvested 1983-1992 Harvested 2002-2003 Unit Examined Infected Examined Infected Examined Infected M1C 138 29 (21.0%) 6 2 (33.3%) 4 0 M4 4 20 1 0 22 0 M5 6 20 2 1 (50%) 1 0 M6 5 20 21 3 (14.3%) 4 0 M8 4 22 (50.0%) 0 0 16 0 M9 1 20 0 0 7 0 M10 0 20 0 0 2 0 Unknown 0 20 2 0 22 0 Total 158 31 (19.6%) 32 6 (18.8%) 78 0 Table 1. Incidence of F. magna infection in harvested moose and unharvested moose in 7 management units in North Dakota. Species No. collected Sites (present/sampled)* Lymnaea caperata 48 4/12 Lymnaea palustris 271 10/12 Lymnaea stagnalis 99 5/12 Combined total 418 10/12 Table 2. Survey data for lymnaeid snails in North Dakota and northwestern Minnesota, 2003-2006. *Includes 78 wetlands at 11 sites in North Dakota and 1 wetland in northwestern Minnesota (see Fig. 3). ALCES VOL. 47, 2011 MASKEY - LIVER FLUKE IN NORTH DAKOTA MOOSE 5 of F. magna in North Dakota. Nonetheless, F. magna appears to be enzootic in moose in eastern North Dakota, although at a much lower prevalence than in nearby northwestern Minnesota. For example, Murray et al. (2006) reported 89% prevalence of F. magna in moose in northwestern Minnesota in the late 1990s, and Karns (1972) reported 87% prevalence in the same region in the 1970s. The failure to detect F. magna in 2002 and 2003 may have been due to the geo- graphic distribution of my sampling. While the majority of historical reports of F. magna infection originated from Unit M1C (Fig. 3), my ability to sample this area was limited. Only 10 moose tag were issued annually in this area in 2002 and 2003, compared to 150 tags issued in 1977-1984; I obtained only 4 samples from unit M1C (Table 1). Nonethe- less, my recent data confirm that F. magna is not highly prevalent in North Dakota moose suggesting that the parasite has not experi- enced a marked increase in prevalence since prior surveys. For example, based on binomial probabil- ity, I had a 95% chance of detecting F. magna in Unit M1C with only 4 samples if the cur- rent prevalence was at least 53%, and a 90% chance if the current prevalence was at least 44%. Also, given my sample size of 25 moose in the 4 units where historical data suggests F. magna occurs (M1C, M5, M6, M8), I had a 95% probability of detecting this parasite even if it occurred at a moderate prevalence (11.5%). In addition, all of the 22 samples from unknown locations were also negative for signs of F. magna infection. Because several of these unknown samples were received in November when only units M5 and M6 (Fig. 2) remained open for hunting, it is probable that a substantial proportion of these livers originated from the eastern part of the state, and F. magna likely infects a relatively small proportion of moose in eastern North Dakota. The 2002-2003 hunter returns provided a more complete sampling of the western part of moose range in the state (M4, M9, M10; n = 31), and these results suggest that preva- lence of F. magna is low in these areas as well. While my surveys for lymnaeid snails were by no means exhaustive, results indicate that at least 3 species of suitable intermediate hosts for F. magna are widespread within the primary range of moose in the state. Natural or experimental infections with F. magna have been reported in L. caperata, L. stagnalis, and L. palustris (Foreyt and Todd 1978, Lausen and Stromberg 1993, Pybus 2001). Although white-tailed deer, the normal host for F. magna, are abundant in North Dakota and at least 3 species of intermediate hosts for F. magna appear to be widely dis- tributed in the state, my results suggest that actual transmission of F. magna to moose is currently limited. This may be due to lack of available wetlands. In central and north- central North Dakota (hunting units M4, M8, M9, and M10) the range of moose lies within the larger “prairie pothole” region of the Great Plains (USFWS 1955) where wetlands are abundant but subject to seasonal dry down and long-term drought cycles (Todhunter and Rundquist 2004). Thus on a seasonal or annual basis, environmental conditions may limit the availability of intermediate hosts or aquatic vegetation, prevent embryonation and hatching of eggs, and reduce survival of meta- cercariae (Swales 1935, Pybus 2001). And based on my data, a large part of the primary range of F. magna in moose in North Dakota (Units M1C, M5, and M6) is within the north- ern Red River Valley that is part of the Lake Agassiz Plain Ecoregion (U.S. Environmental Protection Agency 1996; Fig. 1). This area includes a number of permanent rivers and streams associated with the Red River that are known to support lymnaeid snails (Clarke 1973, Cvancara 1983). However, compared to the prairie pothole region of central and north central North Dakota, the Red River Valley has relatively few permanent or semi-permanent LIVER FLUKE IN NORTH DAKOTA MOOSE - MASKEY ALCES VOL. 47, 2011 6 wetlands. Because riparian habitats and suitable wetlands make up a relatively small proportion of the overall landscape, they may not be capable of sustaining high levels of F. magna infection in cervids. Since recent conditions in eastern North Dakota apparently support only a moderate level of F. magna transmission, the parasite is unlikely to represent a major source of mortal- ity in moose. The historical data reviewed in this study were collected during a period of moose population growth, and the prevalence of F. magna certainly has not increased since that time. Since the completion of the current study, there has been only a single report of an F. magna infected moose in North Dakota, a sick adult cow moose collected in unit M6 (NDGF 2004, unpublished); this moose was also infected with P. tenuis. Additionally, while only 18.8% of moose necropsied as part of targeted surveillance showed signs of F. magna infection, 75.0% were infected with P. tenuis (manuscript in preparation), suggest- ing that other mortality factors may be more important than F. magna. Although Murray et al. (2006) concluded that F. magna was the major source of mortality and morbidity in the declining moose popula- tion in northwestern Minnesota, it should be noted that the prevalence of F. magna had not increased since the pre-decline period (Karns 1972). Further, because the nearly simul- taneous decline of 2 moose populations in close proximity to each other in northwestern Minnesota and in northeastern North Dakota cannot both be attributed to F. magna, other factors common to both areas presumably play a larger role in influencing these populations. As a result, future investigations in North Da- kota and Minnesota should consider how other stressors or pathogens such as P. tenuis affect moose population dynamics in the region. ACKNOWLEDGEMENTS I would like to thank North Dakota EPSCoR, the NDGF, the U.S.D.I. Bureau of Reclamation, the U.S. Fish and Wildlife Service, the North Dakota Chapter of The Wildlife Society, the UND Biology Depart- ment, and the Wheeler Scholarship for funding this project. I would also like to thank Bill Jensen and Roger Johnson for allowing me to collect samples from moose hunters and for providing access to historical data. Thanks also to Scott Peterson for facilitating field work for this project. Additionally, I am grateful to Rick Sweitzer, and to Brett Goodwin, Bob Newman, Brad Rundquist, and Jeff Vaughan for their assistance throughout this research and for their helpful input on this manuscript. Finally, gratitude is expressed to Jason Smith and Eric Pulis for their assistance in the field and lab. REFERENCES Clarke, A. H. 1973. The freshwater mollusks of the Canadian Interior Basin. Malaco- logia 13: 1-509. Cvancara, A. M. 1983. Aquatic mollusks of North Dakota. Report of Investigation Number 78, North Dakota Geological Survey. Kayes Inc., Fargo, North Dakota, USA. Foreyt, W. J,. and A. C. Todd. 1978. Ex- perimental infection of Lymnaeid snails in Wisconsin with Fascioloides magna and Fascioloa hepatica. Journal of Parasitol- ogy 64: 1132-1134. Jensen, W. F. r. e. Johnson, and B. stillings. 2007. Study number C-1: deer population studies. Report number A-172. North Dakota Game and Fish Department, Bismarck, North Dakota, USA. Johnson, R. E. 2002. Moose and elk popu- lation study. North Dakota Game and Fish Department Report Number A-155. North Dakota Game and Fish Department, Bismarck, North Dakota, USA. _____. 2007. Moose and elk population study. North Dakota Game and Fish Department Report Number A-155. North Dakota Game and Fish Department, Bismarck, ALCES VOL. 47, 2011 MASKEY - LIVER FLUKE IN NORTH DAKOTA MOOSE 7 North Dakota, USA. Jones, A., and M. J. PyBus. 2001. Taeniasis and echinococcosis. Pages 150-192 in W. M. Samuel, M. J. Pybus, and A. A. Kocan, editors. Parasitic Diseases of Wild Mammals. Second edition. Iowa State University Press, Ames, Iowa, USA. Karns, P. D. 1972. Minnesota’s 1971 moose hunt: a preliminary report on the biologi- cal collections. North American Moose Conference and Workshop 8: 115-123. Lankester, M. W. 1974. Parelaphostrongy- lus tenuis (Nematoda) and Fascioloides magna (Trematoda) in moose of south- eastern Manitoba. Canadian Journal of Zoology 53: 235-239. _____. 2001. Extrapulmonary lungworms of cervids. Pages 228-278 in W. M. Samuel, M. J. Pybus, and A. A. Kocan, editors. Parasitic Diseases of Wild Mammals. Second edition. Iowa State University Press, Ames, Iowa, USA. _____. 2010. Understanding the impact of meningeal worm, Parelaphostrongylus tenuis, on moose populations. Alces 46: 53-70. _____, and W. M. Samuel. 1998. Pests, para- sites, and diseases. Pages 479-518 in A. W. Franzmann and C. C. Schwartz, editors. Ecology and Management of the North American Moose. Smithsonian Institution Press, Washington D. C., USA. Laursen, J. R., and B. E. StromBerg. 1993. Fascioloides magna intermediate snail hosts: habitat preferences and infection parameters. Journal of Parasitology 79 (6; Supplement): 302. Leighton, F. A. 2001. Fusobacterium necro- phorum infection. Pages 493-496 in E. S. Williams and I. K. Barker, editors. Infec- tious Diseases of Wild Mammals. Third edition. Iowa State University Press, Ames, Iowa, USA. Murray, D. L., E. W. Cox, W. B. Ballard, H. A. WhitlaW, M. S. Lenarz, T. W. Custer, T. Barnett, and T. K. Fuller. 2006. Pathogens, nutritional deficiency, and climate influences on a declining moose population. Wildlife Monographs 166: 1-30. PyBus, M. J. 2001. Liver flukes. Pages 121-149 in W. M. Samuel, M. J. Pybus, and A. A. Kocan, editors. Parasitic Diseases of Wild Mammals. Second edition. Iowa State University Press, Ames, Iowa, USA. Rosza, L., J. Reiczigel, and G. MaJoras. 2000. Quantifying parasites in samples of hosts. Journal of Parasitology 86: 228-232. Smith, J. R., R. A. SWeitzer, and W. F. Jensen. 2007. Diets, movements, and consequenc- es of providing food plots for white-tailed deer in central North Dakota. Journal of Wildlife Management 71: 2719-2726. SWales, W. E. 1935. The life cycle of Fasci- oloides magna (Bassi 1875), the large liver fluke of ruminants of Canada. Canadian Journal of Research, Section D, Zoological Sciences 12: 177-215. Todhuter, P. E., and B. C. Rundquist. 2004. Terminal lake flooding and wetland ex- pansion in Nelson County, North Dakota. Physical Geography 25: 68-85. United States Environmental Protection Agency. 1996. Level III Ecoregions of the Continental United States, Map M-1, various scales. National Health and En- vironmental Effects Research Laboratory, United States Environmental Protection Agency, Corvallis Oregon, USA. United States Fish and WildliFe Service (usFWs). 1955. Wetlands inventory of North Dakota. United States Department of the Interior, Fish and Wildlife Service, Office of River Basin Studies, Billings, Montana, USA.