NECROPSY FINDINGS IN 62 OPPORTUNISTICALLY COLLECTED FREE-RANGING MOOSE (ALCES ALCES) FROM MINNESOTA, USA (2003–13)

Northern Minnesota, US is at the southern edge of the North American moose (Alces alces) range. Between the mid-1980s and early 2000s, a collapse of the northwestern moose population occurred. Similarly, an approximately 50% decline in Minnesota's northeastern population since 2005 raises concern about the survival of Minnesota's moose population (Murray et al. 2006; Lenarz et al. 2010; DelGuidice 2013). Lenarz et al. (2009) indicated that a high proportion of moose mortality in a sample of radio-collared animals was nontraumatic and inferred that pathogens and malnutrition were likely proximate causes of death. In response to the ongoing decline of Minnesota's northeastern population, wildlife agencies increased their efforts to collect entire, freshly dead carcasses for complete necropsies to gain insight into health factors that might affect survival.

We included all entire moose carcasses submitted between 2003 and 2013 to the Minnesota Veterinary Diagnostic Laboratory from Minnesota's northeastern and northwestern moose range in our study (Fig. 1). These moose were either euthanized (via gun shot) by Minnesota Department of Natural Resources (DNR) or tribal personnel due to signs of illness or found dead with no obvious cause. In addition, wildlife agencies submitted animals dying as a result of vehicular accidents since 2009. We grouped the animals in four age classes: calves (<1 yr old), juveniles (≥1 yr to <3 yr old), adults (sexually mature; ≥3 yr old), and undetermined. Calves were identified based on their size and tooth eruption patterns. The peak of calving in Minnesota is mid-May (McGraw et al. 2014). The age of animals >1 yr old was determined by counting cementum annuli at Matson's Laboratory, Milltown, Montana, USA (Sergeant and Pimlott 1959). The incisors of 11 moose were not available for aging. However, we included five of these 11 moose in the adult group due to their antler size or reproductive status. We grouped the remaining six animals in the “undetermined” age group. We recorded the reproductive status of the adult cows as inactive, pregnant (submitted October–May), or lactating (submitted May–October).

Necropsies included gross examination of the entire carcasses and histologic examination of brain, spinal cord, liver, kidney, spleen, lungs, heart, skeletal muscle, abomasum, and intestine. In selected cases, eyes, adrenal glands, thyroid glands, reproductive organs, and skin were examined histologically. We classified the nutritional state as good when adequate fat stores were present, underweight when there was lack of significant internal and subcutaneous adipose tissue without serous atrophy of bone marrow fat and cardiac coronary fat, or cachectic when bone marrow and cardiac coronary fat had undergone serous atrophy. Gross examination of the cranial vault included rinsing of the meninges with 10% buffered formalin to increase the chances of identifying Parelaphostrongylus tenuis nematodes (Slomke et al. 1995). Histologic examination of the brain included three to four coronal sections through the cerebrum, thalamus, and brainstem and one sagittal section through the cerebellum. Fifteen to 40 sections of brain tissue were examined per animal. At least two cross sections of cervical, thoracic, and lumbar spinal cord were examined histologically when possible.

The severity of the winter tick (Dermacentor albipictus) infestation was scored based on the estimated extent of hair loss and the estimated density of winter ticks covering the body. We recorded the tick-associated loss and damage of hair as mild, moderate, or marked, when approximately ≤20%, >20 to ≤50%, and >50%, respectively, of the hair was lost or broken. Only animals submitted between October and early June harbor winter ticks. Only animals submitted between February and early June have winter ticks of sufficient size to be readily noticeable and harmful. Hence only animals submitted during this time interval were evaluated for winter tick infestations.

We sliced the livers at 2–3 cm intervals, and removed and identified flukes when present. We graded the severity of fluke-associated liver lesions as mild, moderate, or marked when approximately ≤15%, >15 to ≤50%, or >50% of the parenchyma was altered by fibrotic bile ducts, nodules, or cavitations with peripheral fibrosis and central opaque brown pasty material and parenchymal fibrosis. We classified fluke-induced liver lesions as “active” when liver flukes (Fascioloides magna) were present in the tissue and as “inactive” when viable liver flukes were not detected. We estimated the number of flukes whenever flukes were present. Fecal material was examined for parasites by floatation analysis. We submitted liver samples to the Diagnostic Center for Population and Animal Health (Michigan State University, East Lansing, Michigan, USA) for inductively coupled mass spectroscopy for selected heavy metals (arsenic, cadminum, lead, and mercury) and mineral elements (i.e., copper, iron, and zinc).

We tested fecal material for Mycobacterium avium subsp. paratuberculosis by PCR and culture. We examined obex and retropharyngeal lymph node for protease-resistant prion protein by immunohistochemistry. Brain samples were tested for rabies virus antigen by direct fluorescent antibody test at the Department of Health, St. Paul, Minnesota (MDH). We submitted a tissue pool (intestine, kidney, lungs, and spleen) for bovine viral diarrhea (BVD) virus-specific PCR. We submitted brain samples to the MDH and the National Veterinary Services Laboratories, Ames, Iowa (NVSL) for PCRs for West Nile virus (WNV), eastern equine encephalitis (EEE) virus, and western equine encephalitis (WEE) virus. Retropharyngeal lymph nodes were cultured for Mycobacterium bovis at NVSL. In addition, aerobic and anaerobic bacterial cultures were performed at the discretion of the pathologists.

We compiled data using Excel (Microsoft, Redmond, Washington, USA) and conducted analyses of summary data of both categorical and continuous variables for a descriptive evaluation using Statistix 10® (Analytical Software, Tallahassee, Florida, USA). We compared the subjectively scored nutritional status with the degree of fluke-induced hepatitis categorized as mild, moderate, and marked as defined earlier. We used a chi-square test to assess the relationship between nutritional status (good, moderately underweight, or cachectic) to fluke-induced hepatitis (negative, mild, or moderate/marked). For animals submitted during winter tick season, February to early June (n = 25), we compared the mean hepatic iron levels (ppm; wet weight) between moose with no or few winter ticks (mild infestation) to moose with moderate to severe winter tick infestations. We conducted a t-test to compare mean liver iron concentrations (ppm; wet weight).

We received 62 moose carcasses with 55 and seven moose originating from the northeastern and northwestern portions of moose range, respectively (Supplementary Tables 1 and 2). There were 21 adults, 10 juveniles, and 25 calves. In addition, six moose of unknown age were submitted belonging either to the juvenile or adult group (four females and two males). We received the majority of the calves (76%) during the winter and spring (December–May). Eight animals were killed in vehicular collisions, 23 were found dead, and 31 were shot after members of the public reported animals in prolonged recumbency or with unusual behavior (e.g., being unafraid of people, running repeatedly into traffic, remaining in one location for long periods, or neurologic signs such as head tilt, circling, and abnormal ear posture or ear twitching). Nine animals were in good nutritional state, including four moose killed in known vehicular accidents. Twenty-eight and 25 moose were cachectic or underweight, respectively (Fig. 2). We considered cachexia without any significant concurrent disease process to be the cause of death in one calf that was submitted in March. Five animals were cachectic without significant concurrent disease process other than marked fluke-induced hepatitis. The remaining cachectic animals had evidence of P. tenuis infection, marked D. albipictus infection, or other significant disease processes such as bacterial infections. There was a tendency for animals submitted during spring and summer to be more often underweight or cachectic than animals submitted during fall and winter (Fig. 2).

The meninges of the brain of one juvenile and one calf harbored one adult nematode, approximately 8 cm long and 1 mm in diameter (consistent with P. tenuis). The brains of all but one animal were suitable for histopathologic examination. Twenty-eight moose (45%), including the two moose with grossly detectable nematodes, had histologic evidence of migration of a nematode in the central nervous system. These cases included six adult cows (28% of the age group), six juveniles (60% of the age group), 15 calves (60% of the age group), and one animal of undetermined age. Thirteen moose had histologically detectable migration tracts without evidence of embryonated ova, larvae, or adult worms. Migration tracts were characterized by a multifocal, randomly distributed round to linear disruption of the parenchyma with hemorrhage, hemosiderosis, glial scarring, or infiltration by gitter cells or eosinophils. Affected brains usually had a mild perivascular and meningeal infiltration with lymphocytes and plasma cells. The brain parenchyma adjacent to the disruption had vacuolar change due to individual swollen myelin sheets and axons. The youngest affected calf was approximately 4 mo old. Besides the migration tracts, embryonated ova or nematode larvae were detected in the meninges of the brain of four moose. Presence of embryonated eggs and larvae suggests that the moose was infected with at least one female and one male P. tenuis. A single adult nematode was detected in the neuropil or the meninges during the histopathologic examination of the brain and rete mirabilis epidurale rostrale in 11 moose. The adult nematodes were 150 to 220 µm in diameter, had a thin cuticle, polymyarian coelomyarian musculature, lateral chords, accessory hypodermal cords, and large multinucleated intestinal cells, consistent with a metastrongyle nematode and more specifically highly suggestive of P. tenuis (Gardiner and Poynton 1999; Anderson 2000). We found lesions of worm migration in the spinal cord in 11 of 43 examined cords. Only, in one affected moose (a calf), lesions of worm migration were limited to the spinal cord (and not detected in the brain). None of the moose killed in vehicular accidents had evidence of P. tenuis infection.

There was marked infestation with D. albipictus in three of five adult moose submitted during winter tick season. One of 10 juveniles had moderate D. albipictus infestation. Ten of 18 calves had moderate to marked winter tick infestation (56%) associated with hair loss and dermatitis/epidermitis. In animals found dead, evidence of anemia was usually associated with moderate or marked winter tick infestation based on pallor of liver, heart, and kidneys. All animals with marked winter tick infestation were underweight or cachectic and had mild to moderate hydropericardium.

Thirty-seven moose (60%) had evidence of active or inactive infection with F. magna. Moderate to marked fluke-induced liver lesions were found in six of eight well-nourished moose (75%), 10 of 24 moderately underweight moose (42%), and 15 of 28 cachectic moose (54%, Fig. 3). Mild (n = 2), moderate (n = 7), and marked (n = 8) fluke-associated lesions were identified in 21 adults. These included three cows that died in vehicular accidents, two of which had moderate and one had marked lesions. Five of 10 juveniles had fluke-associated liver lesions. Two juveniles had moderate lesions and three had marked lesions. Among the 25 calves, two had mild lesions, one had moderate lesions, and seven had marked liver lesions. Two each of the six moose of undetermined age had moderate and marked fluke-induced liver lesions, and one animal of this group had mild lesions. Active infection with F. magna was present in 12 moose (eight calves, three juveniles, one adult). Between one and approximately 20 flukes were present per liver. There was no correlation between the nutritional state of the animals and the degree of fluke-associated hepatitis (chi square 0.0656, P = 0.80; Fig. 2). Multiple animals had evidence of aberrant fluke migration in the lungs and thoracic cavity with chronic fibrous pleuritis.

A variety of internal parasites, interpreted to be incidental findings, were detected, including Setaria sp. in body cavities; hydatid cysts of Echinococcus granulosus in lungs, liver, and kidney; rumen flukes (Paramphistomum sp.); Nematodirus sp. in the small intestine; lungworms, including Protostrongylus sp., Dictyocaulus viviparous, and D. filaria; and cysticerci of Taenia hydatigenea in body cavities or of T. krabbei in skeletal muscle and heart.

Seven of eight moose that died in vehicular accidents had fractured bones and soft tissue hemorrhages, and the eighth moose had subcutaneous and internal hemorrhages without fractures. Nonparasite-related findings in the other 54 moose included a spine fracture in an adult. A juvenile with spine fracture was detected adjacent to railroad tracks. This animal also had parasite migration tracts in the brain. Another adult had focally extensive subcutaneous hemorrhage and hemothorax suggestive of blunt-force trauma. Two adult bulls, submitted during rut season, had each suppurative inflammation with abscesses in the subcutis of the sternal region that extended via the sternum into the thoracic cavity. Trueperella pyogenes was isolated from the abscesses in both cases. Mixed bacterial septicemia was present in three calves, two of which had apparent puncture wounds to the hock region and mandibular region, respectively. These wounds were attributed to large predators (likely the grey wolf, Canis lupus). Pasteurella multocida and beta-hemolytic Streptococcus sp. were isolated from the internal organs of one of these animals, whereas P. multocida and T. pyogenes were isolated from the second animal. Trueperella pyogenes and Proteus vulgaris were isolated from the third calf, which also had fibrinous polyarthritis.

In three moose, the cause of death or the clinical signs were not determined. These moose were underweight but not cachectic and no significant concurrent lesions were detected.

Twenty-eight moose (45%) were positive for intestinal endoparasites by floatation analysis. The feces of 14 calves contained parasite ova (Nematodirus sp., n = 5; Trichuris sp., n = 2; Moniezia sp., n = 2; and F. magna, n = 1). Four calves had infections with two intestinal endoparasites (Nematodirus sp./strongyle type, n = 3 and Nematodirus sp./Moniezia sp., n = 1). The feces of 14 juveniles and adults contained parasite ova (Nematodirus sp., n = 6; strongyle-type ova, n = 4; Trichuris sp., n = 1; and Moniezia sp., n = 3).

Liver arsenic, lead, and mercury concentrations were below detection limits in all moose. The highest cadmium concentration (4.1 ppm, wet weight) was measured in a 14–yr-old cow, the oldest moose in our study. The mean hepatic iron concentration was 495 ppm in moose with no or few winter ticks compared to 122 ppm in moderately or severely affected moose (Supplementary Tables 2 and 3). This difference was significant (P<0.01; Fig. 4).

Mycobacterium avium ssp. paratuberculosis was not detected in any of the 57 moose tested. Protease-resistant prion protein was not detected in any of the 58 examined obex samples. Mycobacterium bovis was not isolated from lymph node samples of any of the 52 moose. Rabies virus antigen was not detected in the 50 moose brains tested. WNV, EEE virus, and WEE virus were not detected in any of the 35 brains tested. Two of 54 tested moose were weakly positive for BVD virus.

The majority of the animals were undernourished. Twenty-eight moose were cachectic (45%) and 25 moose were underweight (40%). The poor nutritional state of these animals is likely in part attributable to the fact that the majority of the animals were submitted during the spring (Schwartz et al. 1987; DelGiudice et al. 2011). During this time, fat stores are frequently depleted particularly in pregnant cows.

Parelaphostrongylus tenuis infection was identified in 28 moose. Excluding the eight animals that died of vehicular accidents, 52% of the submitted carcasses had evidence of P. tenuis infection. This rate of infection is higher than that reported in another study of Minnesota moose (Murray et al. 2006), likely due to the systematic histopathologic examination of the brains in our study. Calves and juveniles were primarily affected, but P. tenuis infection was also prevalent among adults. White-tailed deer (Odocoileus virginianus) are the definitive host of P. tenuis (Lankester 2001). Adult P. tenuis migrate in the meninges of white-tailed deer for years. Infected white-tailed deer shed larvae with the feces. Terrestrial snails become infected with the larvae and serve as intermediate hosts. The snails harboring infective larval stage 3 (L3) larvae are ingested by white-tailed deer and aberrant hosts such as moose (Anderson 1964; Lankester 2001). Parelaphostrongylus tenuis is a suspected cause of the moose population decline (Lankester 2010). Other authors doubt a causative relationship between the presence of P. tenuis and increased moose mortality (Lenarz 2009). The parasite has been reported in neurologic moose in Minnesota before the disease was induced in experimentally infected moose (Fenstermacher 1934; Kurtz et al. 1966). Anderson (1964) infected 1-mo-old moose calves with a high dose of L3 larvae (160 and 200 larvae). Both animals developed neurologic signs severe enough to require euthanasia. Lankester (2002) infected seven 5- to 9-mo-old moose with a low dose of L3 larvae (3–30 larvae). All animals became neurologic about 4 wk after infection. Two animals that received a larger number of larvae (15–30) had to be euthanatized, three moose improved, and two moose recovered completely after 4 mo. Although Lankester (2002) demonstrated that moose are able to survive low dose infections, it is unclear how affected animals fare that are not under human care.

Dermacentor albipictus infestation was a contributing and occasional single-mortality factor in 10 calves and three adults. Winter ticks develop from the nymph stage to the adult on the hosts between October and early June. The female adult ticks are particularly pathogenic, ingesting blood (estimates range from 1 to 8 mL) in addition to causing blood loss at the feeding site (Samuel 2004). Furthermore, winter ticks cause irritation of the skin with widespread hair loss from the animal rubbing against trees. The blood, hair, and energy loss weaken the animals and can cause death due to exposure (Samuel 2004). Quantification of the effects of winter ticks is difficult. We did not attempt to count or calculate the number of ticks found on the animals and only estimated the area of hair loss and the density of ticks. Counting of winter ticks can be misleading. Particularly at the end of winter tick season, moose might no longer carry large numbers of ticks because engorged ticks have dropped off, but the animal can still suffer from blood, hair, and energy loss. The mean hepatic iron concentration of animals with severe winter tick infestation was significantly lower than that of unaffected animals. We used the hepatic iron concentration in our study as a proxy for the degree of blood-loss anemia induced by winter ticks because hematocrit analysis of blood is no longer feasible in a carcass, and the quantification of the degree of anemia at necropsy is unreliable. Furthermore, some moose had been killed by gunshot so that the euthanasia-related acute blood loss masked the true chronic blood loss anemia. The liver (hepatocytes and Kupffer cells) is the principle storage organ for iron (Crichton 2009). Chronic blood-loss anemia caused by blood-feeding ectoparasites results in increased synthesis of hemoglobin (Carlson and Aleman 2009. Over time, the increased demand for iron results in decreased blood iron concentrations and ultimately depletion of iron from the storage organs including liver (Carlson and Aleman 2009; Tvedten 2010; Weiss 2010; Naigamwalla et al. 2012). The lower level of iron in the liver of moose with severe winter tick infestation supports the hypothesis that the animals suffered from chronic blood loss anemia.

Liver lesions as a result of F. magna infection might have been a contributing mortality factor in 13 moose. White-tailed deer are the definitive host of this fluke, and freshwater snails serve as intermediate hosts (Maskey 2011). It is controversial whether F. magna is capable of causing death in moose by itself. Some investigators point out that fluke infestations generally do not cause illness in moose despite having the potential to cause dramatic liver lesions and occasionally lesions in lungs (e.g., “pleuritis”). Lankester and Foreyt (2011) infected two 2-mo-old moose calves and one yearling with metacercaria of F. magna. After 1 yr none of the animals showed evidence of illness and all animals gained weight comparable to unaffected moose despite having marked liver lesions. Similar findings have been described in cattle infected with F. magna (Wobeser et al. 1985; Conboy and Stromberg 1991). Lankester and Foreyt (2011) concluded that F. magna is unlikely to be a critical factor in a moose population decline. Likewise, in our study, several well-nourished moose that died due to vehicular accidents had moderate or marked fluke-induced liver lesions. Nevertheless, fluke-induced hepatitis might become a contributing lethal factor during spring when the added stress of “spring” malnutrition, terminal pregnancy, and concurrent parasitism culminate.

Fecal floatation revealed various nematodes, particularly in calves and juveniles. We did not attempt to quantify the parasite load using fecal floatation. Of the detected nematodes, only Nematodirus spp. are thought to have the potential to be clinically significant pathogens in calves. However, based on our necropsy findings there was no evidence that Nematodirus spp. were a significant contributing cause of disease and death, although one might argue that intestinal endoparasitosis could have contributed to the poor nutritional state of some of the animals.

Hepatic copper concentration varied greatly among animals. However, even animals with low copper concentrations (as low as 3.1 ppm) did not have lesions such as esophageal and gastrointestinal ulceration described in carcasses of moose with “mysterious wasting disease” seen in Sweden (Frank 1998). Furthermore, alopecia and achromotrichia have been reported to be lesions of copper deficiency in moose. The alopecia seen in our study was attributable to winter tick infestation rather than copper deficiency or cachexia, because the alopecic moose were submitted during winter tick season, winter ticks were present, and the skin was severely crusted. Spinal cord demyelination attributed to molybdenum excess in Scandinavia and cobalamin deficiency seen in Nova Scotia was not detected during the histopathologic examination of the spinal cords in our study (Frank 2004).

Caution must be exercised in interpreting our results. This case series is limited and might reflect a biased sample because most animals were reported from road and utility rights of way and other open areas where they would be more visible to the public. Moose infected with P. tenuis and those heavily infested with winter ticks might display signs of illness longer than animals suffering from other disease conditions. However, P. tenuis infection is common in moose from Minnesota and is a significant cause of mortality. There are likely physiologic, nutritional, or health factors that could explain the high prevalence of this disease. The number of white-tailed deer in Minnesota has steadily increased since the 1970s reaching peak populations in 2003 and remaining near historic highs (Minnesota DNR 2011). This increase likely led to increased opportunities for moose to ingest larvae-harboring snails. The cause of the increase of the white-tailed deer population could be attributable to increasingly warmer winters with less snow coverage (Kunkel et al. 2013).

The number of trauma-related deaths was relatively low (17 of 62; 27%). There were eight known vehicular accident cases and five cases of suspected anthropogenic trauma. Furthermore, two cases were attributed to presumptive predator activity. Two bulls died during rut season from suppurative inflammation in the sternal region. The sites and timing of these lesions suggest that they were the result of intraspecies aggression of competing breeding bulls. The relative low number of trauma-related deaths can be explained because in the first 6 yr of the study period, only animals that appeared “sick” or did not have an obvious cause of death were submitted for necropsy. Furthermore, it is unlikely that remains of animals that die due to predation are being submitted in this kind of survey. The DNR is currently undertaking a moose mortality study using collared moose to avoid this “sightability bias” and to avoid a loss of follow up of animals that were victims of predation. Nevertheless, continued examination of moose collected opportunistically will add important information for disease detection.

Our preliminary work identifies the potential role of P. tenuis as a significant mortality factor. Only the systematic histologic examination of the brain and spinal cord allows for an accurate diagnosis of P. tenuis infection. We also provide evidence of the importance of D. albipictus as a mortality factor. A newly emerging disease syndrome was not identified.

We are grateful to Edgar D'Almeida and Marc Schwabenlander for their invaluable and skillful assistance with the necropsies of these animals. We thank Dave Pauly, Lance Overland, and Erik Hildebrandt for technical assistance. Numerous DNR and tribal enforcement and wildlife management staff members assisted with forwarding reports of “sick” moose and retrieval of carcasses from the field. We also thank Jaime Paulin for his help and intellectual input.

Supplementary material for this article is online at http://dx.doi.org/10.7589/2014.02.037.