Mortality in free-ranging Eurasian brown bears (Ursus arctos arctos) in Spain (1998-2018)

Background This work summarizes the confirmed causes of death of Eurasian Results Causes of death were classified based on (i) pathological findings and (ii) caused by “human intervention” or “non human intervention”. In four cases (16%) it was not possible to determine the cause of death due to the bad preservation of found remains or insufficient tissue availability. Based on pathological findings seven out of the 21 (33.3%) brown bears in which the cause of death could be determined died due to infectious diseases (clostridiosis n =4 or infectious canine hepatitis n =3), two (9.5%) due to exertional (degenerative) myopathy (one of those bears also showed clostridiosis) and one due to strychnine poisoning (4.8%), neoplasia (4.8%) or mushroom poisoning (4.8%). The remaining animals died due to traumatic lesions (including fights or infanticide), shooting or wire snare. Mortality data was also classified by deaths caused by “human intervention” or “non human intervention”. The death of one bear showing exertional myopathy after handling was not ascribed to any of the former classifications. Six out of the 20 (30%) brown bears died as a consequence of “human intervention” due to illegal hunting (wire snare hunting n =3 or shooting n =2) and, strychnine poisoning ( n =1). In contrast, fourteen (14/20, 70%) brown bears died by “non-human intervention”; nine of them (9/20, 45%) due to traumatic lesions (fights n =4, traumas n =3 or infanticide n =2), three (3/20, 15%) due to infectious canine hepatitis caused by canine adenovirus type 1 (CAdV-1) infection, one (1/20, 5%) due to cholangiocarcinoma and another one (5%) due to mushroom poisoning. Conclusions This study shows that the main causes of death in Eurasian brown bears are those


Background
In Spain the endangered Eurasian brown bear (Ursus arctos arctos) population is located in the Cantabrian mountain range (northwestern Iberian Peninsula) and represents the southwestern limit distribution for this species in Europe [1]. The population of free-ranging Eurasian brown bear has recovered during the last two decades, from approximately 100 individuals in the nineties to 230-260 presently [1,2]. Brown bears are found dead in nature, sometimes the death is caused by human intervention, i.e. shooting, poisoning or traffic accidents, and on other occasions death is due to nonhuman intervention, i.e. infanticide [3,4].
The identification of causes of mortality in wild natural populations is relevant for the correct design of conservation strategies and management programs. However, knowledge on the mortality among bears is limited because of the difficulty in finding the dead animals in nature.
The aim of the present work is to summarize the causes of confirmed deaths and most significant findings related to deaths reported in twenty-five free-ranging Eurasian brown bears submitted for necropsy in Asturias and Castilla y León (northwestern Spain) over the past 20 years. These data are valuable and may help in the conservation and management of this recovering population.

Necropsy findings
The cause of death and relevant pathological findings in the twenty-five brown bears studied is shown in Table 1. In four (4/25, 16%) cases it was not possible to determine the cause of death due to bad preservation of the remains found or insufficient tissue availability. Usually traumatic death causes such as shooting, wire snares, fighting or infanticide were easier to determine not only based on necropsy and histopathological studies but also using complementary diagnostic techniques (i.e. radiography) or knowledge of the behavior of this species. However other causes were more difficult to discern and are reported below. Some animals (i.e. bear number 1) showed several pathological findings that could lead to death.

Infectious diseases
The five bears (bears number 1, 8, 10, 15 and 23) with gangrenous myositis (Table 1), usually a consequence of wire snare hunting, infanticide, fights or traumas, showed serohemorrhagic edema in the abdominal cavity, thorax, pericardium, and skeletal muscle, and hemorrhages in heart, skeletal muscles, stomach, intestine, liver, spleen, and kidney. Microscopically, vascular damage and hyperacute myodegeneration consisted of myonecrosis, edema, gas, extravasation of fibrin into the interstitial spaces, and lacunar dissolution of myofibers in skeletal muscles were observed in those animals. Clostridium sordellii was identified as the etiological agent of the lesions in four of those brown bears [5]. Clostridium bifermentans was also isolated in one of the former four bears, as well as C. septicum in another one. The presence of Clostridium sordellii was always associated with previous muscle damage (i.e. traumas) that triggered its proliferation.
Gross lesions in the three bears (bears number 11, 14 and 20) with infectious canine hepatitis caused by canine adenovirus type 1 (CAdV-1) consisted of hemorrhages in thymus (in cubs), lungs, heart and mesentery; hemorraghic fluid in thoracic and abdominal cavities; friable and yellowish liver; hepatomegaly; thickening of the gall bladder due to edema and congestion of spleen, kidney and meninges [6]. Microscopically the main pathological findings appeared in liver and gall bladder. Liver showed mild centrolobular multifocal degeneration and necrosis of hepatocytes, with the presence of intranuclear inclusions bodies and low inflammatory infiltration mainly of lymphocytes. The gall bladder showed edema of the wall. Additionally, the brain showed edema, congestion, perivascular cuffing, foci of gliosis, and degeneration of neurons, mainly located in the brainstem. CAdV-1 was confirmed by quantitative polymerase chain reaction (qPCR) and immunohistochemistry.

Exertional (degenerative) myopathy
In two animals exertional myopathy was diagnosed. One of them was a brown bear that died after capture by snare for one week which also showed clostridiosis (bear number 1). An additional female cub died due to exertional myopathy after handling (bear number 9). That cub was found alone in the wild when it was two months old and it was bred in captivity until it was nine months old. Then the cub was transported in order to reintroduce it back into the wild dying during the trip. Both animals showed gross lesions consisted of dry and pale cardiac and some skeletal (mainly intercostals and femoral) muscles (Figs. 1a and 1b). Microscopically severe segmental degeneration of muscles was observed consisted of hypercontracted fibers, extensive Zenker´s hyaline degeneration and coagulative necrosis of myofibers (Figs. 1c to 1 h). Bear number 1 also showed an intensive infiltrate mainly consisting of lymphocytes and macrophages, as well as mineralization in the affected muscles ( Fig. 1f). In that animal necrotic myofibers with surviving satellite cells, invading macrophages and elongating myoblasts, all indicative of events of regeneration, were also observed (Fig. 1d). The cub also showed hypoplasia of adrenal glands (1.7 grams, 0.004% relative weight; physiological relative weight 0.03%) [7].

Mushroom poisoning
Bear number19 showed hemorrhagic gastritis and diffuse hepatic and renal necrosis compatible with mushroom poisoning, likely due to ingestion of poisonous Amanita spp, although that could not be confirmed by toxicological analysis.
Causes of death were classified both based on (i) pathological findings and (ii) caused by "human intervention" or "non human intervention".
Based on pathological findings seven out of the 21 (33.3%) brown bears in which the cause of death could be determined died due to infectious diseases (clostridiosis n = 4 or infectious canine hepatitis n = 3), two (9.5%) due to exertional myopathy (one of those also showed clostridiosis) and one due to strychnine poisoning (4.8%), neoplasia (4.8%) or mushroom poisoning (4.8%). The remaining animals died due to traumatic lesions (including fights and infanticide), shooting or snare.
Mortality data was also stratified by deaths caused by "human intervention" or "non human intervention". The death of bear number 9 showing exertional myopathy after handling was not ascribed to any of the former classifications. Six out of the 20 (30%) brown bears died as a consequence of "human intervention" due to illegal hunting (wire snare hunting n = 3 or shooting n = 2) and, strychnine poisoning (n = 1). In contrast, fourteen (14/20, 70%) brown bears died by "nonhuman intervention"; nine of them (9/20, 45%) due to traumatic lesions (fights n = 4, traumas n = 3 or infanticide n = 2), three (3/20, 15%) due to infectious canine hepatitis caused by CAdV-1 infection, one (1/20, 5%) due to cholangiocarcinoma and another one (5%) due to mushroom poisoning. One of the animals that died due to wire snare hunting and three of those that died due to traumatic lesions showed clostridiosis.

Discussion
This study shows that the main causes of deaths in Eurasian brown bears from the Cantabrian mountain range are those caused by infectious diseases (33.3%), namely clostridiosis (associated to previous muscle damage) or infectious canine hepatitis. In Sweden, where ninety-eight animals were analyzed, no deaths of brown bears were attributed to any infectious disease [4]. Thus, the confirmation of C. sordellii and CAdV-1 (the agent of infectious canine hepatitis in dogs) as the first causes of death in Eurasian brown bear is probably the most outstanding finding in this study [6]. In contrast with previous data on brown bears, for the first time infectious diseases are described as an important cause of death. Moreover, the four fatal cases of clostridial infection described to date, compared to the few descriptions reported worldwide, could show a higher susceptibility of the Cantabrian brown bear population to these bacteria [5]. The putative high susceptibility to infectious pathogens reported here might be a consequence of a weaker immune system due to the lower genetic diversity described for this species in the Cantabrian range subpopulation [9,10]. Another possibility might be a high degree of circulation of the pathogens both in the environment and sympatric species (i.e. CAdV-1 carriers such as wolves) [6].
Two animals died showing exertional myopathy as a consequence of extreme exertion and stress after trapping by a snare or handling. Those are to our knowledge the second and third cases of death by exertional myopathy reported in bears in the literature worldwide. The first case was described by Canada [11]. Those three cases confirm the fact that the prolonged suffering in bears might cause exertional myopathy as occurs in other mammal species, i.e. red fox [12]. Therefore that should be taken into account in the management of bears. The acute presentation of adrenal hypoplasia shown in the cub could be precipitated by the physiologic stress suffered [13].
Despite of the limited number of studied cases (related to the conservation status and behaviour of the species that hampers the detection of moribund or dead bears in nature) the data reported in the present work confirm the persistence and relevance of causes of death directly related to human intervention (30%). The total number of mortalities and causes of death are a conservative figure as unreported mortality is difficult to estimate. Additionally the impossibility of establishing the cause of death in four brown bears submitted for necropsy due to the nature and/or conservation status of available samples must be taken into account when considering the frequency and importance of the different causes of death reported in this paper.
Nevertheless, the confirmation of six out of 25 brown bears submitted to the laboratory for necropsy confirmed as death by shooting, snare and poisoning (4 of them from 1998 to 2008 and two from 2008 onwards) seems to agree with the apparent reduction in illegal killing after the mid-nineties [1,14], suggested as a key factor for the recovery undergone during last two decades [2]. In this regard human actions were responsible of most grizzly bear mortalities in Canada [3]; as well as in Sweden, Mörner et al. (2005) found that human caused mortality stands for 64% of total submissions; meanwhile bears killed by other bears and infanticide were the most frequent cause of natural death (16%) [4].
The detection of four male bears that died after fighting, three due to traumas, three bears that died due to CAdV-1 infection, two cubs that died due to infanticide, one female died due to a cholangiocarcinoma and one male due to mushroom poisoning confirm the existence of deaths attributable to "non-human mediated" causes (70%) usually difficult to detect in nature, and thus highlight the importance of necropsies.
Traffic induced bear mortality has been documented as a common cause of death in bears in other European countries [4,[15][16][17][18][19][20]. In our study we have not reported any road-killed animals, despite one bear that died in a traffic accident (the only one reported in Spain to date) in León in 2008, although necropsy of this bear was not performed, and it is not included in this report [21].
Regarding the limited genetic variability of the Cantabrian brown bear population [2], and taking into account that a free-ranging bear can live about 25 years, we can consider that the three animals found dead at the age of 19-20 years have had the opportunity to widely contribute to the genetic stock of the Cantabrian brown bear population. However, two out of the five necropsied cubs (40%) died due to CAdV-1 infection. Thus, we can consider that an important percentage of future progenitors might die due to that infectious disease among others causes, which can be important for the maintenance of genetic variability of the species, even more in this endangered population.

Conclusion
In summary the data in the present work represent a first review of the death causes reported at necropsy in the Cantabrian brown bear population since 1998. The apparent moderate but steady recovery experienced during last decades provides a new landscape for this endangered population, where recovery and conservation works will have to be accompanied by an increasingly important effort on surveillance and management programs of the species, focused on the control of infectious diseases shared between domestic and wild animals under the one health strategy.

Methods
Twenty-five free-ranging Eurasian brown bears, nine from Castilla y León and 16 from Asturias, of different sexes and ages (see Table 1) were necropsied from 1998 to 2018.