PRION DISEASES

A prion is an abnormal, misfolded form of a normal cell surface protein of neurons; this misfolded protein is infectious in that it can increase its numbers in a host, causing seri­ous disease and death.

The PrP^c is abundant in the brain. This nor­mal cell surface protein of neurons is encoded by a Prnp gene, a gene quite consistent across spe­cies. While the functions of these normal prion proteins are not completely understood, their conversion to prions by ongoing abnormal fold­ing leads to brain damage and eventual death.

Prions cause “transmissible spongiform encephalopathies” (TSEs) in a variety of mam­mals, including scrapie in sheep and goats; bovine spongiform encephalopathy (BSE) or mad cow disease in cattle; transmissible mink encephalopathy in mink (Mustela vison); chronic wasting disease (CWD) in cervids of North America; feline spongiform encephalopathy in cats; ungulate spongiform encephalopathy in oryx (Oryx spp.), greater kudu (Tragelaphus Strepciceros), and nyala (Tragelaphus spp.); as well as several human diseases, including new variant Creutzfeldt-Jakob disease (nvCJD), Gerstmann-Straussler-Scheinker syndrome, and kuru (King et al.

When a prion (e.g., PrP^sc) enters a healthy organism, it acts as a template to guide the mis­folding of normal host (PrP^c) protein into the misfolded prion form (Prusiner 1998, Adriano and Calella 2009). These newly formed prions (PrP^sc) continue converting more normal host proteins themselves, producing ever-increasing amounts of the PrP^sc in the host.

Protein appears to be the sole component of the prion infectious agent (Baskakov and Breydo 2007). It is important to recognize that propagation of the prion (e.g., PrP^sc) depends on the presence of normally folded protein (PrP^c) in which the prion can induce misfolding; ani­mals which do not express the normal form of the prion protein cannot develop nor transmit the disease (Adriano and Calella 2009).

This altered PrP^sc structure is extremely sta­ble and accumulates in infected tissue, causing tissue damage and cell death. Prions are resis­tant to denaturation by chemical and physical agents such as proteinases, formalin, autoclav­ing, or disinfecting. This makes disposal and containment of these particles difficult. The term PrP^res is applied to the remaining prion molecules after treatment with proteinase K (“res” refers to “proteinase K resistant”). PrP^res accumulation in the tissues is an excellent marker for prion infections.

Prions are unprecedented infectious patho­gens that cause invariably fatal neurodegenera- tive diseases by an entirely novel mechanism. Prion diseases may occur as genetic, infec­tious, or sporadic disorders, all of which involve modification of the normal prion protein (PrP^c) (Prusiner 1998). One very important prion disease of wildlife is chronic wasting disease (CWD), which has affected a variety of ungu­lates in North America.

Chronic Wasting Disease (CWD)

causative agent The causative agent for CWD is the misfolded protein sometimes iden­tified as PrP^cwd (Baeten et al. 2007). It is the only known prion found in free-ranging spe­cies (Williams et al. 2002). The first evidence for CWD was a spongiform encephalopathy noticed about 1967 among mule deer and black-tailed deer in Colorado and Wyoming (Williams and Young 1980).

host and transmission In North America, CWD has been found in Rocky Mountain mule deer (Odocoileus hemionus hemionus) (Williams and Young 1980), as well as white­tailed deer (Odocoileus virginianus) (Miller et al. 2000), Rocky Mountain elk (Cervus elaphus nelsoni) (Williams and Young 19 93, Miller et al. 1998), and moose (Alces alces shirasi) (Baeten et al. 2007).

Mule deer fawns are infected by oral inocu­lation, with prifons becoming established in alimentary tract-associated lymphoid tissues (Sigurdson et al. 1999), Among deer with CWD, infectious prions occur in skeletal mus­cle (Angers et al. 2006), as well as the urine, feces, saliva, and blood (Mathiason et al. 2006, Haley et al. 2009, Tamguney et al. 2009); in presymptomatic deer, prions occur in the feces, blood, and saliva (Mathiason et al. 2009, Tamguney et al. 2009). Prions also have been found in elk antler velvet (Angers et al. 2009).

Transmission occurs horizontally from infected to susceptible cervids (Williams et al. 2002, Sigurdson and Aguzzi 2007); shedding in feces or saliva is a likely transmission route (Miller and Williams 2004). Environmental contamination and contact with body fluids also may be important in transmission and maintenance of the disease (Williams and Miller 2002, Miller et al. 2004, Mathiason et al. 2006).

Chronic wasting disease has been enzootic in farmed deer and elk since the early 19 90s (Miller and Williams 2004), and geographic spread of CWD in North America is linked to commerce in infected farmed elk and deer. Cases of CWD occur in at least 17 states, two Canadian provinces, and South Korea (Williams et al.

CLINICAL EFFECTS AND DIAGNOSIS Reported CWD lesions and their anatomic distributions are similar for deer and elk, and broadly resemble those of scrapie, BSE, trans­missible mink encephalopathy, and the human spongiform encephalopathies (Williams and Young 1993); chronic wasting disease is most similar to scrapie and includes early, wide­spread distribution of disease-associated prion protein in lymphoid tissues, with later involve­ment of central nervous system and peripheral tissues; this distribution likely enhances hori­zontal transmission (Williams 2005).

The medulla oblongata, especially the obex, is a consistent PrPcwd-positive tissue. (Peters et al. 2000). Prominent sites for lesions in the brain and spinal cord include the myelen­cephalon, diencephalon, and rhinencephalon; there are no consistent differences in lesions between captive mule deer and naturally occurring cases (Spraker et al. 2002b). While prion diagnosis most commonly has been done with an immunohistochemistry (IHC) assay (O'Rourke et al. 2000), a monoclonal antibody also is available for IHC staining (Spraker et al. 2002a), as is a western blot test (Kreeger et al. 2006). Infectious prion particles also can be identified in blood and urine with a transgenic mouse bioassay (Haley et al. 2009).

Genetic differences are linked with apparent susceptibility to CWD among free-living popu­lations of cervids. Among elk, variability at PrP codon 132 was correlated to susceptibil­ity to CWD (O'Rourke et al.

population effects Early reported preva­lences in Colorado and Wyoming were highest (4.9%) among mule deer, followed by white­tailed deer (2.1%) and elk (0.5%). Based on popu­lation models, it was hypothesized that, in a new population, about 15 to 20 years was required for CWD prevalences to reach 1% and about 37 to 50 years to reach 15%; however, cervid popula­tion declines also were predicted once CWD prevalence exceeded 5% (Miller et al. 2000).

Prevalence of CWD increased among male mule deer until about 5 to 6 years of age, and then declined, but there was no age-related pattern among females; overall, prevalence of CWD continued to increase in the sites moni­tored (Miller and Conner 2005). In Wisconsin, CWD also was more prevalent among male white-tailed deer and increased with age; fawns as young as 5 months tested positive (Grear et al. 2006).

On assessing human land use in Colorado, increased prevalence of CWD occurred among male mule deer in developed areas; there was considerable variation among the study sites, evidence that broader landscape scale factors may be influential (Farnsworth et al. 2005). More recently, prevalences as high as 50% have occurred in free-ranging deer populations, and over 90% in captive deer herds in some areas of the United States and Canada (Gilch et al. 2011).

Infection by CWD prions lowered the mean life spans of 2-year-old deer from 5.2 additional years to i. 6 additional years; it also increased the rate of mountain lions preying on deer by nearly four-fold. Yet despite this selective pre­dation, about one-fourth of the adult deer still remained infected.

special problems Continued decline of deer populations infected with CWD (Miller et al. 2008) continues to be a significant con­cern. A second concern has been expressed about potential transmission of CWD prions to humans (Miller and Williams 2004). Such worries are a natural outcome of the observa­tion that BSE was transmitted in an altered form from cattle to humans, causing a new variant of Creutzfeldt-Jakob disease (Brown et al. 2001).

There are several aspects to consider. Of primary concern is ensuring that no new prion disease affects humans (O'Brien 2000). Another concern is economic; based on the considerable impact of the BSE food scare on the UK economy (Lloyd et al. 2006), suspicion of a public health risk, even if not justified, could sharply curtail deer hunting, with an enormous loss of the essen­tial funding it generates for use by states in wild­life management. Such a change could have far- reaching effects.

Transmission of a form of the CWD prion disease to humans currently seems very remote. There is strong evidence for a molecular-level species barrier that likely limits the suscepti­bility of non-cervid species to the PrP^cwd prion (Raymond et al. 2000, Kong et al. 2005). Yet others argue that food-borne transmission of BSE to humans is already established (Belay et al. 2004), and if transmission ever was fea­sible for a mutated CWD prion, food-borne transmission of CWD could represent a risk for humans, although there is no evidence for this risk; however, infectious prions do occur in the skeletal muscles of deer (Angers et al. 2006) and in elk antler velvet (Angers et al. 2009), both of which are consumed by humans.

control There currently are no reports of effective control of CWD in wild cervid popu­lations. Following an assessment of the BSE events in British cattle herds, it was argued that a simple noninterventionist approach may be most sensible (Dnes 1996).

Based on computer models of selective cull­ing programs among mule deer populations, it was recommended to initiate population con­trol while CWD prevalence is 3.7% annually from 2002 to 2011 (Alba et al. 2013). Those species red- listed by the International Union for Conserva­tion of Nature (IUCN) declined an average of ιι.6% annually. All subsets of data examined showed a declining trend, including species in the IUCN Least Concern category. Thus, amphibian declines may be more widespread and severe than previously realized (Alba et al. 2013).

Concerns about global amphibian declines were expressed in the scientific community in the late 1980s (Wake 1991, 2007), but there is some uncertainty as to when these global declines began. Some argue the declines began in the late 1950s, peaked in the 1960s, and have continued at a reduced rate since (Houlahan et al. 2000). Others use the same data to argue that a significant global decline has occurred only since the 1990s (Alford et al. 2001).

Many causes of amphibian declines have been proposed. Pathogenic organisms, such as bacteria, fungi (Blaustein et al. 1994, Longcore et al. 1999), viruses (Jancovich et al. 1997), and trematode infections (Johnson et al. 1999), are identified as contributing causes. Chemicals, including retinoids (Gardiner and Hoppe 1999, Sessions et al. 1999) and toxic pollutants from anthropogenic chemicals (Ouellet et al. 1997), may be important. Habi­tat changes associated with general weather and global climate change (Pounds et al. 1999), as well as human-caused separations of essential habitats used by different life his­tory stages of a species (habitat split) (Becker et al. 2007), may be significant contributors. Other potential problems include introduced species (Kiesecker and Blaustein 1998), increased predation, UV-B radiation associ­ated with ozone depletion (Blaustein et al. 1997, Blaustein and Belden 2003), as well as interactions among some of these factors (Alford and Richards 1999, Blaustein and Kiesecker 2002). There has been considerable interest and concern among herpetologists and ecologists to better clarify the causes of these declines.

Pathogenic Microorganisms

Pathogenic microorganisms can have major impacts on amphibian populations. The Office International des Epizooties (OIE) ad hoc Group on Amphibian Diseases has recognized two pathogens of particular impor­tance for amphibians: the fungus Batracho- chytrium dendrobatidis (Bd) and viruses of the genus Ranavirus, Family Iridoviridae (Hyatt et al. 2007). Epizootics from both Bd and iri- doviruses (Cunningham et al. 1996, Jancovich et al. 1997, Green et al. 2002) are increasing in frequency (Daszak et al. 1999, Blaustein and Kiesecker 2002).

Chytridiomycosis, infection by the fungus

B. dendrobatidis, is perceived as a significant con­tributor to the worldwide amphibian declines. It has been associated with mortality among many wild populations of frogs and toads, and has been implicated in dramatic population declines of numerous amphibian species, and even complete extinction of some (Daszak et al. 2003, Schloegel et al. 2006). This fungus has been linked to mass mortalities of wild amphib - ian populations in North America (Bradley et al. 2002, Green et al. 2002), Central America (Berger et al. 1998, Lips et al. 2006), the Carib­bean (Lips et al. 2003, Alemu et al. 2008), South America (Ron and Merino 2000, Ron et al. 2003, Lampo et al. 2007), Australia (Berger et al. 1998), New Zealand (Bishop 2000), and Europe (Bosch et al. 2001). The effects of this parasite have been particularly devastating among critically endangered species (Lampo et al. 2007, Alemu et al. 2008). There is a link between greater complexity of amphibian communities and a higher likelihood of infec­tion by B. dendrobatidis (Olson et al. 2013).

Chytridiomycosis also is considered an emerging disease for amphibians (Daszak et al. 1999). An emerging disease can result either from a novel pathogen invading an area or from an endemic pathogen undergoing an increase in host range or pathogenicity (Rachowicz et al. 2005); there has been consid­erable interest in determining the basis for the emergence of chytridiomycosis in amphibians.

Overall, this chytrid fungus likely is a novel pathogen to most areas. In Central America and western North America, researchers have documented amphibian populations healthy in the absence of B. dendrobatidis, but suffering acute mortalities and population declines immediately following detection of the patho­gen (Rachowicz et al. 2005, Lips et al. 2006). At a site in Panama, 6 years of relative stability among amphibian populations was followed by a mass mortality and loss of amphibian biodiversity across eight families of frogs and salamanders subsequent to the rapid appear­ance of B. dendrobatidis (Lips et al. 2006).

In a California study, there was considerable evidence that this fungus was a novel patho­gen based on its low genetic diversity, lack of amphibian-host specificity, little correlation between fungal genotype and geography, local frog extirpation by a single fungal genotype, and evidence of human-assisted fungus migration (Morgan et al. 2007). But in support of endemism, there also were some diverse, recovering populations at a local site; thus neither epidemic spread nor endemism alone explained amphibian decline of that area (Morgan et al. 2007). Also, because of high infection levels among affected species, some apparently healthy amphibian species may serve as reservoirs once B. dendrobatidis becomes present in an area (Johnson 2006).

In Costa Rica, amphibian declines from

B. dendrobatidis were linked to global warm­ing, and it was proposed that global warming exacerbated chytrid outbreaks in Central and South America (Pounds et al. 2006). However, this proposed relationship is disputed, and the outbreaks in Central and South America may be explained better as the result of multiple introductions of B. dendrobatidis, as evidenced by the epidemics moving across the affected regions in a wave-like pattern (Lips et al. 2008). Other amphibian declines in Costa Rica over a 35-year period had little relation to chytrids or climate change (Whitfield et al. 2007). Like­wise, in another study assessing B. dendroba­tidis and weather as likely causes of population declines of boreal toads (Bufo boreas), the popu­lation model provided relatively strong evidence for chytrid-induced mortality, but very little evidence that weather was a significant con­tributor (Scherer et al. 2005). An increase in amphibian commerce also has been perceived to be an important contributor to the spread of

B. dendrobatidis (Picco and Collins 2008).

The innate defense mechanism may be an important aspect of amphibian immunity, including the production of antimicrobial pep­tides in granular glands in the skin that are emptied onto the skin when an amphibian is injured. At least six peptides found in amphib­ians can kill or inhibit growth of the fungi B. dendrobatidis and Basidiobolus ranarum, but not the bacterium Aeromonas hydrophila (Rollins- Smith et al. 2002).

Amphibian immune responses to patho­genic agents, including chytrids, can be exacer­bated by a variety of factors (Carey et al. 1999). Contaminants may exacerbate chytrid out­breaks (Pounds 2001). Increasing UV radiation also has been proposed as a potential co-factor in outbreaks of B. dendrobatidis in the western United States (Blaustein and Kiesecker 2002), but this relationship also has been challenged (Garcia et al. 2006).

Some argue that the fungus is not the main cause for population declines, even in areas where it commonly is reported as infecting amphibians (Burgin et al. 2005, McCallum 2005, Heard et al. 2011). Ranaviruses have a nearly worldwide distribution and can have serious impacts (Lesbarreres et al. 2011); they were the most common cause of amphibian mortality reported among 64 morbidity and mortality events in the United States (Green et al. 2002). However, mortality from rana- viruses affected only widespread and abun­dant amphibian species, and was associated with high population densities; in contrast to chytrid infections, there was no link to any population decline (Green et al. 2002).

Many pathogenic bacteria can cause mortality in amphibians (Worthylake and Hovingh 1989, Garner 2003), and some could contribute to population declines. For example, Aeromonas hydrophila occurs in amphibians (Hird et al. 1981, Garner 2003) and commonly causes mortality through “red-leg” and other syndromes (Dusi 1949, Glorioso et al. 1974, Hubbard 1981, Bravo Farinas et al. 1989, Taylor et al. 1999, Garner 2003). However, these bacteria generally are not associated with ongoing significant population impacts on amphibians.

Amphibian Deformities

Some amphibian declines are associated with amphibian deformities. The widespread appearance of malformed amphibians has been evident since the mid-1990s (Johnson and Sutherland 2003). In 1995, a group of Minnesota school children encountered a large number of northern leopard frogs (Rana pipiens) with extra digits and limbs or missing digits and limbs (Kaiser 1997); these observa­tions captured the interest of both the scientific community and the general public. Most hypotheses on the causes of deformi­ties now fall into two broad categories: trema­tode infections and chemical contaminants (Kiesecker 2002).

Despite the recent attention to amphibian deformities, they are an older problem (Kaiser 2003), and occasional amphibian deformities have been known for centuries (Ouellet 2000). However, they have increased greatly in severity, sometimes affecting 15 to 90% of a population, and involving several species at a single site (Ouellet et al. 1997, Johnson et al. 2002).

The role ofthe digenetic trematode Ribeiroia ondatrae as one cause of amphibian deformi­ties is well established (Johnson et al. 2004), and there are accounts of mass malforma­tions associated with R. ondatrae that go back to the 1940s (Johnson et al. 2003). The reported prevalence of trematode-induced malformations has increased since these ear­lier reports (Johnson et al. 2003), as have the reports of all malformations (McCallum and Trauth 2003).

Many malformed amphibians infected with R. ondatrae die prior to sexual matu­rity, causing some to consider R. ondatrae an important contributor to the widespread declines among amphibian populations (Johnson et al. 2001).

Malformations may result from synergis­tic effects between trematode infections and other factors. For example, salamander limbs exposed to both injury and R. ondatrae infection had three to five times more abnor­malities than those exposed to either factor alone (Johnson et al. 2006b). Further, eutro­phication can change habitat characteristics and may contribute to the emergence and suc­cess of R. ondatrae in amphibians (Johnson et al. 2007).

Malformations also may result from syn­ergistic effects between trematode infection and pesticide exposures (Kiesecker 2002). In some cases, chemical contamination alone may be an important cause of amphibian mal­formations. For example, in a study of several thousand hylid and ranid metamorphs among 42 wetlands in Vermont, the prevalence of limb malformation varied from 0 to 10%; variation in prevalence was most closely linked with prox­imity to agricultural land; chemical toxicants were proposed as the most likely reason (Taylor et al. 2005). There was no evidence for the sig­nificant presence or involvement of R. ondatrae in this same region (Skelly et al. 2007). Some sites with malformations have no evidence of chemical contaminations (Gillilland et al. 2001), and chemicals alone are not adequate to explain many of the deformities observed.

Other Causes of Amphibian Declines

Toxic pollutants have been proposed as pos­sible contributors to amphibian declines, but cause-and-effect relationships have been dif­ficult to establish. Because of the considerable inconsistencies in biogeographical habitats and taxonomic groups studied, the significance of pollution in amphibian declines still cannot be assessed at a global scale or even in regions where most declines occur (Schiesari et al. 2007). There is interest currently in clarify­ing the role of endocrine disruptors as poten­tial contributors to global amphibian declines (Pickford et al. 2007).

Amphibian declines illustrate the complex­ity of factors that may contribute to a wildlife problem, as well as the even greater complexity of potential interactions among these factors. Besides the difficulties in accurately measur­ing declines (Alford et al. 2001), amphibian declines likely are affected by several potential mortality factors that interact with each other and also are influenced by environmental fac­tors. These relations are further complicated if the pathogens are immunosuppressive, or if global warming, chemical contamination, and other environmental changes are stress­ing hosts and reducing their innate or adaptive immune systems, leading to immunosuppres­sion (Carey et al. 1999).

Questions to Consider

1. Based on the information in this case report, and additional knowledge you may have on the topic, summarize in a sentence the key elements you believe to be contrib­uting to global amphibian declines. Briefly justify your response.

2. What additional information do you believe is needed to understand the underlying causes of amphibian declines? Design one study to address that issue.

3. As a manager suspecting an amphibian decline on your refuge, how would you initially address this concern? What would be your long-term plan? Justify your response.

4. What lessons do you believe can be drawn from this case study?

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