**6. Diagnosis of CWD**

Classification of CWD as TSE required histopathological examination of brains from diseased animals [60]. These subsequent analyses [80] were limited to microscopic evaluation of the CNS to detect neuropathological features typical of, but not necessarily exclusive to, prion diseases including neuronal vacuolation, attendant spongiform degeneration of the neuropil, reactive astrocytic gliosis, and florid amyloid plaques.

*Wildlife Population Monitoring*

blockage in these cells [185].

**5.3 Mononuclear phagocytic cells**

of prions when compared to the large intestinal GALT, such as the cecal patches [175–178]. The rectoanal mucosa-associated lymphoid tissues (RAMALT), which also accumulate prion in naturally occurring cases of scrapie in sheep and goats, as well as in CWD in cervids, also displayed reduced incidence of prion accumulation at the earlier stages of disease [96, 179–184]. Taken together, these studies point out to the fact that prions first replicate within the GALT of the upper gastrointestinal tract and then spread to the local lymph nodes and to other SLT, such as the spleen.

A heterogeneous population comprised of monocytes, conventional dendritic cells (DC), and macrophages seem to have also an important role in host infection. From a different lineage of the stromal-derived FDC, conventional DC [157, 158, 186], the antigen-presenting cells par excellence*,* are also involved in the transport of antigens both within Peyer's patches and toward the mesenteric lymph nodes [187–189]. Their strategic position allows them not only to sample their local environment for pathogens and their antigens, processing them, but also to capture and retain unprocessed (native) antigens [190, 191]. Processed or native antigens are then delivered to B and T cells as DC undergo maturation and migrate toward the local SLT to initiate an immune response [190–193]. Prions are wrapped by DC either by complement opsonization, namely, by C1q and C3 components [155, 194], or in a non-specifically manner, through fluid-phase micropinocytosis [144]. Specific chemokines have been reported to play important roles in prion infections by regulating FDC and DC migration within the SLT. CXCL13 chemokine is expressed by FDC and other stromal cells in the B-cell follicles of SLT and recruits CXCR5-expressing cells toward them [195, 196]. On the other hand, CXCL13- CXCR5 signaling mediates the migration of certain populations of DC toward the FDC-containing B-cell follicles [192, 193, 197], and studies have shown that the early accumulation of prions upon FDC in Peyer's patches was impaired and disease susceptibility reduced, in the absence of CXCR5 expression by DC [198]. The interplay between FDC and DC during prion infection was also patent by the finding that early replication of prions upon FDC in the local SLT was inhibited when conventional DC were transiently depleted at the time of exposure [199–202]. Apparently, after being transferred across the gut epithelium either directly by enterocytes or by *M* cells, prions may be then internalized by DC [143, 199] and propagated by them toward FDC in Peyer's patches [198]. The ability of DC to migrate into B-cell follicles [192, 193, 203] suggests that they may also propagate

were able

expression

Additionally, it was observed that FDC in the spleen expressing PrPc

to display high levels of prion replication that was impeded after PrPc

prions to and within SLT, namely, to the mesenteric lymph nodes.

Regardless of FDC and DC roles in the propagation of prions, there are other cell populations that appear to phagocytose and destroy them [204, 205]. In fact, it was observed that tingible body macrophages of the germinal centers display heavy PrPsc accumulations within their endosomal compartments during prion disease [143, 159, 185]. It is speculated that macrophages may scavenge and degrade prions in an attempt to protect the host from infection, as it is suggested by studies that show that macrophage depletion resulted in an enhanced accumulation of PrPsc within SLT [206, 207].

After being acquired orally, prions undergo replication and accumulation upon FDC, reaching a threshold above which neuroinvasion occurs. Experiments

**118**

**5.4 Neuroinvasion**

#### **6.1 Clinical signs and macroscopic lesions**

The main clinical features of progressive CWD disease in adults is weight loss and behavioral changes that typically span weeks or months. Besides weight loss, diseased animals may show a wide range of behavior changes for a long time that include somnolence, lassitude, repetitive walking, polydipsia, and polyuria. Gradually, the affected cervids may exhibit ataxia, mainly in the hind limbs, head tremors, drooped ears, and fixed gaze, and may have difficulty in swallowing. In the end stage, they may show signs of sialorrhea and teeth grinding [235, 236]. The clinical signs may be subtle requiring that the animal keeper/observer is familiar with and has some knowledge of the normal behavior of the species. As a result, the disease is most commonly detected in emaciated cervids hunted, killed, or injured by road accidents or found dead [235].

At necropsy, severe emaciation, poor hair coat condition, megaesophagus, froth or watery rumen contents (often containing sand), abomasal or omasal ulcers, serous atrophy of bone marrow and pericardial fat, enlarged adrenal glands, muscle atrophy [237], and aspiration pneumonia are common findings. As the clinical signs and the macroscopic findings at necropsy are not pathognomonic [49], other diseases must be ruled out, namely, listeriosis, meningoencephalitis, brain abscesses, starvation, nutritional deficiencies or severe parasitism, bluetongue disease, epizootic hemorrhagic disease, meningeal worms (*Parelaphostrongylus tenuis*), and locoweed intoxication [49, 235, 238].

TSEs present very specific diagnostic challenges because of their strain variation, their very long incubation period, and the lack of pathognomonic clinical signs. Moreover, the disease pathogenesis can range both among and within species due to the influence of strain, host genotype, or a combination of the two [239].

#### **6.2 Rapid screening testing**

The initial diagnosis of TSE in all species was based on passive surveillance (e.g., clinical presentation) and assessment via conventional histopathology and subsequently detection of PrPsc by IHC, both time-consuming, technically demanding, and expensive for large surveillance plans [77]. For that reason, the development of commercial rapid immunologically based screening tests (RT) for the detection of PrPsc in tissue homogenates allowed a large-scale active surveillance program for TSE in cattle and small ruminants.

These were originally developed for the detection of BSE in the cattle brain, and extensive formal test evaluation of a wide range of these RT was undertaken at Europe level [240, 241]. Mainly, there are two types of these RT kits allowing the purification, concentration, and detection of PrPsc from samples of tissues obtained from infected animals. Both assays consisted in an immuno-enzymatic technique: one is a sandwich format using two antibodies for the detection of the antigen PrPsc after proteinase K digestion (e.g., TeSeE BioRad®), and the other uses a PrPsc-specific ligand immobilized on the surface of the antigen-capture plate (e.g., HerdCheck IDEXX®). Both are suitable for the surveillance of CWD as diagnostic methods for the detection of the disease [77].

The limited experience in CWD so far in Europe has not allowed us to know the best tissue to be proposed as the most sensitive for surveillance in all circumstances. Sampling only the brain stem could reduce the diagnostic sensitivity for strainhost combinations that are characterized by early lymphoid PrPsc accumulation. Consequently, lymphoid tissues (preferably the tonsils, retropharyngeal lymph nodes, and lymphoid tissues of rectal mucosa) and the brain stem at the level of the obex should be tested to maximize the diagnostic sensitivity in any surveillance program [77].

**121**

*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control*

Like in other recognized animal TSEs, confirmation of an initial CWD "screening positive" (or "suspect") sample can be undertaken by histopathological examination and immunodetection methods either by IHC or Western blot (WB). The former enables PrPsc accumulations to be assessed regarding the types of deposition and anatomical distribution (including cellular) location, while the latter gives some classification data based on the molecular mass and glycosylation profiles of

Histopathologic lesions in the encephalon are similar to those described for ruminant TSEs: perikaryon neuronal vacuoles, microcavitation of the gray matter, astrogliosis, neuronal degeneration and loss, and PrP positively labeled prion deposits and plaques [242]. Duration of clinical disease does not significantly affect the distribution or severity of lesions, and inflammatory cell response is not apparent, unless associated with intercurrent disease [49]. In clinically affected cervids, examination of well-fixed medulla oblongata at the level of the obex is considered sufficient for diagnosis of CWD [80], and sections at this level were used for CWD

Neuropathology varies slightly between deer and elk: elk have more severe lesions in the thalamus and in some white matter areas. Congo red birefringent and PAS-positive amyloid plaques have been seen in the deer brain but not in elk [80]. The cerebral cortex and basal ganglia of the elk with CWD show minimal fine spongiform degeneration and astrogliosis with focal distribution. The spongiform degeneration with astrogliosis is more prominent in the thalamus where it forms clusters of coarse vacuoles. Fine spongiosis, often in small clusters, is present in the molecular layer of the cerebellum, in dorsal nuclei of the pons, and in the substantia gelatinosa of the spinal cord. Occasional large neurons in various nuclei of the pons show a vacuole [243]. Amyloid plaques are relatively common and can be detected on hematoxylin and eosin (HE)-stained brain sections, most prominently and with decreasing frequency, in white-tailed deer, mule deer, and elk [49]. Neuronal loss and astrogliosis are minimal except for the molecular layer of the cerebellum, which shows rarefaction of granule cells with no indication of

The PrPsc immunostaining is consistently present in the cerebral cortex, basal ganglia, and thalamus. In the cerebellum the immunostaining is present in both molecular and granule cell layers as well as in the dentate nucleus. In the pons it is widespread over gray structures, whereas in the spinal cord, it is generally confined to the dorsal part of the dorsal horns [243]. Patterns of PrPsc deposition in CWD-affected cervid brains include perineuronal and perivascular accumulation, extracellular plaques and

Deposition of PrPsc occurs widely in lymphoid tissues (**Figure 5**) during CWD incubation in the absence of histologic lesions in these tissues, resembling classical scrapie in small ruminants [149, 244]. Therefore, lymphoid tissues are very useful for diagnostic purposes and surveillance. Nevertheless, in the referred atypical CWD, no PrPsc was detected in lymphoid tissue [31], similar to that described in heterozygote ARR sheep affected with classical scrapie as well as atypical scrapie. In those cases, lymphoid tissues infectivity should be further studied like it was in atypical scrapie, demonstrating that infectivity can accumulate in lymphoid tissues

granular deposits, and subependymal and subpial deposition [49] (**Figure 5**). Recently, a different neuropathologic phenotype, characterized mainly by intraneuronal deposition of PrPsc and few immunostaining at the dorsal motor of the vagus nerve, was observed in the putative atypical CWD detected in Norwegian

surveillance before availability of immunohistochemistry [49].

*DOI: http://dx.doi.org/10.5772/intechopen.85797*

**6.3 Confirmatory testing**

the PrPsc [77].

apoptosis [243].

moose [31].

even with no detectable PrPsc [245].

*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control DOI: http://dx.doi.org/10.5772/intechopen.85797*

### **6.3 Confirmatory testing**

*Wildlife Population Monitoring*

**6.1 Clinical signs and macroscopic lesions**

locoweed intoxication [49, 235, 238].

**6.2 Rapid screening testing**

TSE in cattle and small ruminants.

methods for the detection of the disease [77].

The main clinical features of progressive CWD disease in adults is weight loss and behavioral changes that typically span weeks or months. Besides weight loss, diseased animals may show a wide range of behavior changes for a long time that include somnolence, lassitude, repetitive walking, polydipsia, and polyuria. Gradually, the affected cervids may exhibit ataxia, mainly in the hind limbs, head tremors, drooped ears, and fixed gaze, and may have difficulty in swallowing. In the end stage, they may show signs of sialorrhea and teeth grinding [235, 236]. The clinical signs may be subtle requiring that the animal keeper/observer is familiar with and has some knowledge of the normal behavior of the species. As a result, the disease is most commonly detected in emaciated cervids hunted, killed, or injured by road accidents or found dead [235]. At necropsy, severe emaciation, poor hair coat condition, megaesophagus, froth

or watery rumen contents (often containing sand), abomasal or omasal ulcers, serous atrophy of bone marrow and pericardial fat, enlarged adrenal glands, muscle atrophy [237], and aspiration pneumonia are common findings. As the clinical signs and the macroscopic findings at necropsy are not pathognomonic [49], other diseases must be ruled out, namely, listeriosis, meningoencephalitis, brain abscesses, starvation, nutritional deficiencies or severe parasitism, bluetongue disease, epizootic hemorrhagic disease, meningeal worms (*Parelaphostrongylus tenuis*), and

TSEs present very specific diagnostic challenges because of their strain variation, their very long incubation period, and the lack of pathognomonic clinical signs. Moreover, the disease pathogenesis can range both among and within species due to the influence of strain, host genotype, or a combination of the two [239].

The initial diagnosis of TSE in all species was based on passive surveillance (e.g., clinical presentation) and assessment via conventional histopathology and subsequently detection of PrPsc by IHC, both time-consuming, technically demanding, and expensive for large surveillance plans [77]. For that reason, the development of commercial rapid immunologically based screening tests (RT) for the detection of PrPsc in tissue homogenates allowed a large-scale active surveillance program for

These were originally developed for the detection of BSE in the cattle brain, and extensive formal test evaluation of a wide range of these RT was undertaken at Europe level [240, 241]. Mainly, there are two types of these RT kits allowing the purification, concentration, and detection of PrPsc from samples of tissues obtained from infected animals. Both assays consisted in an immuno-enzymatic technique: one is a sandwich format using two antibodies for the detection of the antigen PrPsc after proteinase K digestion (e.g., TeSeE BioRad®), and the other uses a PrPsc-specific ligand immobilized on the surface of the antigen-capture plate (e.g., HerdCheck IDEXX®). Both are suitable for the surveillance of CWD as diagnostic

The limited experience in CWD so far in Europe has not allowed us to know the best tissue to be proposed as the most sensitive for surveillance in all circumstances. Sampling only the brain stem could reduce the diagnostic sensitivity for strainhost combinations that are characterized by early lymphoid PrPsc accumulation. Consequently, lymphoid tissues (preferably the tonsils, retropharyngeal lymph nodes, and lymphoid tissues of rectal mucosa) and the brain stem at the level of the obex should be tested to maximize the diagnostic sensitivity in any surveillance

**120**

program [77].

Like in other recognized animal TSEs, confirmation of an initial CWD "screening positive" (or "suspect") sample can be undertaken by histopathological examination and immunodetection methods either by IHC or Western blot (WB). The former enables PrPsc accumulations to be assessed regarding the types of deposition and anatomical distribution (including cellular) location, while the latter gives some classification data based on the molecular mass and glycosylation profiles of the PrPsc [77].

Histopathologic lesions in the encephalon are similar to those described for ruminant TSEs: perikaryon neuronal vacuoles, microcavitation of the gray matter, astrogliosis, neuronal degeneration and loss, and PrP positively labeled prion deposits and plaques [242]. Duration of clinical disease does not significantly affect the distribution or severity of lesions, and inflammatory cell response is not apparent, unless associated with intercurrent disease [49]. In clinically affected cervids, examination of well-fixed medulla oblongata at the level of the obex is considered sufficient for diagnosis of CWD [80], and sections at this level were used for CWD surveillance before availability of immunohistochemistry [49].

Neuropathology varies slightly between deer and elk: elk have more severe lesions in the thalamus and in some white matter areas. Congo red birefringent and PAS-positive amyloid plaques have been seen in the deer brain but not in elk [80]. The cerebral cortex and basal ganglia of the elk with CWD show minimal fine spongiform degeneration and astrogliosis with focal distribution. The spongiform degeneration with astrogliosis is more prominent in the thalamus where it forms clusters of coarse vacuoles. Fine spongiosis, often in small clusters, is present in the molecular layer of the cerebellum, in dorsal nuclei of the pons, and in the substantia gelatinosa of the spinal cord. Occasional large neurons in various nuclei of the pons show a vacuole [243]. Amyloid plaques are relatively common and can be detected on hematoxylin and eosin (HE)-stained brain sections, most prominently and with decreasing frequency, in white-tailed deer, mule deer, and elk [49]. Neuronal loss and astrogliosis are minimal except for the molecular layer of the cerebellum, which shows rarefaction of granule cells with no indication of apoptosis [243].

The PrPsc immunostaining is consistently present in the cerebral cortex, basal ganglia, and thalamus. In the cerebellum the immunostaining is present in both molecular and granule cell layers as well as in the dentate nucleus. In the pons it is widespread over gray structures, whereas in the spinal cord, it is generally confined to the dorsal part of the dorsal horns [243]. Patterns of PrPsc deposition in CWD-affected cervid brains include perineuronal and perivascular accumulation, extracellular plaques and granular deposits, and subependymal and subpial deposition [49] (**Figure 5**).

Recently, a different neuropathologic phenotype, characterized mainly by intraneuronal deposition of PrPsc and few immunostaining at the dorsal motor of the vagus nerve, was observed in the putative atypical CWD detected in Norwegian moose [31].

Deposition of PrPsc occurs widely in lymphoid tissues (**Figure 5**) during CWD incubation in the absence of histologic lesions in these tissues, resembling classical scrapie in small ruminants [149, 244]. Therefore, lymphoid tissues are very useful for diagnostic purposes and surveillance. Nevertheless, in the referred atypical CWD, no PrPsc was detected in lymphoid tissue [31], similar to that described in heterozygote ARR sheep affected with classical scrapie as well as atypical scrapie. In those cases, lymphoid tissues infectivity should be further studied like it was in atypical scrapie, demonstrating that infectivity can accumulate in lymphoid tissues even with no detectable PrPsc [245].

#### **Figure 5.**

*PrPsc immunohistochemistry in CWD affected deer. (A) Perineuronal and granular deposits in neuropil at dorsal vagal nucleus (brainstem), X400; (B) Extracelullar plaque-like deposits (cerebral cortex), X200; (C) perivascular type (brainstem), X200; (D) distribution in both molecular and granular layers of the cerebellum, X200; (E) and (F) presence of PrPsc in several follicules in a lymph node, X100 and X200, respectivelly. (A–D) sections from CWD control samples kindly provided by professor Stefanie Czub (Canadian food inspection agency, National Center for animal diseases) in 2003; (E-F) sections from the CWD proficiency testing 2008 organized by the European reference laboratory for TSEs (APHA, Weybridge). (A-D) specific PrPsc signal was visualized with F99/97.6.1 monoclonal antibody (raised against bovine residues 220–225; VMRD Inc., Pullman, WA; 1:1250 dilution) StreptABC-alkaline phosphatase, new Fuchsin system (DAKO); (E and F) IHC with 2G11 monoclonal antibody (raised against ovine PrP peptide sequence 146-R154-R171–182; Institute Pourquier;1:200 dilution). All tissue sections were counterstained with Mayer's hematoxylin.*

If histopathological examination cannot be carried out due to poor state of the sample and/or IHC results which are not conclusive, then Western immunoblotting is the remaining confirmation method available. This technique relies on detergent extraction followed by treatment with proteinase K to digest any PrPc and detect only PrPsc by a specific antibody presenting bands that correspond to proteins within a range of molecular mass from 17 kDa (unglycosylated PrPsc) to 27 kD (diglycosylated PrPsc) [238].

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*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control*

CH1641 scrapie in sheep and goats and BSE and scrapie [246, 247].

noninvasive methods, such as blood, urine, feces, or saliva. During PMCA, the normal form of PrP (PrPc

WB and IHC allow a comparison of labelling patterns with antibodies that recognize different epitopes of the protein and help to clarify specific proteinase K cleavage inherent of strains type. This approach forms the basis of the discriminatory testing of small ruminant isolates, to differentiate between isolates that can be classified as scrapie, and those that are considered BSE-like by looking at the lower molecular weight for the unglycosylated protein band and the ratio of the monoand diglycosylated fragments, for example, between classical, atypical/Nor98, and

The Norwegian atypical CWD (Nor-16CWD) also presented an unusual PrPsc electrophoretic pattern distinguishable from previous CWD cases and from known ruminant prion diseases in Europe, with the possible exception of sheep CH1641. Transmission studies in several rodent models are ongoing to clarify if this different phenotype could reflect the presence of a new cervid prion strain in moose from

Regarding diagnosis in vivo, more sensitive diagnostic methods are desirable. Using in vitro conversion such as protein misfolding cycling amplification (PMCA) or real-time quaking-induced conversion assay (RT-QuIC), CWD prions are detectable already at a preclinical stage in specimen that can be obtained antemortem by

K-resistant PrP (PrPsc) using small amounts of infectious PrPsc. Continued recruit-

analogous to amplification of DNA by the polymerase chain reaction (PCR). This amplification process enhances detection sensitivity by several orders of magnitude as compared to WB and has been used to confirm the presence of CWD prions in

In the assay referred to as real-time quaking-induced conversion (RT-QuIC) [250], prion seeds are thought to induce recombinant PrP to adopt a β-sheet structure. Thioflavin T, added to the reaction, is incorporated into the growing amyloid causing an altered spectrofluorimetric emission pattern, which is

CWD still represents a challenge in TSE research for which transmission and dissemination remain unchecked. The tools of diagnostic available for identifying infected animals have steadily progressed over time from clinical and pathological descriptions to antibody–antigen-dependent immunoassays and more recently qualitative and quantitative prion amplification techniques. These tools have provided a deep understanding of disease pathogenesis and transmission and allowed animal health technicians to monitor the expanding geographical presence

Of further concern is the fact that CWD is the only transmissible spongiform encephalopathy known to be expanding both geographically and in prevalence [5]. After the confirmation in 2016 of two cases of CWD in a wild reindeer (April) and a wild moose (May) in Norway (the first CWD cases in Europe), this country intensified its surveillance program for CWD in cervids and detected a number

) is converted into protease

by PrPsc are accomplished by sonication in a process

*DOI: http://dx.doi.org/10.5772/intechopen.85797*

Norway [31].

**6.4 New diagnostic methods**

ment and conversion of PrPc

muscle [248] and feces [249].

monitored over time.

of CWD [251].

**7.1 Surveillance**

**7. Surveillance and control of CWD**

*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control DOI: http://dx.doi.org/10.5772/intechopen.85797*

WB and IHC allow a comparison of labelling patterns with antibodies that recognize different epitopes of the protein and help to clarify specific proteinase K cleavage inherent of strains type. This approach forms the basis of the discriminatory testing of small ruminant isolates, to differentiate between isolates that can be classified as scrapie, and those that are considered BSE-like by looking at the lower molecular weight for the unglycosylated protein band and the ratio of the monoand diglycosylated fragments, for example, between classical, atypical/Nor98, and CH1641 scrapie in sheep and goats and BSE and scrapie [246, 247].

The Norwegian atypical CWD (Nor-16CWD) also presented an unusual PrPsc electrophoretic pattern distinguishable from previous CWD cases and from known ruminant prion diseases in Europe, with the possible exception of sheep CH1641. Transmission studies in several rodent models are ongoing to clarify if this different phenotype could reflect the presence of a new cervid prion strain in moose from Norway [31].

#### **6.4 New diagnostic methods**

*Wildlife Population Monitoring*

**122**

**Figure 5.**

*hematoxylin.*

(diglycosylated PrPsc) [238].

If histopathological examination cannot be carried out due to poor state of the sample and/or IHC results which are not conclusive, then Western immunoblotting is the remaining confirmation method available. This technique relies on detergent

*PrPsc immunohistochemistry in CWD affected deer. (A) Perineuronal and granular deposits in neuropil at dorsal vagal nucleus (brainstem), X400; (B) Extracelullar plaque-like deposits (cerebral cortex), X200; (C) perivascular type (brainstem), X200; (D) distribution in both molecular and granular layers of the cerebellum, X200; (E) and (F) presence of PrPsc in several follicules in a lymph node, X100 and X200, respectivelly. (A–D) sections from CWD control samples kindly provided by professor Stefanie Czub (Canadian food inspection agency, National Center for animal diseases) in 2003; (E-F) sections from the CWD proficiency testing 2008 organized by the European reference laboratory for TSEs (APHA, Weybridge). (A-D) specific PrPsc signal was visualized with F99/97.6.1 monoclonal antibody (raised against bovine residues 220–225; VMRD Inc., Pullman, WA; 1:1250 dilution) StreptABC-alkaline phosphatase, new Fuchsin system (DAKO); (E and F) IHC with 2G11 monoclonal antibody (raised against ovine PrP peptide sequence 146-R154-R171–182; Institute Pourquier;1:200 dilution). All tissue sections were counterstained with Mayer's* 

and detect

extraction followed by treatment with proteinase K to digest any PrPc

only PrPsc by a specific antibody presenting bands that correspond to proteins within a range of molecular mass from 17 kDa (unglycosylated PrPsc) to 27 kD

Regarding diagnosis in vivo, more sensitive diagnostic methods are desirable. Using in vitro conversion such as protein misfolding cycling amplification (PMCA) or real-time quaking-induced conversion assay (RT-QuIC), CWD prions are detectable already at a preclinical stage in specimen that can be obtained antemortem by noninvasive methods, such as blood, urine, feces, or saliva.

During PMCA, the normal form of PrP (PrPc ) is converted into protease K-resistant PrP (PrPsc) using small amounts of infectious PrPsc. Continued recruitment and conversion of PrPc by PrPsc are accomplished by sonication in a process analogous to amplification of DNA by the polymerase chain reaction (PCR). This amplification process enhances detection sensitivity by several orders of magnitude as compared to WB and has been used to confirm the presence of CWD prions in muscle [248] and feces [249].

In the assay referred to as real-time quaking-induced conversion (RT-QuIC) [250], prion seeds are thought to induce recombinant PrP to adopt a β-sheet structure. Thioflavin T, added to the reaction, is incorporated into the growing amyloid causing an altered spectrofluorimetric emission pattern, which is monitored over time.

CWD still represents a challenge in TSE research for which transmission and dissemination remain unchecked. The tools of diagnostic available for identifying infected animals have steadily progressed over time from clinical and pathological descriptions to antibody–antigen-dependent immunoassays and more recently qualitative and quantitative prion amplification techniques. These tools have provided a deep understanding of disease pathogenesis and transmission and allowed animal health technicians to monitor the expanding geographical presence of CWD [251].

## **7. Surveillance and control of CWD**

#### **7.1 Surveillance**

Of further concern is the fact that CWD is the only transmissible spongiform encephalopathy known to be expanding both geographically and in prevalence [5].

After the confirmation in 2016 of two cases of CWD in a wild reindeer (April) and a wild moose (May) in Norway (the first CWD cases in Europe), this country intensified its surveillance program for CWD in cervids and detected a number

of other cases of CWD in reindeers and in moose. In the meantime, the European Commission (EC) requested the European Food Safety Agency (EFSA) to recommend surveillance activities and, if necessary, additional animal health risk-based measures to prevent the introduction of the disease and the spread into/within the European Union (EU), specifically Estonia, Finland, Iceland, Latvia, Lithuania, Norway, Poland, and Sweden [252]. Based on EFSA opinion, the EC places in force the Commission Regulation (EU) 2017/1972, amending Annexes I and III of Regulation (EC) No. 999/2001 as regards a surveillance program for chronic wasting disease in cervids in Estonia, Finland, Latvia, Lithuania, Poland, and Sweden and repealing Commission Decision 2007/182/EC (which provided requirements for a survey on CWD in cervids which was carried out from 2007 to 2010). According to this new regulation, the member states (MSs), which have a wild and/or farmed and/or semidomesticated population of moose band/or reindeer (aforementioned MSs), shall carry out a 3-year monitoring program for CWD from 1 January 2018 to 31 December 2020.

The 3-year CWD monitoring program shall cover animals over 12 months of age, estimated on the basis of dentition, obvious signs of maturity, or any other reliable information, of the following cervid species:


According to some authors [252, 253], in order to avoid unbiased prevalence or incidence estimates and to obtain a representative set of animals, a random sampling is always recommended to design monitoring program. This classical approach may be possible to achieve for farmed cervids, but, for wildlife population, random sampling may not be possible to design as sampling frames are not available [252]. However, when disease detection is the first objective, a sampling targeting high-risk animals may be more efficient. That, in fact, was the case of the Norwegian monitoring program that detected the two CWD cases through sampling of "suspect" cervids [252].

Since the aims of the proposed surveillance system were to detect disease in countries where CWD has not yet been detected and to estimate prevalence in areas where disease has been detected, the 3-year surveillance system includes a twostage sampling program (Commission Regulation (EU) 2017/1972):


To date, according to Commission Regulation (EU) 2017/1972, member states shall carry out additional monitoring for TSEs in cervids based on a risk assessment which may take into account the detection of a TSE in cervids in the same or

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*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control*

neighboring regions. Efforts to prevent the introduction of CWD should consider whether prior surveillance data are sufficient to assure that the disease has not

Based on the main provisions in the TSE regulation currently applicable to CWD, TSE-positive cases in cervids must be notified to the commission and the member states (Article 11 of Regulation (EC) No. 999/2001), and all parts of the body of a cervid positive for TSE must be sent to disposal as category 1 materials in accordance with the Animal By-Product Regulation 10 (Article 13.1. (a) of

As it was previously referred (see Section 4), CWD is a very contagious form of TSE. Therefore, control in captive cervids is not easy and even more in the wildlife. According to Uehlinger and colleagues [254], the combination of direct and environmental transmission of CWD prions; the persistence of environmental infectious prion for at least for 2.5 years [255]; the absence of a rapid, practical, and reliable antemortem field tests for detecting preclinical CWD; and the nonavailability of licensed vaccines nor therapies together with the impossibility of quarantine suspicions populations in wild

The management of CWD in affected populations is a proven difficult task, based on population reduction in the case of wild deer or in quarantine and depop-

Measures to control the spread of the disease will depend on whether we are dealing with commercially exploited herds, confined to demarcated areas, or with wild populations of deer. In the first case, and similar to other infectious diseases and other livestock, programs to detect and eliminate positive animals may be used [94]. This task is not easy, however, as a shown by a 2018 study conducted on a private

farm in Colorado where the use of disease control management through rectal biopsy diagnosis was not able to prevent the increase in prevalence suggesting the need to refine management criteria or antemortem diagnostic methods [5].

Thus, the science available to inform effective CWD management and control strategies remains relatively incomplete as it was concluded by different studies [254, 256] during their review on CWD control in North America. Nevertheless, it may be stated that approaches for CWD management generally fall into three categories in order to prevent the introduction of the disease and its spread [252]:

a.Prevention of entry in free regions (where CWD is assumed absent or is not

Regarding preventive and containment strategies, it was referred that these measures tend to focus on regulations (e.g., bans on movements of live animals, carcasses, or specified risk materials) and efforts to prevent the introduction of CWD should consider whether prior surveillance data are sufficient to assure that

Haley and collaborators [94] suggest that outer mechanisms of disease control could be useful like the development of simple antemortem test to use prior to animal movements among farms, the breeding for resistant genotypes, the installation of effective fences to prevent transmission between commercial

b.Containment procedures of a CWD focus to avoid disease spread

c.Control/suppression in an affected herd or population

the disease has not already spread into the area of interest [252].

cervids make the control of this disease in wild populations very difficult.

*DOI: http://dx.doi.org/10.5772/intechopen.85797*

already spread into the area of interest.

ulation in commercially reared animals.

believed to occur)

Regulation (EC) No. 999/2001).

**7.2 TSE control**

*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control DOI: http://dx.doi.org/10.5772/intechopen.85797*

neighboring regions. Efforts to prevent the introduction of CWD should consider whether prior surveillance data are sufficient to assure that the disease has not already spread into the area of interest.

Based on the main provisions in the TSE regulation currently applicable to CWD, TSE-positive cases in cervids must be notified to the commission and the member states (Article 11 of Regulation (EC) No. 999/2001), and all parts of the body of a cervid positive for TSE must be sent to disposal as category 1 materials in accordance with the Animal By-Product Regulation 10 (Article 13.1. (a) of Regulation (EC) No. 999/2001).

### **7.2 TSE control**

*Wildlife Population Monitoring*

1 January 2018 to 31 December 2020.

• Roe deer (*Capreolus capreolus*)

• Red deer (*Cervus elaphus*)

• Moose (*Alces alces*)

reliable information, of the following cervid species:

• White-tailed deer (*Odocoileus virginianus*)

• Eurasian tundra reindeer (*Rangifer tarandus tarandus*)

• Finnish forest reindeer (*Rangifer tarandus fennicus*)

of other cases of CWD in reindeers and in moose. In the meantime, the European Commission (EC) requested the European Food Safety Agency (EFSA) to recommend surveillance activities and, if necessary, additional animal health risk-based measures to prevent the introduction of the disease and the spread into/within the European Union (EU), specifically Estonia, Finland, Iceland, Latvia, Lithuania, Norway, Poland, and Sweden [252]. Based on EFSA opinion, the EC places in force the Commission Regulation (EU) 2017/1972, amending Annexes I and III of Regulation (EC) No. 999/2001 as regards a surveillance program for chronic wasting disease in cervids in Estonia, Finland, Latvia, Lithuania, Poland, and Sweden and repealing Commission Decision 2007/182/EC (which provided requirements for a survey on CWD in cervids which was carried out from 2007 to 2010). According to this new regulation, the member states (MSs), which have a wild and/or farmed and/or semidomesticated population of moose band/or reindeer (aforementioned MSs), shall carry out a 3-year monitoring program for CWD from

The 3-year CWD monitoring program shall cover animals over 12 months of age, estimated on the basis of dentition, obvious signs of maturity, or any other

According to some authors [252, 253], in order to avoid unbiased prevalence or incidence estimates and to obtain a representative set of animals, a random sampling is always recommended to design monitoring program. This classical approach may be possible to achieve for farmed cervids, but, for wildlife population, random sampling may not be possible to design as sampling frames are not available [252]. However, when disease detection is the first objective, a sampling targeting high-risk animals may be more efficient. That, in fact, was the case of the Norwegian monitoring program that detected the two CWD cases through sampling of "suspect" cervids [252]. Since the aims of the proposed surveillance system were to detect disease in countries where CWD has not yet been detected and to estimate prevalence in areas where disease has been detected, the 3-year surveillance system includes a two-

• At the first stage, a random sampling is applied for wild/semidomesticated cervids [primary sampling (PSU)] corresponding to geographical areas containing cervid populations, whereas for farmed cervids they will correspond to farms.

• At the second stage, a convenience sampling is applied targeting high-risk

To date, according to Commission Regulation (EU) 2017/1972, member states shall carry out additional monitoring for TSEs in cervids based on a risk assessment which may take into account the detection of a TSE in cervids in the same or

stage sampling program (Commission Regulation (EU) 2017/1972):

animals of the selected species within PSU (**Figure 6**).

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As it was previously referred (see Section 4), CWD is a very contagious form of TSE. Therefore, control in captive cervids is not easy and even more in the wildlife. According to Uehlinger and colleagues [254], the combination of direct and environmental transmission of CWD prions; the persistence of environmental infectious prion for at least for 2.5 years [255]; the absence of a rapid, practical, and reliable antemortem field tests for detecting preclinical CWD; and the nonavailability of licensed vaccines nor therapies together with the impossibility of quarantine suspicions populations in wild cervids make the control of this disease in wild populations very difficult.

The management of CWD in affected populations is a proven difficult task, based on population reduction in the case of wild deer or in quarantine and depopulation in commercially reared animals.

Measures to control the spread of the disease will depend on whether we are dealing with commercially exploited herds, confined to demarcated areas, or with wild populations of deer. In the first case, and similar to other infectious diseases and other livestock, programs to detect and eliminate positive animals may be used [94].

This task is not easy, however, as a shown by a 2018 study conducted on a private farm in Colorado where the use of disease control management through rectal biopsy diagnosis was not able to prevent the increase in prevalence suggesting the need to refine management criteria or antemortem diagnostic methods [5].

Thus, the science available to inform effective CWD management and control strategies remains relatively incomplete as it was concluded by different studies [254, 256] during their review on CWD control in North America. Nevertheless, it may be stated that approaches for CWD management generally fall into three categories in order to prevent the introduction of the disease and its spread [252]:


Regarding preventive and containment strategies, it was referred that these measures tend to focus on regulations (e.g., bans on movements of live animals, carcasses, or specified risk materials) and efforts to prevent the introduction of CWD should consider whether prior surveillance data are sufficient to assure that the disease has not already spread into the area of interest [252].

Haley and collaborators [94] suggest that outer mechanisms of disease control could be useful like the development of simple antemortem test to use prior to animal movements among farms, the breeding for resistant genotypes, the installation of effective fences to prevent transmission between commercial

#### **Figure 6.**

*Surveillance program for CWD in cervids. Schematic presentation of two-stage sampling approach of the 3-year surveillance programme for CWD in cervids in Estonia, Finland, Latvia, Lithuania, Poland and Sweden in cervids defined on commission regulation (EU) 2017/1972.*

and wild populations, and equipment cleaning and quarantine prior to animal introduction.

Free-ranging deer population reduction is empirically effective in reducing a disease with direct contact transmission, but several studies have shown that in the case of CWD, the results are below expected [94, 257].

Capturing, testing, and recapturing positive free-ranging mule deer did not reduce prevalence in the female population [258].

Concerning the prevention of entry in free regions, according to Regulation (EC) No. 999/2001, in the EU, the feeding to cervids of proteins derived from animals is prohibited, with the exception of milk and milk products, eggs and egg products, hydrolyzed proteins from nonruminants or from ruminant hides and skins, and gelatine and collagen from nonruminants (Article 7 and Annex IV). Also, at import into the EU, an attestation is required for meat and meat products from wild and farmed cervids coming from the USA or Canada (Chapter F of Annex IX), confirming that the products:


As a result of the detection of the first case in Norway, in Europe, in addition to the permanent measures as described in the aforementioned regulation, the Commission Implementing Decision (EU) 2016/1918 enforced temporary safeguard measures in relation to CWD (until December 31, 2017). According to this

**127**

*TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control*

decision, the movement of live cervids from Norway into the European Union was prohibited with several derogations permitting its movements under certain circumstances (e.g., movements of live cervids from Norway to Sweden or Finland for direct slaughter; movements of live reindeer for seasonal grazing from Norway

According to the European Food Safety Agency [252], the derogations that lay down in this decision present a risk of introduction of CWD into the EU, since it was concluded that the most likely pathway of introduction of CWD into the EU is the movement of live cervids, either by deliberate transportation or by the movement of wild animals across the border of Norway to Sweden or Finland. Additionally, these authors present other measures to reduce the probability of introduction of CWD into the EU, namely, the use of natural cervid urine lures and awareness campaigns targeting both local Norwegian hunters and hunters visiting Norway from (and returning to) other countries regarding the personal protective equipment (PPE), disinfection, the safe dressing of carcasses, and the appropriate

In general, according to several authors [252, 254, 256], other measures can be implemented in order to contain and/or to control/suppress CWD in a region or country where the disease is present. Some of those measures may include:

• Reducing environmental contamination (e.g., interdict the use of dead cervids to feed wildlife nor to use as baits for hunting, unless they have been tested

• Reduction of animal-to-animal contact (e.g., ban artificial feeding, lick blocks

• Decrease population densities by definition of strategic plan for hunting management policies (e.g. increasing harvest permits and/or hunting season)

Regarding the last measure (decrease population), Uehlinger and colleagues

• Culling of wildlife is often unpopular. For that reason, any control program that includes culling of animals must take into account the public acceptance

Since dispersion of infected deer may not be limited by political boundaries, regional and interstate/interprovincial cooperation and collaboration will be an important part of any successful management strategy. Also, it is always important to evaluate the costs in order to understand the feasibility of the control strategy

This review gathers known and recent features of this progressive and fatal neurodegenerative disease affecting cervid species, contributing for chronic wasting disease awareness among scientific community and stakeholders. Conscious that research in this field has a long way to go to answer many of the questions that remain open, we believe that this review will contribute to the understanding of the

• To date, the evidence is unclear that increased nonselective culling pressure has

or other congregating management procedures).

a beneficial effect on the spread or prevalence of CWD.

proposals (when developing any intervention program) [254].

and hunters' attitudes and behaviors.

occurrence of prion diseases in wildlife.

*DOI: http://dx.doi.org/10.5772/intechopen.85797*

to some listed areas in Sweden and Finland).

disposal of carcass trimmings.

negative, prior to use).

and culling practices.

[254] alert that:

### *TSE Monitoring in Wildlife Epidemiology, Transmission, Diagnosis, Genetics and Control DOI: http://dx.doi.org/10.5772/intechopen.85797*

decision, the movement of live cervids from Norway into the European Union was prohibited with several derogations permitting its movements under certain circumstances (e.g., movements of live cervids from Norway to Sweden or Finland for direct slaughter; movements of live reindeer for seasonal grazing from Norway to some listed areas in Sweden and Finland).

According to the European Food Safety Agency [252], the derogations that lay down in this decision present a risk of introduction of CWD into the EU, since it was concluded that the most likely pathway of introduction of CWD into the EU is the movement of live cervids, either by deliberate transportation or by the movement of wild animals across the border of Norway to Sweden or Finland. Additionally, these authors present other measures to reduce the probability of introduction of CWD into the EU, namely, the use of natural cervid urine lures and awareness campaigns targeting both local Norwegian hunters and hunters visiting Norway from (and returning to) other countries regarding the personal protective equipment (PPE), disinfection, the safe dressing of carcasses, and the appropriate disposal of carcass trimmings.

In general, according to several authors [252, 254, 256], other measures can be implemented in order to contain and/or to control/suppress CWD in a region or country where the disease is present. Some of those measures may include:


Regarding the last measure (decrease population), Uehlinger and colleagues [254] alert that:


Since dispersion of infected deer may not be limited by political boundaries, regional and interstate/interprovincial cooperation and collaboration will be an important part of any successful management strategy. Also, it is always important to evaluate the costs in order to understand the feasibility of the control strategy proposals (when developing any intervention program) [254].

This review gathers known and recent features of this progressive and fatal neurodegenerative disease affecting cervid species, contributing for chronic wasting disease awareness among scientific community and stakeholders. Conscious that research in this field has a long way to go to answer many of the questions that remain open, we believe that this review will contribute to the understanding of the occurrence of prion diseases in wildlife.

*Wildlife Population Monitoring*

and wild populations, and equipment cleaning and quarantine prior to animal

*Surveillance program for CWD in cervids. Schematic presentation of two-stage sampling approach of the 3-year surveillance programme for CWD in cervids in Estonia, Finland, Latvia, Lithuania, Poland and* 

case of CWD, the results are below expected [94, 257].

*Sweden in cervids defined on commission regulation (EU) 2017/1972.*

reduce prevalence in the female population [258].

confirming that the products:

suspected

• Exclude the offal and spinal cord

Free-ranging deer population reduction is empirically effective in reducing a disease with direct contact transmission, but several studies have shown that in the

Capturing, testing, and recapturing positive free-ranging mule deer did not

Concerning the prevention of entry in free regions, according to Regulation (EC) No. 999/2001, in the EU, the feeding to cervids of proteins derived from animals is prohibited, with the exception of milk and milk products, eggs and egg products, hydrolyzed proteins from nonruminants or from ruminant hides and skins, and gelatine and collagen from nonruminants (Article 7 and Annex IV). Also, at import into the EU, an attestation is required for meat and meat products from wild and farmed cervids coming from the USA or Canada (Chapter F of Annex IX),

• Are derived from animals tested for CWD with negative results

• Are derived from animals which do not come from a herd (for farmed animals) or a region (for wild animals) where CWD has been confirmed or officially

As a result of the detection of the first case in Norway, in Europe, in addition to the permanent measures as described in the aforementioned regulation, the Commission Implementing Decision (EU) 2016/1918 enforced temporary safeguard measures in relation to CWD (until December 31, 2017). According to this

**126**

introduction.

**Figure 6.**
