**5.2 Follicular dendritic cells (FDC) and B and T lymphocytes**

Several studies show that in prion-infected hosts, high accumulations of disease-specific PrP are detectable within the B-cell follicles of the SLT [145–149]. After internalization, evidence suggests that prions accumulate and replicate in the follicular dendritic cells (FDC) that lie in the B-cell follicles and germinal centers of SLT [150–156]. FDC possess multiple slender and long dendritic processes, differentiate from ubiquitous perivascular precursor cells (pericytes), and are a distinct lineage from bone marrow-derived conventional dendritic cells (DC) [157, 158]. FDC extend throughout the B-cell follicle being able to trap and retain large amounts of native antigens upon their surfaces in the form of immune complexes composed by antigen–antibody and/or opsonizing complement components. Apparently, prions can be bound by opsonizing complement components [152–155], internalized, and replicated within FDC, as it was demonstrated that they accumulate upon PrPc -expressing FDC in the SLT of experimentally infected mice, sheep with natural scrapie, cervids with CWD, and patients with vCJD [145–149]. Subsequently, immunohistochemistry studies identified FDC as the first sites of prion conversion and replication in experimentally infected mice, in which increased prion labeling was observed on the plasma membranes of FDC in the Peyer's patch germinal centers [143]. Furthermore, evidence suggests that FDC maturation and regression cycle are impaired by disease-associated prion protein, thus affecting immune function [159]. Despite this, some prion strains can establish infection within SLT regardless of FDC, as it was demonstrated by experimental studies in mice, where infection occurred associated to the high endothelial venules in the lymph nodes [160].

The role of B and T cells in prion disease pathogenesis remains uncertain. Experimental studies showed that mice lacking mature B and T cells were not susceptible to peripheral infection with prions [145, 161–163], despite prion disease pathogenesis being unaffected in T-deficient mice [162, 164, 165]; however, it seems that prions are not capable of replicating within B and T cells [166, 167]. These cells appear to be intermediary actors in prion diseases, by providing physical and humoral support to other cells, thus contributing to the homeostatic state of the immune system. However, B cells, in particular, seem to play a more active role during prion infection. The ability of secreting tumor necrosis factor-α (TNF-α) and lymphotoxins (LT) turns B cells pivotal in keeping FDC in their differentiated state, since in the absence of stimulation from these cytokines, FDC rapidly dedifferentiate [168]. In Peyer's patches, B cells migrate to the mesenteric lymph nodes and then return to the circulation [169]. It is this constant rotation between Peyer's patches and SLT that may help B cells to disseminate prions acquired from FDC as they both migrate together through the germinal centers [170, 171]. Additionally, the accumulation of prions in the spleen and subsequent neuroinvasion are significantly decreased in the specific absence of B cells [162]. It is also noteworthy that prions were detected in B cells from the blood of sheep with scrapie [172] and deer with CWD [173].

As it was already mentioned, FDC in the Peyer's patches are major sites of early prion replication in orally infected animals, both experimentally [137–140, 174] and in the natural forms of the disease. The study of natural scrapie cases in sheep showed that GALT of the upper gastrointestinal tract exhibited early accumulation

*Wildlife Population Monitoring*

transmission of CWD [77].

molecular shapes of PrPsc and PrPc

that compensate the absence of PrPc

Pertaining the immune system, PrPc

protein backbones [136].

dams.

PrPc

**5.1** *M* **cells**

CWD did not result in brain accumulation of PrPsc during primary passage in ovinized mice (Tg338). One factor that may have contributed to the lack of transmission could be varying levels of PrPsc and PrPCWD between brain homogenates

Non-Tg mice have been described as resistant to CWD infection [115], while the VM/Dk inbred strain of mice infected with wapiti CWD prions reported limited infection [29], suggesting the existence of some variable species barriers to the

Nalls et al. [93] have demonstrated mother-to-offspring transmission and disease progression to a viable cohort of offspring that were born to CWD-infected

**5. Pathogenesis of CWD: the interaction of prions and immune system**

The unique features of prions as infectious agents are also blatant when considering its interaction with the host's immune system. As stated previously, PrPc is involved in important neurologic functions; however, the depletion of neuronal

 in experimental models, such as mice, cows, and goats, did not result in altered phenotypes [11]. This suggests the existence of redundant mechanisms

and stromal cells. This abundant expression may explain why PrPsc does not elicit specific immune responses despite its accumulation in high levels within the host's secondary lymphoid tissues (SLT) in the initial phases of the infection. In particular, gut-associated lymphoid tissues (GALT), such as the Peyer's patches in the small intestine, seem to be pivotal for the infection and disease process. As in most natural occurring prion diseases, CWD of cervids is acquired peripherally, mainly after oral exposure, after which prions replicate in GALT. The importance of GALT in the pathogenesis of prion diseases is well documented by several studies that revealed impaired neuroinvasion in the absence of Peyer's patches [137–140].

Within GALT, studies suggest that prions depend on host *M* cells to enter the organism, as they seem to be involved in the initial transfer of the prion agent across the gut epithelium. These are specialized epithelial cells widely spread among the follicle-associated epithelium, which are involved in the transcytosis of particulate antigens and microorganisms from the gut lumen into the GALT [141]. *M* cells are essential to the gut immune system being fundamental to antigen recognition and presentation of pathogens or commensal microflora. As such, factors that increase *M*-cell density in the gut epithelium (coinfections or miscellaneous inflammatory conditions) may also increase susceptibility to orally acquired prion infections. A study by Wyckoff et al. [142] mentions the binding of prions to certain types of soil particles that may result in an increased uptake of prions from the gut lumen, but it is not yet clear if this uptake is mediated by *M* cells. Host infection by

Several researchers noted that dissimilarity between PrP sequences alone does not accurately predict the transmissibility between species and other factors are under investigation. One that was used in recent studies was the conformational selection model, which proposes that compatibility between the three-dimensional

is more important than the sequences of the

, albeit its ubiquitous cellular expression.

is expressed in lymphocytes, granulocytes,

from animals with scrapie and CWD, respectively [119].

**116**

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. Additionally, it was observed that FDC in the spleen expressing PrPc were able to display high levels of prion replication that was impeded after PrPc expression blockage in these cells [185].

#### **5.3 Mononuclear phagocytic cells**

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 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].

#### **5.4 Neuroinvasion**

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

**119**

**6. Diagnosis of CWD**

amyloid plaques.

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

concurrent infections, and hosts' age and immune status [144].

The onset of neurodegeneration is preceded by the activation of glial cells, microglia, and astrocytes [225, 226]. Microglial cells, in particular, by intervening in synaptic remodeling and in the removal of dead and dying cells, are important for the course of neurological changes during prion infection. Organotypic cerebellar culture devoid of microglia exhibited enhanced prion accumulation [227], whereas CNS prion disease pathogenesis was exacerbated in vivo in the absence of microglia [228]. Studies revealed that during prion infections, a cytokine response mediated by TGF-β and PGE2 develops within the CNS, stimulating microglia to adopt an anti-inflammatory status [223, 229, 230]. Evidence suggests that this anti-inflammatory ambiance may be beneficial for the host, as CNS prion infection in mice deficient in the anti-inflammatory cytokines interleukin (IL)-4, IL-10, and IL-13 displays an aggravated disease pathogenesis [231, 232]. Other studies supported this hypothesis by the finding that blocking colony-stimulating factor (CSF) receptor-1 resulted in the expression of selective anti-inflammatory microglial markers, slowing the development of neuropathology and extending survival times [233]. Activated microglia scavenge and clear prions and prion-affected cells, but neural degeneration seems to occur when prion accumulation is associated to an inflammatory response in the microglia [234]. Together, these data point out to the existence of both anti-inflammatory and pro-inflammatory responses by microglial cells during CNS prion infections [226, 234] contributing to the symptoms of this disease.

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

in which immunohistochemical tracing was performed revealed that prions then infect enteric nerves and spread along efferent fibers of both the sympathetic (e.g., splanchnic nerve) and parasympathetic (e.g., vagus nerve) nervous systems, spreading within them to the CNS, where they ultimately cause neurodegeneration [143, 208–212]. The initial transfer of prions from FDC to the peripheral nerves continues surrounded by knowledge gaps, but it appears that tunneling nanotubes (TNT) may play an important role at this stage of the disease. These are slender membrane-bound cylinders of cytoplasm that connect cells to enable cell-to-cell communication and the intercellular transfer of plasma membrane or cytoplasmic components [213]. Since mononuclear phagocytes and DC are in close proximity with enteric nerves [214–217] and in vitro studies showed that prions can be transferred between DC and neurons via endolysosomal vesicles within TNT [215–218], it is possible that the same transfer also occurs in vivo. Another hypothesis is that the transfer of prions may also occur in association with small endosomal-derived vesicles, termed exosomes [219], as it was demonstrated experimentally. Studies supporting this theory are based on the observation that DC infected with prions can release exosomes that in turn may infect neighboring cells [220]. In the CNS, prion aggregates accumulate to a point where they overcome the structures within it, starting from the synapses and then proceeding to axons, where axonal retraction is observed, and then to the cell body, in which atrophy and neuronal loss are lastly observed [221–224]. The abovementioned process is, however, slowly and progressive, depending on factors such as prion agent strains, the existence of

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

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

in which immunohistochemical tracing was performed revealed that prions then infect enteric nerves and spread along efferent fibers of both the sympathetic (e.g., splanchnic nerve) and parasympathetic (e.g., vagus nerve) nervous systems, spreading within them to the CNS, where they ultimately cause neurodegeneration [143, 208–212]. The initial transfer of prions from FDC to the peripheral nerves continues surrounded by knowledge gaps, but it appears that tunneling nanotubes (TNT) may play an important role at this stage of the disease. These are slender membrane-bound cylinders of cytoplasm that connect cells to enable cell-to-cell communication and the intercellular transfer of plasma membrane or cytoplasmic components [213]. Since mononuclear phagocytes and DC are in close proximity with enteric nerves [214–217] and in vitro studies showed that prions can be transferred between DC and neurons via endolysosomal vesicles within TNT [215–218], it is possible that the same transfer also occurs in vivo. Another hypothesis is that the transfer of prions may also occur in association with small endosomal-derived vesicles, termed exosomes [219], as it was demonstrated experimentally. Studies supporting this theory are based on the observation that DC infected with prions can release exosomes that in turn may infect neighboring cells [220]. In the CNS, prion aggregates accumulate to a point where they overcome the structures within it, starting from the synapses and then proceeding to axons, where axonal retraction is observed, and then to the cell body, in which atrophy and neuronal loss are lastly observed [221–224]. The abovementioned process is, however, slowly and progressive, depending on factors such as prion agent strains, the existence of concurrent infections, and hosts' age and immune status [144].

The onset of neurodegeneration is preceded by the activation of glial cells, microglia, and astrocytes [225, 226]. Microglial cells, in particular, by intervening in synaptic remodeling and in the removal of dead and dying cells, are important for the course of neurological changes during prion infection. Organotypic cerebellar culture devoid of microglia exhibited enhanced prion accumulation [227], whereas CNS prion disease pathogenesis was exacerbated in vivo in the absence of microglia [228]. Studies revealed that during prion infections, a cytokine response mediated by TGF-β and PGE2 develops within the CNS, stimulating microglia to adopt an anti-inflammatory status [223, 229, 230]. Evidence suggests that this anti-inflammatory ambiance may be beneficial for the host, as CNS prion infection in mice deficient in the anti-inflammatory cytokines interleukin (IL)-4, IL-10, and IL-13 displays an aggravated disease pathogenesis [231, 232]. Other studies supported this hypothesis by the finding that blocking colony-stimulating factor (CSF) receptor-1 resulted in the expression of selective anti-inflammatory microglial markers, slowing the development of neuropathology and extending survival times [233]. Activated microglia scavenge and clear prions and prion-affected cells, but neural degeneration seems to occur when prion accumulation is associated to an inflammatory response in the microglia [234]. Together, these data point out to the existence of both anti-inflammatory and pro-inflammatory responses by microglial cells during CNS prion infections [226, 234] contributing to the symptoms of this disease.
