In 1995, Shaw (1995)
suggested that the occurrence of a shorter incubation period for Creutzfeldt–Jakob disease (CJD) in farmers could have come from ‘breathing the dust from feed containing prion’. The present studies were done to simulate the inhalation of airborne particles and the direct contamination of the nasal passages with CWD prions. The finding that CWD prions can be transmitted via inhalation (perhaps even more effectively than by nostril contact only), while unique for CWD infection, extends precedent for transmission of prions via the respiratory system. Sheep and hamsters inoculated with scrapie intranasally (Hamir et al., 2008
; Sbriccoli et al., 2008
) and hamsters inoculated with TME extranasally (Kincaid & Bartz, 2007
) have been shown to develop TSE. Results in the latter study suggested that the nasal passages may even be more effective than the oral route in transmitting prion disease (Kincaid & Bartz, 2007
In the present study, 86
% (six of seven) of Tg(cerPrP) mice exposed to CWD via aerosol developed CWD versus 22
% (two of nine) of IN-exposed mice (Fisher's exact test, P
-value=0.0406). It may be argued that the early deaths of four mice in the initial CWD-aerosol exposure group could bias the statistical significance of the results. After modifications to the exposure procedure, early mortalities were eliminated. Moreover, two of two surviving mice in the initial exposure and four of five mice in the second exposure study developed TSE.
One possible explanation for the enhanced infectivity after aerosol exposure might be the disruption and dispersion of infectious PrPCWD
aggregates during aerosolization to yield more small infectious particles or seeds (Silveira et al., 2005
). These smaller aggregates might be more readily taken up by lymphoid or distal airway epithelial cells not typically accessible by either nasal contact or drop-wise instillation of prions.
Another potential explanation for enhanced infection after aerosolization could be that a larger prion dosage was delivered by aerosol versus nasal exposure. Based on a random distribution of infectivity in the aerosol material, the respiratory tidal volume of mice and the average anaesthetized respiratory rate for a 4 min period, we calculated that aerosol-exposed mice would receive approximately 1.2
% of the available inoculum, or a maximum of 24 μg of particulate inoculum deposited in the respiratory system. Mice inoculated intranasally received 10 μl of a 10
% brain homogenate or 100 μg of particulate inoculum – four times that delivered by aerosol. Given that only the nostril region (<5 mm) of each mouse was exposed during aerosolization, we estimate that even if up to an additional 15 μl of inoculum were to be ingested due to nasal region grooming, the total dosage would not surpass (and more likely would never attain) that delivered intranasally.
Clearly, inoculation by either the aerosol or nasal route would result in inoculum entering the alimentary tract via the nasopharynx. Thus, oral exposure and potential uptake cannot be avoided or excluded. However, oral inoculation of Tg(cerPrP) mice with 100 μg CWD particulate brain homogenate failed to transmit CWD infection or disease after >700 days of observation (D. M. Seelig, G. L. Mason, G. C. Telling & E. A. Hoover, unpublished results) (Table ). Thus, it is unlikely that CWD transmission by the aerosol or nasal routes reflects infection via the alimentary tract.
Summary of CWD inoculation results in Tg(cerPrP) mice
Mice are an excellent model for studying airborne and direct nasal contact transmissions because they are obligate nasal breathers (Klemens et al., 2005
). Odorants and particles inhaled into the nasal passage are subject to a number of cell surfaces. Thus, multiple sites of CWD prion entry are plausible, including the NALT, the mucosal associated macrophages and/or dendritic cells, respiratory epithelium, olfactory epithelium and VNO. The NALT typically incorporates the retropharyngeal lymph nodes, palatine and lingual tonsil (Kuper et al., 1992
) and is similar in structure and function to the gut-associated lymphoid tissue (GALT) in that it is responsible for antigen uptake and presentation by M cells, B cells and follicular dentritic cells (Heritage et al., 1997
; Kuper et al., 1992
). The GALT, especially the Peyer's patches, is considered to be the primary site of PrPSc
uptake for BSE, variant CJD and scrapie (Beekes & McBride, 2000
; Fox et al., 2006
; Heggebo et al., 2002
; Press et al., 2004
; Spraker et al., 2002b
; van Keulen et al., 2008
Mice do not have tonsils or retropharyngeal lymph nodes per se, but rather a bi-symmetrical NALT structure that lines the floor of the nasal cavity (Heritage et al., 1997
). Kincaid & Bartz (2007)
found that hamsters inoculated via uptake of droplets of TME inoculum via the external nares had shorter incubation periods using a lower dose of infectious prions than those inoculated orally. Studies in deer inoculated orally or naturally exposed to CWD indicate that the primary structures that accumulate PrPCWD
early in infection are the retropharyngeal lymph nodes and tonsils (Keane et al., 2008
; Sigurdson et al., 1999
; Spraker et al., 2004
). Nevertheless, in the present study we were unable to demonstrate PrPCWD
in the NALT of early, pre-terminal or terminal Tg(cerPrP) mice after aerosol or nasal exposure to CWD.
Another seemingly probably site for prion entry and infection is the olfactory mucosal epithelium, which contains odour receptors that provide a direct neural connection from the nasal cavity to the olfactory bulbs of the brain. Previous IHC studies of deer terminally infected with CWD have demonstrated PrPCWD
depositions in the olfactory bulbs, which also show marked spongiform degenerative changes (Spraker et al., 1997
; Williams & Young, 1992
). However, in deer, sequential PrPCWD
accumulation in the brain appears to occur in a caudal (brainstem) to rostral (frontal cortex) fashion as the disease progresses (Spraker et al., 2002a
). In the present study, PrPCWD
was not detected in the olfactory bulbs of terminal CWD-infected Tg(cerPrP) mice, although aggregates were identified in the frontal cortex immediately dorsal to the olfactory bulbs in some mice. This was especially surprising since the olfactory bulb glomeruli are sites of substantial cervid PrPC
expression in naïve Tg(cerPrP) mice.
Given that CWD prions have been demonstrated in saliva (Mathiason et al., 2006
), urine (Haley et al., 2009
; Kariv-Inbal et al., 2006
) and soil (Johnson et al., 2006
), it is possible that prion entry could involve the VNO – a region of the anterior ventral nasal passages specialized to detect non-volatile molecules such as pheromones by a process known as the Flehmen response (Thorne & Amrein, 2003
; Kelliher et al., 2001
; Meredith & O'Connell, 1979
). Nevertheless, neither we nor DeJoia et al. (2006)
were able to detect PrPRES
in the VNO of terminal CWD-inoculated Tg(cerPrP) mice or TME-inoculated hamsters, respectively. Thus, while all of the above exposure studies failed to identify PrPCWD/RES
in mucosal sites, it remains likely that early prion trafficking involves relatively few potential protease-sensitive oligomeric molecules, which may not be identifiable with the detection methods used.
Our inability to detect PrPCWD
outside the central nervous system in the present studies was somewhat perplexing. This finding could reflect a more limited peripheral expression of PrPC
expression in the Tg(cerPrP) mice versus deer, although other studies in our laboratory have demonstrated PrPC
in many peripheral tissues of Tg(cerPrP) mice (D. M. Seelig, G. L. Mason, G. C. Telling & E. A. Hoover, unpublished results). Additionally, overfixation of our tissues could eliminate possible PrPCWD
aggregates. An alternative explanation would be that TSE induced by nasal exposure to CWD prions in Tg(cerPrP) mice in mediated largely by non-lymphoreticular system pathways. Such a pathway is supported by the recent work of Bessen et al. (2009)
, demonstrating that IN inoculation of RML scrapie into immunodeficient transgenic mice transmits TSE without lymphoreticular system involvement.
In summary, the present study demonstrates the transmissibility of prions via aerosolization. Several aspects of respiratory transmission of CWD prions remain to be refined and no evidence of a peripheral or lymphoid phase of the infection was detected. The results suggest that prion exposure via the respiratory system merits consideration in prion transmission and biosafety.