Laboratory of Virology, lstituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy1
Author for correspondence: Maurizio Pocchiari.Fax +39 06 49903012. e- mail pocchia{at}virus1.net.iss.it
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Abstract |
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Introduction |
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The appearance of new variant CJD (nvCJD) in the UK (Will et al. , 1996 ), resulting from exposure to the agent of bovine spongiform encephalopathy (BSE; Bruce et al., 1997
), may lead to an increased risk, with respect to sporadic CJD, of accidental transmission from man-to-man by medical procedures, including dental work. This is because there is evidence that the level of infectivity outside the CNS is higher in nvCJD patients than in cases of the sporadic disease. Preliminary data have shown that tonsils from patients with nvCJD are loaded with PrPsc while those of patients with sporadic CJD are not (Hill et al., 1999
).
In the present paper we used scrapie-infected hamsters to measure the level of infectivity in oral tissues commonly involved in dental procedures and the efficiency of the intradental route as a way of TSE transmission.
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Methods |
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Before use, samples were thawed and vigorously shaken by vortexing. One group of animals was injected i.p. (0·1 ml) into the lower left quadrant of the abdomen by using sterile syringes with 26 gauge needles; another group was injected intradentally (i.d., 0·005 ml) into the lower (mandibular) left incisor. lntradental injection was performed with a Hamilton syringe on anaesthetized animals (Thiopental 0·750·9 mg per animal) after the incisor crown was dissected and the pulpar root cavity opened with sterile slowly rotating diamond burrs.
Animals were housed 612 per cage, observed for 5 days per week, and scored for the presence of clinical signs as previously described (Pocchiari et al., 1987 ).
Dissection of tissues.
lntraperitoneally or intradentally scrapie-infected hamsters were sacrificed with chloroform. Brains, cervical spinal cord, trigeminal ganglia, gingival tissues and pulps of the four incisor teeth were removed by different sets of dissection instruments to minimize cross- contamination between samples. Each set was sterilized by steam autoclave at 132 °C for two cycles of 1 h. The gingival sample was taken by curetting tissue from buckle and lingual side of the frontal alveolar arch and from the palate by a scalpel blade Bard Parker 21. The pulps were removed from the apexes of extracted incisors using a K-File endodontic instrument. Samples were immediately frozen at -70 °C.
Assay of infection.
Recipient hamsters (n=812) were inoculated intracerebrally (i.c.) with 0·05 ml of a 10% brain, cervical spinal cord, gingival tissue, tooth pulps or trigeminal ganglia suspension. Each sample was obtained by pooling tissues from 810 donor animals and was homogenized with Ultraturrax in sterile PBS with different tips for each sample. Incubation periods (mean±SEM) were measured and infectivity titres were estimated by applying these values to a dose incubation curve drawn after an end- point titration (Pocchiari et al., 1989 ). An inverse relationship exists between dose and incubation period of the 263K strain in hamsters, which gives an average incubation period of 155·5 days for 1 LD50 i.c. unit in 0·05 ml of a 10% brain (and we assumed other tissues) homogenate (Pocchiari et al., 1989
).
Recipient hamsters were observed daily for clinical signs of scrapie disease for 400 days after i.c. infection.
PrP assay.
The protease-resistant fraction of PrPsc (PrP2730) was purified from brain and trigeminal ganglia as previously described (Xi et al., 1994 ), electrophoresed on 15% SDSpolyacrylamide gels, and then electrotransferred to a nitrocellulose membrane. After non-specific binding was blocked with 3% fish gelatin, the membrane was processed with rabbit polyclonal antibody against hamster PrP2730 (P8-1, 1:2000; Xi et al. , 1994
), then processed with a goat anti-rabbit IgG conjugated with alkaline phosphatase (Bio-Rad) and finally stained with naphtol/fast red solution.
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Results |
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However, at the end of the clinical course the levels of infectivity of cervical spinal cord and brain were almost the same (Table 1 ); the mean incubation periods were 59·7±1·0 days (n=7) for animals inoculated with the cervical spinal cord and 63·5±0·4 days (n=10) for animals inoculated with brain material. Though delayed in timing, the growth curve of the scrapie agent in trigeminal ganglia followed the same pattern as that found in the brain, with no infectivity detectable before the 50th day and a sudden increase in the second half of the incubation period. Recipient animals inoculated with trigeminal ganglia taken at the end of the incubation period developed scrapie disease with a mean incubation period of 69·5±1·4 days ( n=19), which is only 6 days longer than the mean incubation period observed in hamsters receiving brain taken at the same time- point.
Finally, the levels of infectivity in the gingival tissue and pulps during the clinical stage of scrapie disease were 7·2 (estimated from the mean incubation period of recipient animals, 79·0±0 days, n=7) and 5·6 (96·9±0·9 days, n=10) log LD50 i.c. units/g of tissue, respectively. Although these values were clearly lower than those found in the trigeminal ganglia, they were nonetheless higher than expected and clearly showed that oral tissues may harbour a good amount of infectivity. It remains, however, to be clarified why gingival tissue had a higher level of infectivity than the pulp.
Presence of PrP2730 in a nervous peripheral station
Aliquots (less than 0·5 ml) of the homogenized trigeminal ganglia taken either 7 days after inoculation or at the end of the clinical course were used to detect PrP2730. In accordance with the results obtained in the measurement of infectivity, no PrP2730 was found in the 7-day-sample, while a great amount was seen in the 105-day-sample (Fig. 2). This result adds further evidence to the role played by the peripheral neural cells in supporting replication of the scrapie agent.
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Discussion |
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After i.p. inoculation, the onset of scrapie replication in the trigeminal ganglia occurs about 10 days later than in the brain and 20 days later than in the cervical spinal cord, suggesting a centrifugal spread of the scrapie agent from the sensory nuclei of the trigeminal system [extended from the medulla oblongata up to the mesencephalon and, in i.p. CJD-infected mice, showing early PrPsc deposits (Muramoto et al. 1993 )] to the trigeminal ganglia. These findings support the view that after intraperitoneal inoculation the scrapie agent moves first centripetally to the CNS (Kimberlin & Walker, 1979
, 1982
; Baldauf et al., 1997
) and then centrifugally to the peripheral nervous system (Kimberlin et al., 1983a
), including trigeminal ganglia.
The other important result obtained by this study is that the injection of scrapie into the tooth pulp of Syrian hamsters is an efficient route of infection which gives a mean incubation period ranging between those observed after intraocular (about 130 days; Buyukmihci et al., 1983 ; Kimberlin & Walker, 1986
) and intrasciatic (about 180190 days; Kimberlin & Walker, 1986
) inoculations. Temporal differences are probably due to the relative distances between the injection sites and the brain, rather than to different efficiencies of these peripheral routes. The rate of spread of scrapie infection to the CNS, as measured following intraperitoneal inoculation (Kimberlin & Walker, 1979
, 1982
; Kimberlin et al., 1983a
), oral administration (Beekes et al. , 1996
) or injection of sciatic (Kimberlin et al., 1983b
) or optic (Kimberlin & Walker, 1986
; Scott & Fraser, 1989
) nerves, is always equivalent to about 1 mm/day, consistent with the slowest rate of axonal transport. The same rate of scrapie propagation was estimated between teeth and trigeminal ganglia. Teeth are innervated by bipolar sensory neurons whose cell bodies are in the homolateral trigeminal ganglion. Thirty days after intrapulpal injection there was infectivity and PrP2730 accumulation in the homolateral trigeminal ganglion but not in the controlateral one, suggesting that the infection had spread through the mandibular branch (about 25 mm long in Syrian hamsters) of the trigeminal nerve at an approximate rate of 1 mm/day.
Although these results cannot be directly applied to humans affected by TSEs, they raise the possibility that surgical instruments used during major dental procedures in CJD patients and inadequately decontaminated may represent a means of man-to-man transmission of TSEs. People incubating nvCJD are of particular concern, because they carry higher level of PrPsc, and likely infectivity, in peripheral tissues than sporadic CJD cases (Hill et al., 1999 ). Concern will further increase if upcoming studies show that trigeminal ganglia of nvCJD patients are more affected than those of sporadic CJD cases.
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Acknowledgments |
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References |
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Received 15 June 1999;
accepted 21 July 1999.