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Structure and ultrastructure of intra-vitam parasitic destruction of the external dental tissue in the fish, Anarhichas lupus L.
STRUCTURE AND ULTRASTRUCTURE OF INTRA-VITAM PARASITIC DESTRUCTION OF THE EXTERNAL DENTAL TISSUE IN THE FISH, ANARHICHAS LUPUS L. B. KEREBEL, MARIE-T&R&E LE CABELLE~‘and LISE-MARR KEREBEI F.R.A. INSERM.
Faculte de Chirurgie Dentaire. I Place Alexis Ricordeau.
44042 Nantes.
France
Summary--Three specimens of Anurhichas lupus L. with teeth presenting burrowing lesions were studied with light microscopy, SEM. and high resolution TEM. Findings suggested that the lesions were due to fungoid invasion by Mrc,r/ires os.@~ga\. The burrowing lesions always originated in the more highly mineralized external tooth layer, thus distinguishing them from post-mortem soil changes which may start in any part of the tooth. The channels produced by the lesion had no particular orientation to the dentinal vascular canals. It is suggested that enzymes are secreted locally by the causal organism: burrows were thus not found at a distance from the fungus whose numerous microvilli were in close contact with the mineralized walls of the channels; this distinguishes the lesion from dental caries. Needle-shaped crystals are found within the cytoplasm or adherent to the plasma membrane of the burrowing organism. Other crystals in the immediate neighbourhood of the organism were hollow. and thus suggestive of an organism feeding on mineral material. Bacterial invasion was sometimes in association with the burrowing activity. and may play a part in it. but often appeared to be a later invasion.
INTRDDU(‘TION
and polarized light. Six other conical and four other molariform teeth from upper and lower jaws were demineralized in 10 per cent formalin and 5 per cent nitric acid in distilled water. embedded in Paraplast. sectioned at 5-811rn on a microtome and stamed by haematoxylin and eosin. Other teeth were coated successively with carbon and gold and examined under the scanning electron microscope. some of them directly, others after the organic material had been removed in sodium hypochlorite. Fragments of teeth were post-fixed in 2 per cent osmic acid with cacodylate buffer at pH 7.4. dehydrated in a graded series of methanol concentrations and embedded in methacrylate. Undemineralized sections were cut on an ultramicrotome with a diamond knife and examincd under the transmission electron microscope.
Peyer (1926) described fungoid invasion by Mycelitrs ossifragus in the teeth of Raiu clmufu. Arsuffi (1939) found elongated boring organisms containing granules deeply staining with azocarmine in the external vitrodentine layer of Tetrodon mucu[atus. Kohlenberger (1940) observed at the surface of the teeth of Anarhic+ras lupus L. a whitish microcrystalline material which Schmidt (1954) assumed to be the result of fungal attack by Mrc~litus ossifiugus. He had noted some whitish lesions on the occlusal surfaces of molar-shaped teeth and on the tips of conical teeth of an adult specimen of Anrrrhichas lupus L. Ground sections in ordinary and polarized light showed these chalky areas to be penetrated by bore canals similar to those attributed to attack by Mrcelires ossifiugus in fossil bone. Schmidt (1962a) also observed a similar microcrystalline crusty layer and bore canals in the spines of living specimens of sea-urchins. All this information was obtained by light microscopy only. The understanding of the mechanism of hard tissue destruction, either parasitic or microbial, seemed to us sufficiently important to warrant a thorough study of this parasitic process.
RF.SI’l.TS Whitish burrowing lesions, clearly visible against the yellowish background of the teeth. were found in both younger specimens and the older one. The lesions were on the tips of the conical anterior teeth and either on the tips or above the approximal areas of the molariform teeth. which are in close contact. Ground sections in ordinary light showed lesions, 0.26mm deep and 1.31 mm wide. hollowing out the external tooth layer (Figs. I 4). At the periphery of the lesion there was a front of closely packed burrows parallel with the general direction of the dentinal vascular canals (Fig. 5). Some of the channels in the advancing front changed direction abruptly (Figs. 2 and 3). Their progress wit,hin the dentinal tissue seemed to be unconnected,wtth the course of the vascular canals (Fig. 4).
%IATERIAI.SAI*;D METHODS Three specimens of ilnctrhichus lupxs L. were used. two young ones, 50 cm long, and an older one, 120 cm long. The animals were caught off the coasts of Newfoundland, and their jaws immediately fixed in 9 per cent neutral formalin. Ground sections. 60jtm thick, some stained with Toluidine blue. of 6 conical and 6 molariform teeth from upper and lower jaws were observed in ordinary 147
148
B. Kerebel. Marie-Thkrtse Le Cabellec and Lise-Marie Kerebel
The average diameter of the channels was about 4 pm, whereas the average diameter of the dentinal vascular canals was about IOpm. The channels were sometimes blunt-ended (Fig. 4) or presented terminal bulbs (Fig. 2). Roughly spherical structures, 30pm dia, were observed in the external areas of demineralized sections (Fig. 6). The external dental tissue was disintegrated. Within some channels, structures presumed to be the shrunken organisms were found (Fig. 7). With SEM, the attacked tooth surface appeared to be hollowed out with clear-cut cavities, whereas the mineralized material between the burrows remained undamaged (Fig. 8). The channels, running parallel to each other, presented ring-like grooves in the walls (Fig. 9). On surfaces from which the organic material had not been lost, some segmented filamentous structures, about 3.5pm in diameter, were observed, associated with spherical bodies of about 2.5pm diameter (Fig. IO). Undemineralized sections with TEM showed the burrowing organisms in situ. Sometimes a narrow space, tilled with many small vesicles. remained between the parasite and the mineralized walls of the burrowed-out channel (Fig. 11). Sometimes the burrowing organism appeared to fill the lumen of the channel completely (Fig. 12). There were then many membrane-bound vesicle-like bodies in close contact with the mineralized walls (Fig. 13). The vesicle-like bodies originated from the microvilli of the cytoplasmic membrane of the microorganism (Fig. 14). Dentine mineral crystals were visible outside the plasma membrane, tangential or perpendicular to it. Similar crystals were also scattered within the cytoplasm (Fig. 15). The dentinal tissue in contact with the burrowing organism contained some crystals with hollow centres (Fig. 16). Adjacent to channels completely filled with the organism, there were others in which only a parasite ghost remained, together with scattered crystals (Fig. 12). Bacteria were found within channels where a space remained between the parasite and the mineralized walls (Figs. 17 and 18). Sometimes, they occupied exclusively spaces where hard tissue had been destroyed (Fig. 17). DISCUSSION
The burrowing lesions were located in the external dental tissue. Some workers, using light microscopy (Le Cabellec, Daculsi and Geistdoerfer, 1978) and X-ray diffraction (Kerebel ef u/., 1978). describe the external dental tissue (called durodentine by Schmidt, 1954) as better mineralized than the rest of the dentine. Initially, the burrowing lesions appeared to be located in the external tooth layer exclusively, in agreement with Schmidt’s observations (1954) and contrary to those of Thomasset (1931). Starting from the surface of the better mineralized dental tissue, the organisms proceeded into the dentinal tissue, avoiding the course of the vascular canals which would be likely to provide an easier pathway for organisms feeding on organic material. Burrowed-out channels and vascular canals can be easily differentiated by their dimensions. The sharply cut-out lesions seemed to be produced by direct contact of the organism with the dentine
which, at the periphery of the channels, remained undamaged, as seen with SEM. With SEM the ends of the pits bored by the parasite appeared slightly concave. The walls were marked by a series of ring-like grooves, as if the lesion had been progressively pitted. Although the destructive process was strictly localized to the area in direct contact with the organism, the great number of vacuoles within the cytoplasm suggests an associated enzyme activity. The burrowing process is quite different from dental caries. In caries bacterial enzyme-containing secretions diffuse within the dentine and dentinal tubules so that damage is always found at a distance from the initial site of attack. In caries, the material within dentinal tubules is known to be due to a diffused process of demineralization and proteolysis, whereas the burrowing lesions were always found on surfaces in direct contact with the burrowing organism. In agreement with Schmidt (1954) we found no burrowing lesions in mucous-covered tooth surfaces or unerupted teeth. TEM examination of the lesions showed that no demineralization occurred at a distance from the burrowing organism. However, many vesicles were found in close contact with the undamaged tissue, at the interface with the organism. These vesicles were interpreted as sections of the numerous microvilli of the plasma membrane. The needle-shaped crystals. found either within the cytoplasm or adherent to the plasma membrane, whereas others seemed to be torn from the periphery of the dental tissue, all in the immediate neighbourhood of the burrowing organisms, are suggestive of an organism feeding on mineral material. This is in contradictjon with Schmidt’s findings on post-mortem attack by Mycelites ossifragus (Schmidt and Keil, 1971). Schmidt (1954) noted that in Anarhichas lupus L. intra-vitam attack by Mycelites ossifragus did not affect teeth covered by the oral mucosa, the vascular canals and the pulpal cavity, i.e. areas containing organic material. However, Schmidt’s conclusions were based on light microscopy only. TEM observations provide more precise and reliable information. Bone and tooth burrowing lesions in living and fossil species have been attributed to fungoid invasion by organisms called collectively Mwelites ossifragus (Peyer, 1968). Post-mortem changes of this kind in human teeth long buried in soil have been described (Paltauf and Haberda, 1.927; Werner, 1937; Soggnaes, 1950, 1955; Hammarlund-Essler, 1952; Werelds, 1961. 1967). Burrowing lesions in the teeth of living fishes are similar in many details to the changes in fossil bone and teeth (selachians, teleosts, reptiles) attributed to Myc’elites ossifrugus (Roux, 1887; Jaekel, 1890; Bauer, 1898; Weiler, 1926: Peyer, 1926, 1945, 1968; Thomasset. 1931: Schmidt, 1954. 1962a.b; Schmidt and Keil, 1971). Our findings here suggest that the burrowing progresses from the tooth surface towards the inside. On the contrary, post-mortem destruction starts anywhere in the mineralized tissue and the pulp surface of teeth is commonly affected (Thomasset. 193 1; Clement, 1963: Soggnaes. 1963). On this difference, Peyer (1968) and Schmidt and Keil (1971) drew a distinction between post-mortem and intravitam attacks. Immersion in sea-water is not a necessary condition for fungal attack by Mwe/ite.s os.sij?ayus. According
Intra-vitam
parasitic
destruction
(1864). human teeth soaked in fresh water showed invasion of mycelial filaments and spores starting from the surface. Wedl’s drawings are very like our photomicrographs of the parasitic lesions in .4narhichas lupus L. Light microscopy of the injured dental tissue of Anarhichas lupus L. showed spore-like bodies outside the lesions, whereas hyphal structures were found within the burrowed-out channels. These observations were corroborated by SEM which showed filaments, suggestive of a fungal hypha, penetrating into the burrow and related to spores outside the pit. Segments visible on the filament are suggestive of the transverse septa described in certain fungal species. The hyphae seen with SEM penetrating burrows and filling it completely can be assumed to be the initial agent responsible for the hard tissue destruction. Where destruction was deep, bacteria were found either together with the parasite or alone, suggesting that bacterial invasion occurs either together with the fungal attack or after it; bacteria may play some part in the destructive process, Thomasset (193 I). investigating fungoid invasion in the teeth of several fish species. noted that Mycelites oss(/i~~g~cs is originally iocalized in bone tissue and penetrates the teeth only where the dental tissue has a structure and hardness similar to bone. There is some evidence that Mycelites ossifrugus may have a special affinity for calcium carbonate. Our investigato Wed1
tions do not corroborate this. X-ray diffraction (Kerebel c’f rrl.. 1978) shows that the external dental tissue of Anurhic~ha.~ lupus L. consists chiefly of calcium
phosphate
and magnesium. We found that the burhad a greater affinity for this more mineralized tissue than for the underlying dentine which possesses a more or less amorphous phase of calcium phosphate (Kerebel et (I/., 1978). AI Anarhichus lupus L. feeds on echinoderms and molluscs which are infested by Mj.celites ossifragus (Schmidt. 1954). this is likely to explain how the organism comes into direct contact with the teeth. Attrition facets of tooth surfaces of Anarhichas hqxc.\ L. are frequently observed in the SEM (Le Cabellec er ul., 1978). However. initial wear lesions are not necessary for fungoid invasion because burrowing lesions were present on the sides of molariform teeth which, not being in occlusion, are not subject to functional damage. rowing
organism
~(,~)~oM./R/~YI~IP~II\ The research
investigation
grant from INSERM. Indivtduelle 77 I 00 74.
ATP
was supported by and Aide
53 77 85
of the teeth of .4ntrrhichus
Arsuffi E. 1939. Beitrage zur vergleichenden Histologic und Histogenese der Zahne. Untersuchungen am Gebiss van Labridae, Sparidae und Gymnodontes. Z. Zellforsch. 29, 670-693. Bauer F. 1898. Die lchthyosaurier des oberen weissen Jura. Polronroqruph~ 44, 283- 328. Clement A. J. 1963. Variations in the microstructure and biochemtstry of human teeth. In: Drnrul Anthropolq)
L.
14’)
(Edited by Brothwell D. R.) Vol. 5, pp. 245 269. Pergamon Press, Oxford. Hammarlund-Essler E. 1952. Histologisk Undersokning av Tander oc Kakben fran Medeltidsskelett. St.ensk TlrnGkwe-Tidskr$.
45,
275- 290.
1890. Gange van Fadenpilzen IM\,c,c,/ifc>* O.MIC;e.\. fruyus Roux) in Dentinbildungen. Sitrufic&~ Nurur$ frrunde Berlin, pp. 92-94. Kerebel B.. Le Cabellec M. T.. Daculsi G. and Kerebel L. M. 1978. Osteodentine and vascular osteodentine of .4nurhic,hw Iupu~ L. Crs// Tiu. Rrs. 187. 135 146. Kohlenberger H. 1940. Zur Kenntnis dea Vasodentins. 1. mikrosk.-unur. Fowh. 48, 461 477. Le Cabellec M. T.. Daculsi G. and Geistdoerfer P. lY78. Rapports de la morpholog,ie et de l’histologie dentaires d‘ Anurhic,huslupus L. (Potsson. Teltosteen Perciforme) avec son mode d’alimentation: Apport de la microradiographie et du marquape par la tetracycline. (~~~a..I %r&. 56. 1103 1109. Paltauf A. and Haberda A. 1927. Der Zahn m forensischer Beziehung. In: Hundhucl: der Zuhnhrilkunde (Edited by SchefB. Urban .and Schwarzcnberg. Berlin and Wien. Peyer B. 1926. Uber einen Fall von Caries an einem Rochengebiss: Crrh. ,sc,/tweiz. ,iuturf. Gc,.s. 107. 242. Peycr B. 1945. llber Algen turd Pilre in tirrischen Hartsubstanzen. rlrch. Jftlrir~ K/cut\-Sri/r. I we+l‘rww/~ 20. 496 546. Peyer B. 1968. Corttpurtrtirr O~ionroloyt~ (Edited by Zangerl R.l. The University of Chicago Press. Chicago. Roux W. 1887. Uber einen m Knochen lebende Gruppe von Fadenpilzen (M~crlirrs o.uifrtuqlts). Z. WI\.,. Lo,)/. 45. 227-254. Schmidt W. J. 1954. Uber Bau und Entwtcklung der Zahne des Knochenfisches Anarrhichus lupus L. und ihren Befall mit “Mycrlitrs ossifraguc”. Z. Zellftirwh. Mikrork Anar. 40, 25 48. Schmidt W. J. 1962a. M~cr/irr.~befall an Stacheln von lebenden Seeigeln. Zoo/. An:. 169. 2455252. Schmidt W. J. 1962b. Uber Mtcelitr+Befall an Zahnen fossiler Haie. Inr. Rww grs. Hrtlrohiol. I-fycfrrqr. 47. 587.-601. Schmidt W. J. and Keil A. 1971. Polurizim/ ~Lflc~rrwop\~ of Dr~~tul Tiuws. Pergamon Press. Oxford. Sognnaes R. F. 1950. Histological studies of anctent and recent teeth with special regard to differentral diagnosis between irtrru rituui and post mortem characteristtcs.
Jaekel
Am.
0.
J. PhJs.
Anthrop.
8. 269. 270.
Sognnaes R. F. 1955. the teeth of ancient Sognnaes R. F. 1963. special reference to
Post mortem microscoptc defects m man. Awhr Prrth. 59. 559 570 Dental hard tissue destruction with idiopathic erosions. In: .Mrd~mi,w~ of Hurt) Tiwtr Dr.struction (Edited by Sognnacs R. F.1. pp. 91 -153. Am. Ass. Adv. Sci.. Washington. Thomasset J.-J. 1931. Sur un champipnon fossile. .Vl~~,~c/irc,,\ os$rc~ya.\ (Roux). Bull. SOC. GoI. E‘r. I. 597 6113. Wedl C. 1864. Uber einen im Zahnhem und im Knochen ketmenden Pilr. Sher. dr. A/&. Uiss. 50. I71 IYJ Weiler W. 1976. Mitteilungen ubrr die Wtrhcltterrestc aus dem Mittelpliozan dea Natrontales. VII. Sclachit und
Acanthopterygii. REFERENCES
lupu\
Sh~r. c/t. A!&. U’is. 217 340.
Werelds R. J. 1961. Observations macroscopiquea et microscopiques sur certaines alterations post mortem dcs dents. Bull. Grpmr. inr. Rw/I. Scrcwt. Sfomtrr. 4. 7 60 Werelds R. J. 1967. Du moment oil apparaisscnt dans les dents humaines les alterations post mortem en forme d’evidements canaliculaires. Presence de Iesionu dcnmires identiques in viva chez des Potssons. Hill/. (;rpait. i,zr. Rt& Scir,ir. Src~rziar. 10. 419 447. Werner H. 1937. Scheinbare und wirkliche Karies on prahistorischen Ztihnen. t. Rrr.~wh. 5. 70 7Y
B. Kerebel,
I50
Marie-The&e
Le Cabellec
Plate Fig.
and Lise-Marie
Kerebel
I.
I. Burrowing lesion (0.26mm deep and 1.31 mm wide) hollowing out the external tooth layer above the approximal area of a molar-shaped tooth. Ground section, ordinary light. x 45
Fig. 2. Burrowing
channel
(bc) changing
direction abruptly. Terminal ordinary light. x 520
bulbs (arrows).
Fig. 3. Channels
(arrows)
Fig. 4. Burrows
(bc) unconnected with the course of the dentinal vascular canals channel (arrow). Ground section, ordinary light. x 550
Fig. 5. Advancing Fig. 6. External
within the external tooth layer (e) parallel with dentine. Ground section, ordinary light. x 450
Ground
the tooth
surface
layer with approximately spherical structures (arrows); section. Haematoxylin and eosin. x 175
Fig. 7. Detail of disintegrated within the burrows
(s). d,
(vc). Flat-ended
front of closely-packed burrows (bc) parallel with the general direction vascular canals (vc). Ground section, ordinary light. x 520 tooth
section,
of the dentinal
d. dentine.
Demineralized
external tooth layer (e) with shrunken burrowing organisms (bc). Demineralized section. Haematoxylin and eosin. x 450
(arrows)
Plate 2 Fig. 8. Cross-sections Fig. 9. Walls of channels Fig.
10. Hyphal Fig. Il.
structures Burrowing
of burrows (bc) marked
(h) related organism
Fig. 12. Burrowing organism Arrow indicates
on the tooth by ring-like
to spore-like bodies X 1900
(bo) in situ within
surface.
grooves
(s). Arrows
a burrow
SEM.
x 7000
(arrows).
SEM.
indicate
in dentine
x 2100
segmentation.
(d). TEM.
SEM.
x 21,000
(bo) filling completely the lumen of a channel. Vesicles (v), dentine a parasite ghost with scattered crystals (c). TEM. x 16.000
(d).
Plate 3. Fig. 13. Detail of membrane-bounded Fig.
14. Microvilli
Fig, 15. Dentine
vesicles (v) in close contact with the mineralized Dentine (d). TEM. x 90,000
(m) of the plasma crystals
membrane
of a burrowing x 80.000
tangential or perpendicular organism (bo). Dentine
Fig. 16. Crystals Fig. 17. Bacteria
with hollow
(b) within
Fig. 18. The bacteria
(bo). Dentine
to the plasma membrane (d). TEM. x 158,000
centres
(arrows).
the burrowed-out under
organism
higher
TEM.
channels.
resolution.
TEM.
wall of a channel.
(mb) of a burrowing
x 600,000 TEM.
(d). TEM.
x 22,500
x 128,000
Plate 1.
152
B. Kerebel, Marie-Th&se
Le Cabellec and Lise-Marie Kerebel
Plate 2.
Intra-vitam
parasitic destruction
of the teeth of Anarhichas lupus L.
Plate 3.
153
Faculte de Chirurgie Dentaire. I Place Alexis Ricordeau.
44042 Nantes.
France
Summary--Three specimens of Anurhichas lupus L. with teeth presenting burrowing lesions were studied with light microscopy, SEM. and high resolution TEM. Findings suggested that the lesions were due to fungoid invasion by Mrc,r/ires os.@~ga\. The burrowing lesions always originated in the more highly mineralized external tooth layer, thus distinguishing them from post-mortem soil changes which may start in any part of the tooth. The channels produced by the lesion had no particular orientation to the dentinal vascular canals. It is suggested that enzymes are secreted locally by the causal organism: burrows were thus not found at a distance from the fungus whose numerous microvilli were in close contact with the mineralized walls of the channels; this distinguishes the lesion from dental caries. Needle-shaped crystals are found within the cytoplasm or adherent to the plasma membrane of the burrowing organism. Other crystals in the immediate neighbourhood of the organism were hollow. and thus suggestive of an organism feeding on mineral material. Bacterial invasion was sometimes in association with the burrowing activity. and may play a part in it. but often appeared to be a later invasion.
INTRDDU(‘TION
and polarized light. Six other conical and four other molariform teeth from upper and lower jaws were demineralized in 10 per cent formalin and 5 per cent nitric acid in distilled water. embedded in Paraplast. sectioned at 5-811rn on a microtome and stamed by haematoxylin and eosin. Other teeth were coated successively with carbon and gold and examined under the scanning electron microscope. some of them directly, others after the organic material had been removed in sodium hypochlorite. Fragments of teeth were post-fixed in 2 per cent osmic acid with cacodylate buffer at pH 7.4. dehydrated in a graded series of methanol concentrations and embedded in methacrylate. Undemineralized sections were cut on an ultramicrotome with a diamond knife and examincd under the transmission electron microscope.
Peyer (1926) described fungoid invasion by Mycelitrs ossifragus in the teeth of Raiu clmufu. Arsuffi (1939) found elongated boring organisms containing granules deeply staining with azocarmine in the external vitrodentine layer of Tetrodon mucu[atus. Kohlenberger (1940) observed at the surface of the teeth of Anarhic+ras lupus L. a whitish microcrystalline material which Schmidt (1954) assumed to be the result of fungal attack by Mrc~litus ossifiugus. He had noted some whitish lesions on the occlusal surfaces of molar-shaped teeth and on the tips of conical teeth of an adult specimen of Anrrrhichas lupus L. Ground sections in ordinary and polarized light showed these chalky areas to be penetrated by bore canals similar to those attributed to attack by Mrcelires ossifiugus in fossil bone. Schmidt (1962a) also observed a similar microcrystalline crusty layer and bore canals in the spines of living specimens of sea-urchins. All this information was obtained by light microscopy only. The understanding of the mechanism of hard tissue destruction, either parasitic or microbial, seemed to us sufficiently important to warrant a thorough study of this parasitic process.
RF.SI’l.TS Whitish burrowing lesions, clearly visible against the yellowish background of the teeth. were found in both younger specimens and the older one. The lesions were on the tips of the conical anterior teeth and either on the tips or above the approximal areas of the molariform teeth. which are in close contact. Ground sections in ordinary light showed lesions, 0.26mm deep and 1.31 mm wide. hollowing out the external tooth layer (Figs. I 4). At the periphery of the lesion there was a front of closely packed burrows parallel with the general direction of the dentinal vascular canals (Fig. 5). Some of the channels in the advancing front changed direction abruptly (Figs. 2 and 3). Their progress wit,hin the dentinal tissue seemed to be unconnected,wtth the course of the vascular canals (Fig. 4).
%IATERIAI.SAI*;D METHODS Three specimens of ilnctrhichus lupxs L. were used. two young ones, 50 cm long, and an older one, 120 cm long. The animals were caught off the coasts of Newfoundland, and their jaws immediately fixed in 9 per cent neutral formalin. Ground sections. 60jtm thick, some stained with Toluidine blue. of 6 conical and 6 molariform teeth from upper and lower jaws were observed in ordinary 147
148
B. Kerebel. Marie-Thkrtse Le Cabellec and Lise-Marie Kerebel
The average diameter of the channels was about 4 pm, whereas the average diameter of the dentinal vascular canals was about IOpm. The channels were sometimes blunt-ended (Fig. 4) or presented terminal bulbs (Fig. 2). Roughly spherical structures, 30pm dia, were observed in the external areas of demineralized sections (Fig. 6). The external dental tissue was disintegrated. Within some channels, structures presumed to be the shrunken organisms were found (Fig. 7). With SEM, the attacked tooth surface appeared to be hollowed out with clear-cut cavities, whereas the mineralized material between the burrows remained undamaged (Fig. 8). The channels, running parallel to each other, presented ring-like grooves in the walls (Fig. 9). On surfaces from which the organic material had not been lost, some segmented filamentous structures, about 3.5pm in diameter, were observed, associated with spherical bodies of about 2.5pm diameter (Fig. IO). Undemineralized sections with TEM showed the burrowing organisms in situ. Sometimes a narrow space, tilled with many small vesicles. remained between the parasite and the mineralized walls of the burrowed-out channel (Fig. 11). Sometimes the burrowing organism appeared to fill the lumen of the channel completely (Fig. 12). There were then many membrane-bound vesicle-like bodies in close contact with the mineralized walls (Fig. 13). The vesicle-like bodies originated from the microvilli of the cytoplasmic membrane of the microorganism (Fig. 14). Dentine mineral crystals were visible outside the plasma membrane, tangential or perpendicular to it. Similar crystals were also scattered within the cytoplasm (Fig. 15). The dentinal tissue in contact with the burrowing organism contained some crystals with hollow centres (Fig. 16). Adjacent to channels completely filled with the organism, there were others in which only a parasite ghost remained, together with scattered crystals (Fig. 12). Bacteria were found within channels where a space remained between the parasite and the mineralized walls (Figs. 17 and 18). Sometimes, they occupied exclusively spaces where hard tissue had been destroyed (Fig. 17). DISCUSSION
The burrowing lesions were located in the external dental tissue. Some workers, using light microscopy (Le Cabellec, Daculsi and Geistdoerfer, 1978) and X-ray diffraction (Kerebel ef u/., 1978). describe the external dental tissue (called durodentine by Schmidt, 1954) as better mineralized than the rest of the dentine. Initially, the burrowing lesions appeared to be located in the external tooth layer exclusively, in agreement with Schmidt’s observations (1954) and contrary to those of Thomasset (1931). Starting from the surface of the better mineralized dental tissue, the organisms proceeded into the dentinal tissue, avoiding the course of the vascular canals which would be likely to provide an easier pathway for organisms feeding on organic material. Burrowed-out channels and vascular canals can be easily differentiated by their dimensions. The sharply cut-out lesions seemed to be produced by direct contact of the organism with the dentine
which, at the periphery of the channels, remained undamaged, as seen with SEM. With SEM the ends of the pits bored by the parasite appeared slightly concave. The walls were marked by a series of ring-like grooves, as if the lesion had been progressively pitted. Although the destructive process was strictly localized to the area in direct contact with the organism, the great number of vacuoles within the cytoplasm suggests an associated enzyme activity. The burrowing process is quite different from dental caries. In caries bacterial enzyme-containing secretions diffuse within the dentine and dentinal tubules so that damage is always found at a distance from the initial site of attack. In caries, the material within dentinal tubules is known to be due to a diffused process of demineralization and proteolysis, whereas the burrowing lesions were always found on surfaces in direct contact with the burrowing organism. In agreement with Schmidt (1954) we found no burrowing lesions in mucous-covered tooth surfaces or unerupted teeth. TEM examination of the lesions showed that no demineralization occurred at a distance from the burrowing organism. However, many vesicles were found in close contact with the undamaged tissue, at the interface with the organism. These vesicles were interpreted as sections of the numerous microvilli of the plasma membrane. The needle-shaped crystals. found either within the cytoplasm or adherent to the plasma membrane, whereas others seemed to be torn from the periphery of the dental tissue, all in the immediate neighbourhood of the burrowing organisms, are suggestive of an organism feeding on mineral material. This is in contradictjon with Schmidt’s findings on post-mortem attack by Mycelites ossifragus (Schmidt and Keil, 1971). Schmidt (1954) noted that in Anarhichas lupus L. intra-vitam attack by Mycelites ossifragus did not affect teeth covered by the oral mucosa, the vascular canals and the pulpal cavity, i.e. areas containing organic material. However, Schmidt’s conclusions were based on light microscopy only. TEM observations provide more precise and reliable information. Bone and tooth burrowing lesions in living and fossil species have been attributed to fungoid invasion by organisms called collectively Mwelites ossifragus (Peyer, 1968). Post-mortem changes of this kind in human teeth long buried in soil have been described (Paltauf and Haberda, 1.927; Werner, 1937; Soggnaes, 1950, 1955; Hammarlund-Essler, 1952; Werelds, 1961. 1967). Burrowing lesions in the teeth of living fishes are similar in many details to the changes in fossil bone and teeth (selachians, teleosts, reptiles) attributed to Myc’elites ossifrugus (Roux, 1887; Jaekel, 1890; Bauer, 1898; Weiler, 1926: Peyer, 1926, 1945, 1968; Thomasset. 1931: Schmidt, 1954. 1962a.b; Schmidt and Keil, 1971). Our findings here suggest that the burrowing progresses from the tooth surface towards the inside. On the contrary, post-mortem destruction starts anywhere in the mineralized tissue and the pulp surface of teeth is commonly affected (Thomasset. 193 1; Clement, 1963: Soggnaes. 1963). On this difference, Peyer (1968) and Schmidt and Keil (1971) drew a distinction between post-mortem and intravitam attacks. Immersion in sea-water is not a necessary condition for fungal attack by Mwe/ite.s os.sij?ayus. According
Intra-vitam
parasitic
destruction
(1864). human teeth soaked in fresh water showed invasion of mycelial filaments and spores starting from the surface. Wedl’s drawings are very like our photomicrographs of the parasitic lesions in .4narhichas lupus L. Light microscopy of the injured dental tissue of Anarhichas lupus L. showed spore-like bodies outside the lesions, whereas hyphal structures were found within the burrowed-out channels. These observations were corroborated by SEM which showed filaments, suggestive of a fungal hypha, penetrating into the burrow and related to spores outside the pit. Segments visible on the filament are suggestive of the transverse septa described in certain fungal species. The hyphae seen with SEM penetrating burrows and filling it completely can be assumed to be the initial agent responsible for the hard tissue destruction. Where destruction was deep, bacteria were found either together with the parasite or alone, suggesting that bacterial invasion occurs either together with the fungal attack or after it; bacteria may play some part in the destructive process, Thomasset (193 I). investigating fungoid invasion in the teeth of several fish species. noted that Mycelites oss(/i~~g~cs is originally iocalized in bone tissue and penetrates the teeth only where the dental tissue has a structure and hardness similar to bone. There is some evidence that Mycelites ossifrugus may have a special affinity for calcium carbonate. Our investigato Wed1
tions do not corroborate this. X-ray diffraction (Kerebel c’f rrl.. 1978) shows that the external dental tissue of Anurhic~ha.~ lupus L. consists chiefly of calcium
phosphate
and magnesium. We found that the burhad a greater affinity for this more mineralized tissue than for the underlying dentine which possesses a more or less amorphous phase of calcium phosphate (Kerebel et (I/., 1978). AI Anarhichus lupus L. feeds on echinoderms and molluscs which are infested by Mj.celites ossifragus (Schmidt. 1954). this is likely to explain how the organism comes into direct contact with the teeth. Attrition facets of tooth surfaces of Anarhichas hqxc.\ L. are frequently observed in the SEM (Le Cabellec er ul., 1978). However. initial wear lesions are not necessary for fungoid invasion because burrowing lesions were present on the sides of molariform teeth which, not being in occlusion, are not subject to functional damage. rowing
organism
~(,~)~oM./R/~YI~IP~II\ The research
investigation
grant from INSERM. Indivtduelle 77 I 00 74.
ATP
was supported by and Aide
53 77 85
of the teeth of .4ntrrhichus
Arsuffi E. 1939. Beitrage zur vergleichenden Histologic und Histogenese der Zahne. Untersuchungen am Gebiss van Labridae, Sparidae und Gymnodontes. Z. Zellforsch. 29, 670-693. Bauer F. 1898. Die lchthyosaurier des oberen weissen Jura. Polronroqruph~ 44, 283- 328. Clement A. J. 1963. Variations in the microstructure and biochemtstry of human teeth. In: Drnrul Anthropolq)
L.
14’)
(Edited by Brothwell D. R.) Vol. 5, pp. 245 269. Pergamon Press, Oxford. Hammarlund-Essler E. 1952. Histologisk Undersokning av Tander oc Kakben fran Medeltidsskelett. St.ensk TlrnGkwe-Tidskr$.
45,
275- 290.
1890. Gange van Fadenpilzen IM\,c,c,/ifc>* O.MIC;e.\. fruyus Roux) in Dentinbildungen. Sitrufic&~ Nurur$ frrunde Berlin, pp. 92-94. Kerebel B.. Le Cabellec M. T.. Daculsi G. and Kerebel L. M. 1978. Osteodentine and vascular osteodentine of .4nurhic,hw Iupu~ L. Crs// Tiu. Rrs. 187. 135 146. Kohlenberger H. 1940. Zur Kenntnis dea Vasodentins. 1. mikrosk.-unur. Fowh. 48, 461 477. Le Cabellec M. T.. Daculsi G. and Geistdoerfer P. lY78. Rapports de la morpholog,ie et de l’histologie dentaires d‘ Anurhic,huslupus L. (Potsson. Teltosteen Perciforme) avec son mode d’alimentation: Apport de la microradiographie et du marquape par la tetracycline. (~~~a..I %r&. 56. 1103 1109. Paltauf A. and Haberda A. 1927. Der Zahn m forensischer Beziehung. In: Hundhucl: der Zuhnhrilkunde (Edited by SchefB. Urban .and Schwarzcnberg. Berlin and Wien. Peyer B. 1926. Uber einen Fall von Caries an einem Rochengebiss: Crrh. ,sc,/tweiz. ,iuturf. Gc,.s. 107. 242. Peycr B. 1945. llber Algen turd Pilre in tirrischen Hartsubstanzen. rlrch. Jftlrir~ K/cut\-Sri/r. I we+l‘rww/~ 20. 496 546. Peyer B. 1968. Corttpurtrtirr O~ionroloyt~ (Edited by Zangerl R.l. The University of Chicago Press. Chicago. Roux W. 1887. Uber einen m Knochen lebende Gruppe von Fadenpilzen (M~crlirrs o.uifrtuqlts). Z. WI\.,. Lo,)/. 45. 227-254. Schmidt W. J. 1954. Uber Bau und Entwtcklung der Zahne des Knochenfisches Anarrhichus lupus L. und ihren Befall mit “Mycrlitrs ossifraguc”. Z. Zellftirwh. Mikrork Anar. 40, 25 48. Schmidt W. J. 1962a. M~cr/irr.~befall an Stacheln von lebenden Seeigeln. Zoo/. An:. 169. 2455252. Schmidt W. J. 1962b. Uber Mtcelitr+Befall an Zahnen fossiler Haie. Inr. Rww grs. Hrtlrohiol. I-fycfrrqr. 47. 587.-601. Schmidt W. J. and Keil A. 1971. Polurizim/ ~Lflc~rrwop\~ of Dr~~tul Tiuws. Pergamon Press. Oxford. Sognnaes R. F. 1950. Histological studies of anctent and recent teeth with special regard to differentral diagnosis between irtrru rituui and post mortem characteristtcs.
Jaekel
Am.
0.
J. PhJs.
Anthrop.
8. 269. 270.
Sognnaes R. F. 1955. the teeth of ancient Sognnaes R. F. 1963. special reference to
Post mortem microscoptc defects m man. Awhr Prrth. 59. 559 570 Dental hard tissue destruction with idiopathic erosions. In: .Mrd~mi,w~ of Hurt) Tiwtr Dr.struction (Edited by Sognnacs R. F.1. pp. 91 -153. Am. Ass. Adv. Sci.. Washington. Thomasset J.-J. 1931. Sur un champipnon fossile. .Vl~~,~c/irc,,\ os$rc~ya.\ (Roux). Bull. SOC. GoI. E‘r. I. 597 6113. Wedl C. 1864. Uber einen im Zahnhem und im Knochen ketmenden Pilr. Sher. dr. A/&. Uiss. 50. I71 IYJ Weiler W. 1976. Mitteilungen ubrr die Wtrhcltterrestc aus dem Mittelpliozan dea Natrontales. VII. Sclachit und
Acanthopterygii. REFERENCES
lupu\
Sh~r. c/t. A!&. U’is. 217 340.
Werelds R. J. 1961. Observations macroscopiquea et microscopiques sur certaines alterations post mortem dcs dents. Bull. Grpmr. inr. Rw/I. Scrcwt. Sfomtrr. 4. 7 60 Werelds R. J. 1967. Du moment oil apparaisscnt dans les dents humaines les alterations post mortem en forme d’evidements canaliculaires. Presence de Iesionu dcnmires identiques in viva chez des Potssons. Hill/. (;rpait. i,zr. Rt& Scir,ir. Src~rziar. 10. 419 447. Werner H. 1937. Scheinbare und wirkliche Karies on prahistorischen Ztihnen. t. Rrr.~wh. 5. 70 7Y
B. Kerebel,
I50
Marie-The&e
Le Cabellec
Plate Fig.
and Lise-Marie
Kerebel
I.
I. Burrowing lesion (0.26mm deep and 1.31 mm wide) hollowing out the external tooth layer above the approximal area of a molar-shaped tooth. Ground section, ordinary light. x 45
Fig. 2. Burrowing
channel
(bc) changing
direction abruptly. Terminal ordinary light. x 520
bulbs (arrows).
Fig. 3. Channels
(arrows)
Fig. 4. Burrows
(bc) unconnected with the course of the dentinal vascular canals channel (arrow). Ground section, ordinary light. x 550
Fig. 5. Advancing Fig. 6. External
within the external tooth layer (e) parallel with dentine. Ground section, ordinary light. x 450
Ground
the tooth
surface
layer with approximately spherical structures (arrows); section. Haematoxylin and eosin. x 175
Fig. 7. Detail of disintegrated within the burrows
(s). d,
(vc). Flat-ended
front of closely-packed burrows (bc) parallel with the general direction vascular canals (vc). Ground section, ordinary light. x 520 tooth
section,
of the dentinal
d. dentine.
Demineralized
external tooth layer (e) with shrunken burrowing organisms (bc). Demineralized section. Haematoxylin and eosin. x 450
(arrows)
Plate 2 Fig. 8. Cross-sections Fig. 9. Walls of channels Fig.
10. Hyphal Fig. Il.
structures Burrowing
of burrows (bc) marked
(h) related organism
Fig. 12. Burrowing organism Arrow indicates
on the tooth by ring-like
to spore-like bodies X 1900
(bo) in situ within
surface.
grooves
(s). Arrows
a burrow
SEM.
x 7000
(arrows).
SEM.
indicate
in dentine
x 2100
segmentation.
(d). TEM.
SEM.
x 21,000
(bo) filling completely the lumen of a channel. Vesicles (v), dentine a parasite ghost with scattered crystals (c). TEM. x 16.000
(d).
Plate 3. Fig. 13. Detail of membrane-bounded Fig.
14. Microvilli
Fig, 15. Dentine
vesicles (v) in close contact with the mineralized Dentine (d). TEM. x 90,000
(m) of the plasma crystals
membrane
of a burrowing x 80.000
tangential or perpendicular organism (bo). Dentine
Fig. 16. Crystals Fig. 17. Bacteria
with hollow
(b) within
Fig. 18. The bacteria
(bo). Dentine
to the plasma membrane (d). TEM. x 158,000
centres
(arrows).
the burrowed-out under
organism
higher
TEM.
channels.
resolution.
TEM.
wall of a channel.
(mb) of a burrowing
x 600,000 TEM.
(d). TEM.
x 22,500
x 128,000
Plate 1.
152
B. Kerebel, Marie-Th&se
Le Cabellec and Lise-Marie Kerebel
Plate 2.
Intra-vitam
parasitic destruction
of the teeth of Anarhichas lupus L.
Plate 3.
153