KEVIN R. KAZACOS1 AND JOHN J. TuREK2

Transcription

1 Proc. Helminthol. Soc. Wash. 50(1), 1983, pp. 36^2 Scanning Electron Microscopy of the Eggs of Baylisascaris procyonis, B. transfuga, and Parascaris equorum, and Their Comparison with Toxocara canis and Ascaris suuin KEVIN R. KAZACOS1 AND JOHN J. TuREK2 1 Department of Veterinary Microbiology, Pathology and Public Health and 2 Electron Microscopy Laboratory, School of Veterinary Medicine, Purdue University, West Lafayette, Indiana ABSTRACT: The surface structure of the eggs of Baylisascaris procyonis, B. transfuga, and Parascaris equorum was studied using light and scanning electron microscopy, and found to be almost identical. By light microscopy, the egg shells of these species appear to be of three layers, and the surface is finely granulated or particulate. By scanning EM, the surface of these eggs is irregularly granular; by higher magnification, the many granules are part of a fine three-dimensional reticular framework or lattice of fibrils. There is no evidence of openings or sutures in these eggs, and the eggs are not mammillate or pitted as described by others. A fine reticular substructure is also seen in the surface of Toxocara canis eggs by high-magnification SEM, but not in Ascaris situm eggs. The operculumlike region described at one pole of A. sunm eggs is confirmed. The surface morphology of nematode eggs is a useful characteristic for identification as well as for assessing taxonomic relationships (Hartwich, 1962). Scanning electron microscopy (SEM) has been used successfully in studying and comparing the fine-structural morphology of various nematode eggs (Ishii, 1972; Ishii and Habe, 1973; Specian et al., 1973; Ubelaker and Allison, 1975; Barus et al., 1979; Wharton, 1979). Baylisascaris procyonis and B. columnaris, the common ascarids of raccoons and skunks, respectively, have surfaced as important causes of visceral larva migrans and fatal cerebrospinal nematodiasis in animals (see Kazacos et al., 1981, and Reed et al., 1981, for references). Because both species also possess marked zoonotic potential (Kazacos, 1981; Kazacos et al., 1981), it is important to recognize properly Baylisascaris eggs from feces or soil. Few descriptions of Baylisascaris eggs exist; most are based on light microscopic observations, and are inconsistent and oftentimes contradictory (Goodey and Cameron, 1923; McClure, 1933; Mclntosh, 1939; Stefariski and Zarnowski, 1951; Sprent, 1953, 1968, 1970; Okoshi et al., 1962; Kikuchi et al., 1979; Uni and Takada, 1981). By light microscopy, the eggs of Baylisascaris spp. resemble those of Parascaris equorum, although lighter in color (pers. obs.). In this paper we report on the surface structure of Baylisascaris procyonis, B. transfuga, and Parascaris equorum eggs, as determined by light and scanning electron microscopy. Comparisons are made to the eggs of Toxocara canis and Ascaris sunm, and new information is presented on the fine structure of these eggs. Materials and Methods Adult female Baylisascaris procyonis were collected from the intestines of raccoons live-trapped in West Lafayette, Indiana. Eggs were removed from the terminal uteri of the worms and fixed in cold 3% glutaraldehyde in phosphate buffer (ph 7.4). Eggs of B. transfuga were collected from formalin-fixed worms 36

2 OF WASHINGTON, VOLUME 50, NUMBER 1, JANUARY previously collected from black bears at a local zoo. Paras car is e quorum, Toxocara canis, and Ascaris siium adults were collected locally from horses, dogs, and swine, respectively, and the eggs fixed as described for B. procyonis. Following fixation, the eggs were processed by routine methods. They were rinsed in 0.1 M phosphate buffer (ph 7.4), postfixed for 1 hr in 1% osmium tetroxide in phosphate buffer (ph 7.4), and rinsed again in buffer. They were then dehydrated through a graded series of ethanol, critical-point dried with carbon dioxide as the transition fluid, sputter coated with gold, and examined in an ISI Super MINI-SEM. Results The eggs of Baylisascaris procyonis, B. transfuga, and Parascaris equorum are rounded-oval in shape. By light microscopy the shell appears to be of three layers: an outer surface layer, a middle shell, and an inner lipid layer (Fig. 1). By light microscopy the surface of the eggs of B. procyonis, B. transfuga, and P. equorum is finely granulated or particulate, the numerous granules or particles being irregular in size and shape and with no apparent pattern (Fig. 2). This surface morphology is confirmed and further elucidated with SEM, which reveals that the surface of the eggs of these three species is almost identical (Figs. 3-8), and very different from that of T. canis and A. suum eggs (Figs. 9-12). By lower magnification SEM, the surface of Baylisascaris and Parascaris eggs is covered by many irregularly shaped granules or particles, which give the impression of being debris adherent to the eggs (Figs. 3, 5, 7). The granules are readily apparent at higher magnification, and are found to be part of the egg shell and to consist of one to many smaller granules or particulate subunits (Figs. 4, 6, 8). Also apparent at this magnification, the surface consists of a fine three-dimensional reticular framework or lattice of fibrils, which interconnect and branch with each other and with the granules, which are a part of the structure. The entire surface has this arrangement, and there is no evidence of any openings or sutures in the eggs of these species. By SEM, the eggs of Toxocara canis (Figs. 9, 10) are basically similar to what was seen by Ishii and Habe (1973) and Ubelaker and Allison (1975), except that the surface network of ridges and depressions is not nearly as irregular in pattern or distribution as that depicted and described by those workers. Also, under high magnification, a fine reticular substructure is seen in both the ridge and depression areas (Fig. 10). The eggs of Ascaris suum (Figs. 11, 12) are essentially as depicted previously (Ishii and Habe, 1973; Ubelaker and Allison, 1975), with a surface of large ridges and depressions. The operculumlike area seen by Ubelaker and Allison (1975) at one pole of these eggs was also observed (Fig. 11). Under high magnification, the fine reticular substructure seen in the T. canis eggs is not seen in the A. suum eggs (Fig. 12). Discussion The eggs of Baylisascaris spp. nematodes have been variously described by different investigators. Mclntosh (1939) felt that the eggs of B. schroederi were mammillated, whereas Goodey and Cameron (1923) and McClure (1933) felt that those of B. columnaris and B. procyonis, respectively, were finely mammillated.

3 38 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY. 4 t>v.- *m &- v X>?.ii.k**.«.-^ >-: n^ - I r»*s.'?'> ^ps'fc*' *V 7 *- AV L ^T %T.****:~&*4 * ^%.^H' -^ -» - W*f»-^V*'A * ""% ^ ^f*' %<!* %^*? *^^^ ^-^Lv i>/f- Light and scanning electron micrographs of Baylisascaris procyonis and B. transfuga Figures 1-6. eggs. 1, 2. Baylisascaris procyonis, light microscopy. Figure 2 shows surface detail. x800. 3, 4. Zfoylisascaris procyonis, SEM. Note irregularly granular surface and reticular framework substructure of granules and fibrils. x925 and x9,100, respectively. 5, 6. Baylisascaris transfuga, SEM. Structure similar to Figures 3 and 4. x950 and x9,100, respectively. Sprent (1953) described the eggs of B. devosi as covered with minute pits and elevations, but "evidently sticky as the eggs usually become coated with particulate matter." In his description of the genus Baylisascaris, Sprent (1968) stated that the eggs were finely pitted; this is also stated in the key of Hartwich (1974). Sprent (1970), however, indicated that the eggs of B. tasmaniensis were smooth and without pits, but with faint corrugations on their surface. Okoshi et al. (1962) stated that the egg of B. transfuga "resembles that of Ascaris lumbricaides," which is not really supported by their light-microscopic photographs, our work, or the work of others (Uni and Takada, 1981). Our light and SEM studies indicate that the surface of the eggs of B. procyonis, B. transfuga, and P. equorum is finely granulated, as it is comprised of many irregular granules on a lattice substructure (Figs. 1-8). The surface is not mam-

4 OF WASHINGTON, VOLUME 50, NUMBER 1, JANUARY Figures Scanning electron micrographs of Parascaris equorum, Toxocara canis and Ascaris suum eggs. 7, 8. Parascaris equorum, SEM. Structure similar to Figures 3 6. x950 and x8,925, respectively. 9, 10. Toxocara canis, SEM. Note regular surface patterning and fine reticular substructure. x925 and x!3,725, respectively. 11, 12. Ascaris suum, SEM. Note surface patterning, operculumlike area (arrow), and lack of visible reticular substructure. X975 and x9,200, respectively. millate or pitted as in the eggs of Ascaris and Toxocara, respectively (Webster's Third New International Dictionary, 1971; Figs. 9-12), as described by others. Our results are in agreement with Stefariski and Zarnowski (1951), who by light microscopy found the eggs of B. procyonis to be oval "a coque irregulierement granuleuse." An irregularly fine granular outer shell on B. procyonis eggs was

5 40 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY also noted by Hartwich (1962), although in his key he described Baylisascaris eggs as being finely pitted (Hartwich, 1974). Using SEM, Kikuchi et al. (1979) described the eggs of B. schroederi as covered by a proteinaceous coat upon which minute protuberances have formed (translation). Uni and Takada's (1981) excellent SEM photographs of B. transfuga eggs agree exactly with ours, but they described the eggs as being uneven and crepuscular (?). It is probable that the coating of B. devosi eggs with particulate matter that was noted by Sprent (1953) was actually a reflection of the irregularly granular outer surface itself, rather than adherent foreign material. However, some variation in egg structure may exist among Baylisascaris species, such as for B. tasmaniensis (Sprent, 1970) versus those reported here, because intrageneric variation in eggs is known for both Toxocara and Ascaris (Ubelaker and Allison, 1975). The operculumlike area seen at the pole of Ascaris eggs by Ubelaker and Allison (1975) was confirmed by Uni and Takada (1981) and in this study. No such area was seen by Ubelaker and Allison (1975) in Toxocara canis or T. felis eggs, nor has one been seen by others, including us in this study. Regarding Baylisascaris eggs, Uni and Takada (1981) indicated that "under light microscopy we believe the operculum-like area was probably there, as seen in one pole of the egg shell in some eggs." However, no SEM evidence of such a structure on Baylisascaris eggs was presented by them, nor was one seen by us in any of the Baylisascaris or Parascaris eggs examined by light microscopy or SEM. We therefore doubt the existence of such a structure on these eggs. With regard to the differences we saw in the regularity and structure of the ridges and depressions on T. canis eggs as compared to what was seen by other workers (Specian et al., 1973; Ubelaker and Allison, 1975), we attribute these differences to artifacts in their eggs, which showed marked shrinkage, distortion, and wrinkling. For the preparation of helminth eggs for our SEM studies, routine methods worked well, and the special glycerol preparative procedures, etc. advocated by Specian et al. (1973) apparently are not necessary. Based on the transmission EM studies by Poor (1967), coupled with their own SEM studies, Ubelaker and Allison (1975) suggested that the surface sculpturing present on many ascarid eggs (Ascaris, Toxocara, etc.) was due to outfolding and modification of the chitinous layer of the eggs, rather than directly due to uterine secretions (Monne and Honig, 1954) or to surface tension properties of coating proteins (Christenson et al., 1950). This was strongly supported by Wharton (1979), who found that the Toxocara-like surface of Porrocaecum ensicaudatum eggs was formed by variations in the thickness of the chitinous layer. Wharton (1979) suggested that this sculpturing would increase the structural strength of the shell, thus increasing the protection afforded to the embryo or larva within. From our studies, it can be seen that the surface structure of Baylisascaris and Parascaris eggs is very different from that of Ascaris or Toxocara. It is not known whether the eggs' chitinous layer contributes to the granular surface or reticular lattice in Baylisascaris or Parascaris, or whether the structure of these eggs is simply a reflection of the external uterine layer, and the result of a congealing or condensation of uterine protein secretions, somewhat along the lines of what Christenson et al. (1950) had in mind. With regard to Baylisascaris and Parascaris eggs, this question of shell makeup and contribution of the chitinous layer should be resolved by transmission EM studies. If the surface morphology of eggs is a valid characteristic in the assess-

6 OF WASHINGTON, VOLUME 50, NUMBER 1, JANUARY ment of taxonomic relationships, then Baylisascaris and Parascaris would appear perhaps to have some historical affinities. However, based upon their major differences in definitive hosts and life cycles, especially the utilization of intermediate hosts by most species of Baylisascaris (Sprent, 1953, 1954, 1973; Tiner, 1953a, b), their close similarities in eggshell surface structure are probably the result of parallel evolution in eggshell formation. Acknowledgments This work was supported in part by grants no. IND and IND073029V from the United States Department of Agriculture, and is journal paper number 8933 of the Purdue University Agricultural Experiment Station. Literature Cited Barns, V., F. Tenora, and R. Wiger Scanning electron microscopy of eggs of European species of the genus Syphacia Seurat, 1916 (Nematoda). Folia Parasitol. (Praha) 26: Christenson, R. O., L. Jacobs, and B. G. Chitwood Nemic ova. Pages in B. G. Chitwood and M. B. Chitwood, eds. Introduction to Nematology. University Park Press, Baltimore. Poor, W. E Ultrastructural aspects of oocyte development and shell formation in Ascaris lumbricoides. J. Parasitol. 53: Goodey, T., and T. W. M. Cameron Observations on the morphology and life history of Ascaris colutnnaris Leidy, a nematode parasite of the skunk. J. Helminthol. 1:1-8. Hartwich, G Uber den Waschbarenspulwurm Ascaris procyonis Stefanski et Zarnowski 1951, und seine Stellung im System der Ascaroidea (Nematoda). Cslka Parasitol. 9: (In German) Hartwich, G Keys to the genera of the Ascaridoidea. In R. C. Anderson et al., eds. CIH Keys to the Nematode Parasites of Vertebrates, No. 2. Commonwealth Agricultural Bureaux, Farnham Royal. 15 pp. Ishii, Y Morphology of helminth ova through the scanning electron microscope. Fukuoka Acta Med. 63: (In Japanese) Ishii, Y., and S. Habe Scanning electron microscopy of helminth ova. (15) Ascaris lumbricoides, Ascaris lumbricoides suum and Toxocara canis. Igaku no Ayumi 84: (In Japanese) Kazacos, K. R Animal and public health implications of the nematode genus Baylisascaris. 26th Annual Proc. Am. Assoc. Vet. Parasitol., St. Louis, Mo., p. 21. Kazacos, K. R., W. L. Wirtz, P. P. Burger, and C. S. Christmas Raccoon ascarid larvae as a cause of fatal central nervous system disease in subhuman primates. J. Am. Vet. Med. Assoc. 179: Kikuchi, S., T. Oshima, K. Saito, and Y. Okuyama Scanning electron microscopy of an ascarid Baylisascaris schroederi (Mclntosh, 1939) Sprent, 1968 from the giant panda. Jpn. J. Parasitol. 28: (In Japanese) McClure, G. W Nematode parasites of mammals. From specimens collected in the New York Zoological Park, Zoologica (N.Y.) 15: Mclntosh, A A new nematode, Ascaris schroederi, from a giant panda, Ailuropoda inelanoleuca. Zoologica (N.Y.) 24: Monne, L., and G. Honig On the properties of the egg envelopes of various parasitic nematodes. Ark. Zool. 7: Okoshi, S., M. Takashio, and S. Nakagawa Toxascaris transfuga (Rudolphi, 1819) Baylis et Daubney 1922, found in captive bears in Japan. Jpn. J. Vet. Sci. 24: Reed, W. M., K. R. Kazacos, A. S. Dhillon, R. W. Winterfield, and H. L. Thacker Cerebrospinal nematodiasis in bobwhite quail. Avian Dis. 25: Specian, R. D., V. F. Allison, J. E. Ubelaker, and J. H. Martin Preparation of amyl acetate and acetone labile eggs from parasitic nematodes for scanning electron microscopy. 31st Ann. Proc. Electron Microsc. Soc. Am., New Orleans, La., pp

7 42 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY Sprent, J. F. A On the life history of Ascaris devosi and its development in the white mouse and the domestic ferret. Parasitology 42: Sprent, J. F. A The life cycles of nematodes in the family Ascarididae Blanchard J. Parasitol. 40: Sprent, J. F. A Notes on Ascaris and Toxascaris, with a definition of Baylisascaris gen. nov. Parasitology 58: Sprent, J. F. A Baylisascaris tasmaniensis sp. nov. in marsupial carnivores: heirloom or souvenir? Parasitology 61: Sprent, J. F. A Observations on migratory behaviour and development of Baylisascaris tasmaniensis. Parasitology 67: Stefariski, W., and E. Zarnowski Ascaris pracyonis n. sp. z jelita szopa (Procyon lotor L.). Ascaris procyonis n. sp. provenant de 1'intestin de Procyon lotor L. Ann. Mus. Zool. Pol. 14: (In French) Tiner, J. D. 1953a. Fatalities in rodents caused by larval Ascaris in the central nervous system. J. Mammal. 34: Tiner, J. D. 1953b. The migration, distribution in the brain, and growth of ascarid larvae in rodents. J. Infect. Dis. 92: Ubelaker, J. E., and V. F. Allison Scanning electron microscopy of the eggs of Ascaris lumbricoides, A. suum, Toxocara canis, and T. mystax. J. Parasitol. 61: Uni, S., and S. Takada Comparison of scanning electron microscopy on Baylisascaris transfuga, Toxascaris leonina and Ascaris lumbricoides (Nematoda: Ascarididae). Jpn. J. Parasitol. 30: Webster's Third New International Dictionary of the English Language, Unabridged G. & C. Merriam Co., Springfield, Mass pp. Wharton, D. A The structure of the egg-shell of Porrocaecum ensicaudatum (Nematoda: Ascaridida). Int. J. Parasitol. 9: Survey or Taxonomic Papers Authors submitting manuscripts of a survey or taxonomic nature for publication in the Proceedings of the Helminthological Society of Washington are urged to deposit representative specimens in a recognized depository such as the National Parasite Collection at Beltsville, Maryland and include the accession numbers in the manuscript.