Sexual Transmission of Spotted Fever Group Rickettsiae by

Transcription

1 INFECTION AND IMMUNITY, Feb. 1980, p Vol. 27, No /80/ /05$02.00/0 Sexual Transmission of Spotted Fever Group Rickettsiae by Infected Male Ticks: Detection of Rickettsiae in Immature Spermatozoa of Ixodes ricinus STANLEY F. HAYES,`* WILLY BURGDORFER,' AND ANDRE AESCHLIMANN2 National Institute ofallergy and Infectious Diseases, Epidemiology Branch, Rocky Mountain Laboratories, Hamilton, Montana 59840,1 and Zoological Institute of the University of Neuchdtel, Neuchdtel 2000, Switzerland2 Electron microscopic evidence is provided showing that the newly described "Swiss agent," a spotted fever group rickettsia, is incorporated into the reproductive cells of its male tick vector, Ixodes ricinus. Rickettsiae were found in spermatogonia, spermatocytes, and maturing spermatids. The potential significance of these observations is briefly discussed in relation to published data on sexual transmission of rickettsiae by ticks. Wolbach, as early as 1919 (16), suggested the presence of spotted fever rickettsiae in spermatozoa of the tick vector, Dermacentor andersoni, and in 1933, Philip and Parker (11) demonstrated the occurrence of sexual transmission of Rickettsia rickettsii by infected males to noninfected tick females. These observations led to the speculation that sexual transmission represented an important ecological factor in the maintenance of spotted fever rickettsiae in nature. Burgdorfer and Canaris (cited by Burgdorfer and Varma) (4) also noted the transfer of R. rickettsii during copulation of normal D. andersoni females with infected males but found no evidence of rickettsiae in the tissues of spermiophores per se; the organisms were primarily located in the spermatophore and spermiophore fluids. Recent electron microscopic investigations of a new spotted fever group rickettsia in Ixodes ricinus from Switzerland (1; W. Burgdorfer, A. Aeschlimann, 0. Peter, S. F. Hayes, and R. N. Philip, Acta Trop., in press) revealed massive rickettsial infections throughout the testicular tissues of ticks. The presence of this rickettsial agent (Swiss agent) in spermatogonia and spermatocytes and its incorporation into maturing spermatids of L ricinus are the subjects of this paper. (5) or with fluorescein isothiocyanate-labeled immunoglobulin against the Swiss agent. Segments of tissues from the other testicular lobe were used for electron microscopy. For this purpose, tissues were fixed according to previously described methods (7), dehydrated in acetone, and embedded in Spurr medium (13). Silver and silver-gold sections were obtained with a Du Pont diamond knife on a Reichert OM-U2 ultramicrotome, placed on naked 300-mesh copper grids, and poststained with 2% aqueous uranyl acetate (ph 3.9) for 5 min and then with Reynolds lead citrate or Venable and Coggeshall lead stain (12, 15) for 4 min. They were then examined with a Hitachi 11-EU electron microscope at 75 kv. RESULTS Light microscopy. Gim6nez- and FA-stained smears of testicular tissues revealed typically stained rickettsiae throughout the supportive tissues as well as in spermatogonia and spermatocytes (Fig. 1, 2). In spermatids, rickettsiae could no longer be seen. Occasionally there were, within spermatids, concentrations of chromatic granules similar to rickettsial organisms in size and color. However, these granules were found also in control ticks and subsequently were identified as mitochondria. Rickettsial organisms were not present in tissues of control ticks. Electron microscopy. Examination of testicular tissue containing spermiophore cysts MATERIALS AND METHODS composed of germinal cells, primary spermatocytes, and early spermatids revealed apparently Freshly molted male I. ricinus from the progeny of dividing rickettsiae in the epithelial cells of the females infected with the Swiss agent were used. Male ticks from a colony free of rickettsiae served as controls. For removal of the testicular tissues, the ticks the spermiophore cysts (Fig. 4), and within de- testes wall (Fig. 3), within the flattened cells of were dissected in brain heart infusion broth by methods described previously (7). Impression and tissue and 6). Sections prepared from normal control veloping spermatocytes and spermatids (Fig. 5 smears were then prepared from one of the two testicular lobes and were stained by the method of Gim6nez Within early spermatocytes, the rickettsiae ticks were devoid of these organisms. 638

2 .ik / -ibe lel Sr t a S0 *1**.- Aw a'r 71 a I 0~~~~~~~~~~~~~~~~H 'C,~~~~~~~~~~~~~~~~~~N W xi.-~~~~~~~~~~~~~~~i FIG. 1. Testicular tissue of I ricinus showing the Swiss agent within the cyst wall connective tissue cells (Gimeinez stain, 1,900x). FIG. 2. Early spermatocyte infected with the Swiss agent. Early spermatids may be seen in the background (see arrows) (Gimenez stain, 1,900x). FIG. 3. Thin section profile of Swiss agent in tissues of the outer testicular wall (16,500X). FIG. 4. The Swiss agent in the cyst wall tissues of a testicular cyst. Developing spermatocytes exhibiting early cisternal ring development are adjacent to the infected tissue (12,300x). 639

3 /.E -.9 '4'..;v,,. 6 AX 7.t'$1.:..' 4h... -A*::.,. ;;Tl. I.,.,#A.. V-,..*. - x ~~~~~~~~~~~/.(4. '-,.: IV. -AW FIG. 5. Early spermatocyte infected with the Swiss agent (16,500x). FIG. 6. Rickettsiae located within the cytoplasmic region of a developing spermatid. Mitochondria (arrows) are seen adjacent to the internal cisternal ring (27,OOOx). FIG. 7. Cross-section profile of an elongating spermatid infected with the Swiss agent. The rickettsiae are located adjacent to the elongated nucleus. Mitochondria may also be seen within the central cytoplasmic area of the spermatid (36,OOOx). FIG. 8. Ultrastructural appearance of the Swiss agent in I. ricinus testicular wall tissue (33,OOOx). 640

4 VOL. 27, 1980 were present throughout the cytoplasm where they often appeared closely associated with developing subplasmalemmal cysternae and mitochondria. Frequently they were also close to the elongated nucleus (Fig. 7) located near the outer, posterior surface of the developing spermatid. No rickettsiae were observed to inhabit the cytoplasm of intercellular bridges connecting adjacent spermatids to one another. The rickettsial organisms (Fig. 8) possessed an ultrastructure described previously for the Swiss agent (Burgdorfer et al., in press), i.e., a trilaminar cell wall, a microcapsular layer, a prominent plasma membrane that encloses a cytoplasm containing randomly dispersed (not prominent) ribosomes, and a finely amorphous ground substance. Most organisms had a splotchy, almost vacuolar, appearance caused by some degree of reticulation of the nuclear material. A discrete halo zone (slime layer) was not always associated with each organism. The rickettsiae were never found within any form of membrane-limited vacuole. Despite occasionally heavy rickettsial invasion of testicular tissues, there was no histopathological evidence of degeneration of spermatids. DISCUSSION The demonstration by electron microscopy of the rickettsial Swiss agent in spermatogonia, spermatocytes, and early spermatids of L ricinus provides definite evidence that rickettsial agents are capable of invading the germ cells of male ticks. However, since this rickettsial infection has not been followed during the entire process of spermiogenesis (the final part of which takes place in the genital system of the female tick), we have not determined whether or not such rickettsiae-laden spermatozoa are capable of infecting the eggs of a normal L ricinus female. Our observations supported the idea that other rickettsiae such as the spotted fever agent, R. rickettsii, might also be found in sperm cells of its tick vector, D. andersoni, as postulated by Wolbach. Indeed, preliminary electron microscopy of testicular tissues of infected wood ticks confirmed the presence of R. rickettsii, not only in spermatogonia and spermatocytes, but also in developing spermatids. Thus, it appears that previous failure (cited by Burgdorfer and Varma) (4) to detect R. rickettsii in maturing sperm cells may be attributed to the fact that the stains used (Gim6nez and direct fluorescentantibody technique applied to smear preparations) are not capable of penetrating the sheath of spermatids. By using these methods we failed to detect the Swiss agent in spermatids of I. ricinus. SPOTTED FEVER GROUP RICKETTSIAE 641 Copulation of L ricinus, unlike that of D. andersoni, takes place independently of a blood meal and may occur shortly after a female reaches the adult stage, long before the tick is ready for a blood meal (2, 6-10, 14). Therefore, we speculate that rickettsiae transferred via spermatids as well as with the spermatophore and spermiophore fluids may have sufficient time to cause a massive infection of the ovary and possibly also of other tissues. According to Philip and Parker (11), sexual transmission of R. rickettsii by infected D. andersoni males occurred in four of 11 tests. In three of these, the female ticks when allowed to engorge on guinea pigs proved infectious, suggesting invasion of salivary glands by the rickettsiae transferred during copulation. Since copulation of D. andersoni takes place only during the feeding process, Philip and Parker allowed female ticks to partially feed, removed them for copulation with infected males in pill boxes, and after 3 to 5 days, again placed them on guinea pigs for at least 9 days or until complete engorgement had been accomplished. It was not determined whether the transfer of R. rickettsii occurred by infectious male secretions or via infected spermatozoa Ȧlthough the transfer of R. rickettsii during copulation has repeatedly been confirmed, there is as yet no conclusive evidence that this process under natural feeding conditions will lead to a generalized infection of female ticks. Also, all tests to demonstrate rickettsial infection in the progeny of such females have so far been negative (Burgdorfer et al., in press). The above data suggest that rickettsiae now known to be present not only in male seminal fluids, but in the spermatids as well, may not be present in sufficient quantities to produce a generalized infection. Physiological changes may be induced in the rickettsiae, as the developing spermatozoa undergo spermatogenesis, spermiogenesis, capacitation, and involvement in the fertilization process (3, 9), to the extent that they are rendered avirulent and nonpathogenic. It may also be speculated that they are eliminated with the shedding of excess cytoplasm during capacitation and elongation of the maturing spermatozoa (9) Ṫhe mechanisms of sexual transmission and the significance of this phenomenon as a means of infecting ticks with rickettsiae are far from being resolved. However, research is now in progress at both the Rocky Mountain Laboratories and the Zoological Institute of the University in Neuchatel to define the physiological behavior and development of the Swiss agent and of R. rickettsii in spermatozoa before and

5 642 HAYES, BURGDORFER, AND AESCHLIMANN during the fertilization processes of their respective tick vectors. ACKNOWLEDGMENT This work is supported in part by a grant from the Swiss Foundation for Scientific Research (no ). LITERATURE CITED 1. Aeschlimann, A., W. Burgdorfer, H. Matile, 0. Peter, and R. Wyler Aspects nouveaux du r6le de vecteur joue par Ixodes ricinus L. en Suisse. Acta Trop. 36: Balashov, Y. S Nutrition and course of spermatogenesis in male ixodids. Dokl. Akad. Nauk SSSR 110: Brinton, L. P., W. Burgdorfer, and J. H. Oliver, Jr Histology and fine structure of spermatozoa and egg passage in the female tract of Dermacentor andersoni Stiles (Acari-Ixodidae). Tissue Cell 6: Burgdorfer, W., and M. G. R. Varma Transstadial and transovarial development of disease agents in arthropods. Annu. Rev. Entomol. 12: Gimenez, D. F Staining rickettsiae in yolk-sac cultures. Stain Technol. 39: Graf, J.-F Copulation, nutrition et ponte chez Ixodes ricinus L. (Ixodoidea: Ixodidae), 2e partie. Bull. Soc. Entomol. Suisse 51: Hayes, S. F., and W. Burgdorfer Ultrastructure of Rickettsia rhipicephali, a new member of the spotted fever group rickettsiae in tissues of the host vector INFECT. IMMUN. Rhipicephalus sanguineus. J. Bacteriol. 137: Oliver, J. H., Jr Symposium on reproduction of arthropods of medical and veterinary importance. J. Med. Entomol. 11: Oliver, J. H., Jr., and L. P. Brinton Sperm maturation in ticks: an example of capacitation in invertebrates? p In W. Junk (ed.), Proceedings of the Third International Congress on Acarology. Prague. 10. Oliver, J. H., Jr., and L. P. Brinton Cytogenetics of ticks (Acari: Ixodoidea). 7. Spermatogenesis in the Pacific Coast tick, Dermacentor occidentalis Marx (Ixodidae). J. Parasitol. 58: Philip, C. B., and R. R. Parker Rocky Mountain spotted fever: investigation of sexual transmission in the wood tick Dermacentor andersoni. Public Health Rep. 48: Reynolds, E. A The use of lead citrate at a high ph as an electron opaque stain for cell microscopy. J. Cell. Biol. 1: Spurr, A. R A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26: Tuzet, O., and T. Millot Recherches sur la spermiogeneses des Ixodes. Bull. Biol. Fr. Belg. 71: Venable, J. H., and R. Coggeshall A simplified lead citrate stain for use in electron microscopy. J. Cell. Biol. 25: Wolbach, S. B Studies on Rocky Mountain spotted fever. J. Med. Res. 41:1-197.