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Detection of Theileria sergenti schizonts in bovine lymph node
Inrernarumnl Journal/or C 1997 Australian Sooety
Pergamon
PII: SOO20-7519(97)00092-l
RESEARCH
Porositdog~y, Vol. 21, No. 1 I. pp. 1375.-1378. 1997 for Parasmlogy. Published by Elsevier Science Ltd Printed in Great Britam 002&7519/97 $17.00+0.00
NOTE
Detection of Theileria sergenti Schizonts in Bovine Lymph Node KATSURO HAGIWARA,*§ SATOSHI KAWAMOTO,? *School
KOUJI TAKAHASHI,* TAKASHI KUROSAWA,* CHIAKI ISHIHARA*
HIROYUKI KAZUYOSHI
and
of Veterinary Medicine, Rakuno Gakuen University, Bunkyoudai, Ebetsu,
Hokkaido 069, Japan Shintoku Animal Husbandry Experimental Hokkaido 081, Japan $Section of Serology, Institute of Immunological Science, Hokkaido Hokkaido 060, Japan THokkaido
TANIYAMA,* IKUTAS
Prefectural
Station, Shintoku, University,
Sapporo,
(Received 4 March 1997; accepted 10 July 1997)
Abstract-Hagiwara K., Takahashi K., Taniyama H., Kawamoto S., Kurosawa T., Ikuta K. & Ishihara C. 1997. Detection of Theileria sergenti schizonts in bovine lymph node. International Journalfor Parasitology 27: 1375-1378. The growth of Theileria serge& schizonts in bovine lymph nodes was examined by experimental infection. Six naive Holstein calves were inoculated with a sporozoite stabilate into the superficial cervical and subiliac lymph nodes. Biopsy samples from the inoculated calves were collected every 24 h for lOdays. Histological examination demonstrated the schizont of T. sergenti on day 4 post-inoculation. The growth of schiionts in the lymph node was observed on days 4-g post-inoculation. Electron microscopic observation disclosed that the core of the schizont has a high density central core surrounded by numerous electrondense spherules. 0 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd.
Key words: Theileria sergenti; Schizonts; lymph node. Theileriosis is one of the more serious infectious diseases of cattle in Japan. The full life-cycle of TheiZerin sergenti remained obscure for a long time, since the intracellular parasites are very small and many of their morphological details are unrecognisable by light microscopy. By histological observation, the patterns of asexual reproduction within the salivary glands of the vector ticks (Higuchi, 1987; Kawai et al., 1993; Takahashi et al., 1993) and within the erythrocytes of cattle (Kawamoto et al., 1990a, 1990b) were identified with certainty. Histological study for T. sergenti schizonts in experimentally infected calves was reported (Sato et al., 1993, 1994); however, the development of schizont growth in cattle remained unknown. Some progress towards sub-unit vaccine develop-
§To whom correspondence should be addressed. Tel: +81-(11)-386-11ll;Fax: +81-(ll)-387-5890.
ment for Theileriaparva has been made (Musoke et al., 1992; Nene et al., 1995). These studies focused on the recombinant sporozoite vaccine antigen, ~67. Immunity against T. sergentiinfections is likely to be directed at both the sporozoite and the schizont lifecycle stages. Lack of an in vitro cultivation system for the schizont-stage parasite and the difficulty of detection of T. sergenti schizonts in lymphoid cells in infected cattle has hampered progress towards vaccine development. Here, we focus on the detection of schizont growth in calf lymph nodes, by inoculating with sporozoite stabilate. Sporozoite stabilate of T. sergenti (Chitose strain; Takahashi, 1976) was used for animal infection. The stabilate was derived from the nymphal ticks (Huemaphvsalis mageshimaensis) picked up from a calf infected with T. sergenti(parasitaemia of 15%). Four months after feeding, the ticks were fed on rabbits at 20°C for 3days. Thereafter, the salivary glands of
1315
1376
K. Hagiwara et al.
these ticks were removed and were stained with methyl green pyronin to examine the appearance of sporozoites, according to the method described by Takahashi et al. (1993). The sporozoite stabilate was suspended at approximately 300 infected salivary gland acini in 1 ml of phosphate buffered saline (PBS) pH 7.2. For inoculation of sporozoite stabilate to calves, six naive Holstein calves (3-5 months old) were inoculated with sporozoite stabilate (0.25 ml of sporozoite stabilate/one lymph node) into superficial cervical and subiliac lymph nodes, respectively. As a control, the same dose of uninfected tick salivary gland acini suspension was inoculated into a lymph node of control calves. For histological preparation, lymph nodes from those calves were collected by excision biopsies at 24-h intervals for lOdays. All the animals were treated according to the Laboratory Animal Control Guidelines of the Rakuno Gakuen University. Tissues fixed in 4% phosphate-buffered paraformaldehyde (pH 7.4) were prepared for histological or immunohistochemical studies. The tissues were dehydrated in ethanol and embedded in paraffin. Histological sections (&6-pm thick) were processed for H & E staining, or used for immunohistochemistry. None of the calves showed any clinical symptoms throughout the experiments. The development of T. sergenti merozoites was observed in the erythrocytes from all of the inoculated calves at approximately 15 days post-inoculation. The lymph nodes from these inoculated calves were collected by biopsy for 10 days post-inoculation. Histological observation revealed that there were no inflammatory reactions and no schizont-like structures in the lymph nodes from inoculated calves on days l-3, and from the control lymph nodes throughout the experiments. However, early stages of the schizonts, approximately 55 x 44pm, were observed in lymph nodes on day 4 postinoculation. The cells had round- or oval-shaped basophilic cores and poor cytoplasm (Fig. 1A). Significant growth of the schizont cells was observed on days 5-6 post-inoculation (Fig. 1B), approximately 166x 1lOpm on day 5 and 266x 166pm on day 6, respectively (Fig. IC). Along with the development of the schizont cells, the cells had enlarged and irregular shaped basophilic cores, and were rich in cytoplasm having basophilic granules. On day 8, the core disappeared from the enlarged schizont cells and the cells were filled with numerous basophilic granules (Fig. ID). By days 9 and 10, the granule cells had disappeared from the lymph node (data not shown). To verify the localisation of the theilerial antigens, the avidin-biotin complex method was used. Histological sections from lymph nodes fixed in 4% phosphate-buffered paraformaldehyde (pH 7.4) were incu-
Fig. 1. The development of T. sergenti schizonts. (A) Parasitic cell has a large basophilic core and a small amount of cytoplasm on day 4 after inoculation. Scale bar = 5 pm. (B) Developing schizont cell has an enlarged basophilic core with irregular shape and a large amount of cytoplasm containing many basophilic granules peripherally on day 5 after inoculation. Scale bar=5 pm. (C) An enlarged schizont has a segmental core and an abundant cytoplasm containing a large number of basophilic granules on day 6 after inoculation. Scale bar = 20 pm. (D) A most developed schizont has a large amount of cytoplasm filled with numerous basophilic granules without basophilic core on day 8 after inoculation. Scale bar = 20 pm.
bated initially in 2% normal goat serum in PBS pH 7.4. The primary antibodies, anti-T. sergenti bovine serum, which were collected from T. sergentiinfected cattle, were applied next; these included antiT. sergenti antibodies (IFA titer: 1: 10 000) to identify theilerial antigen. After washing, the appropriate secondary antibodies consisting of biotinylated antibovine immunoglobulin were applied. This was followed by application of a horseradish peroxidase (HRP)-linked avidin-biotin complex (Histofine ABC kit, Nichirei, Japan). Application of diaminobenzidine/hydrogen peroxide to the bound HRP enzyme caused a brown precipitate. Immunohistochemical examinations disclosed that the baso-
1377
Fig. 2. Photomicrograph of T. sergenfi schizont on day 6 after inoculation. The basophilic granules in the cytoplasm of schizont cells reacted positively with anti-T. sergenri bovine serum. Avidin-biotin-peroxidase complex stain with methyl green counterstain. Scale bar = 10 pm. Inset: all basophilic granules in the schizont show a positive immunoreactivity with anti-T. sergenti antibody. Scale bar =40pm.
philic granules in the cytoplasm of schizont cells reacted positively with anti-K sergenti bovine serum (Fig. 2). For electron microscopic observation, lymph node tissues from sporozoite stabilate-inoculated calves were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C. These tissues were washed with phosphate buffers, refixed in 2% osmium tetroxide in phosphate buffers at 4°C dehydrated in a graded series of ethanols followed by propylene oxide and embedded in Quetole 812. The ultra-thin sections were stained with uranyl acetate and subsequently lead citrate. The prepared specimens were examined with an electron microscope (JEM-l200EX, Nihon Denshi). Electron microscopic observation of the schizont cells in lymph node on day 6 post-inoculation showed that the core of the enlarged schizont cell had consisted of internal low electron-dense material and marginal high electron-dense granules, approximately 3310pm in diameter (data not shown). These spherules had a thin membrane and inner electrondense granules. Protrusions of the membranes of the spherules (approximately l-2 pm in diameter) suggested that they are budding (Fig. 3). Inoculation of a sporozoite stabilate into calf lymph nodes generated enlarged schizonts on days 48 postinoculation. Histological and immunohistochemical examination revealed that the schizonts consisted of central core structures and numerous basophilic granules in the cytoplasm. The granules reacted positively
with anti-T. sergenri antibodies. Electron microscopic observation showed that the enlarged schizonts have a central core and marginal granules which contain electron-dense granules. The granules are presumed to be the macroschizont or microschizont according to previous reports (Schein et al., 1978; Sato ef al., 1993). Although schizonts of T. parva and T. annduta are formed in lymphocytes or monocytes (DeMartini & Moulton. 1973), in the results of our experiment, schizonts of T. sergenti were observed in lymph nodes but not in the peripheral blood mononuclear cells obtained from all of the calves examined. Interestingly, infiltration of inflammatory cells was not observed in the inoculated lymph nodes from all the calves. As described, no schizonts were detected in the lymph node sections within 3 days post-inoculation. Thus, the sporozoites probably invade immediately into the host cells to escape from the host immunity as described in T. parl;a (Fawcett et al., 1982). Immunohistochemical examination showed that there is no T. sergenti antigen on the surface of the enlarged schizont cells. The T. sergenti sporozoite might invade the host cell during the early incubation period. Subsequently, schizont growth may occur in the lymph node coating with the host cell membrane. Therefore, investigation of the early stage of the schizont in the lymph node is important to identify the host cell. Although further study will be needed to clarify the schizont growth in the host cells, we could easily detect the schizont growth in a time-dependent manner.
K. Hagiwara
1378
Fig. 3.
Electron
er al.
micrograph of T. sergenti schizont on day 6 after inoculation. The spherule contains highly granules and has a protrusion of membrane suggesting budding (arrow). Scale bar = 1 pm.
Acknowledgements-The authors wish to thank the late Professor K. Takahashi, Dr M. Tsuji and MS M. Takahashi (Rakuno Gakuen University) for their helpful advice and encouragement. This work was supported by Research Fellowships of the Japan Society for the promotion of Science for Young Scientists.
REFERENCES DeMartini J. C. & Moulton J. E. 1973. Response of the bovine lymphatic system to infection by Theileriaparua. I. Histology and ultrastructure of lymph nodes in experPathimentally-infected calves. Journal of Comparative ology 83: 28 l-298. Fawcett D. W.. Doxsey S., Stagg D. A. & Young A. S. 1982. The entry of sporozoites of Theileria paraa into bovine lymphocytes in vitro. Electron microscopic observations. European Journal qf Cell Biologv 27: l&21. Higuchi S. 1987. Development of Theileria sergenti in the midgut of the tick, Haemaphvsalis longicornis. Japanese Journal of Veterinary Science 49: 341-347. Kawai S.. Takahashi K., Kurosawa T. & Sonoda M. 1993. Light and electron-microscopical observation of the early development stage of Theileria sergenti in the gut of Haemaphysalis longicornis. The Journal of Veterinary Medical Science 55: 837-839. Kawamoto S., Takahashi K., Kurosawa T.. Sonoda M. & Onuma M. 1990. Intraerythrocytic schizogony of Theileria sergenti in cattle. Japanese Journal of Veterinary Science 52: 1251-1259. Kawamoto S., Takahashi K., Onuma M., Kurosawa T. & Sonoda M. 1990. Invasion of bovine erythrocytes by
electron-dense
Theileria sergenti piroplasms in r’itro. Japanese Journal q/ Veterinary Science 5t: 126 1-l 263 Musoke A., Morzaria S., Nkonge C.. Jones E. & Nene V. 1992. A recombinant sporozoite surface antigen of Theileria parva induces protection in cattle. Proceedings of the National Academql of Sciences U.S.A. 89: 514518. Nene V.. Inumaru S.. McKeever D., Morzaria S.. Shaw M. & Musoke A. 1995. Characterization of an insect cellderived Theileria paroa sporozoite vaccine antigen and immunogenicity in cattle. Infection atzd Immunity 63: 503508. Sato M., Kamio T.. Kawazu S., Taniguchi T.. Minami T. & Fuiisaki K. 1993. Histological observations on the schizonts in cattle infected with Japanese Theileria sergenti. The Journal of Veterinarv Medical Science 55: 571L 574. Sato M., Kamio T.. Kawazu S.. Tanaka S., Taniguchi T. & Fujisaki K. 1994. Development of Theileriu sergenti schizonts in the lymph node of experimentally infected cattle. The Journal of Veterinary Medical Science 56: 715722. Schein E., Mehlhorn H. & Warnecke M. 1978. Electron microscopic studies on the schizogony of four Theileria species of cattle (T. naroa, T. lawrencei, T. annulata and T. &tans). Para&lo&a 14: 337-348. Takahashi K. 1976. Studies on the infection and immunitv of Theileria sergenti. Journal of the College of Dairying 8: 179-278. Takahashi K., Kawai S., Yaehata K.. Kawamoto S., Hagiwara K., Kurosawa T., Tajima M. & Sonoda M. 1993. Spologony of Theileria sergentiin the salivary glands of the tick vector Haemaphysalis longicornis. Parasito1og.v Research 79: 1-7.
Pergamon
PII: SOO20-7519(97)00092-l
RESEARCH
Porositdog~y, Vol. 21, No. 1 I. pp. 1375.-1378. 1997 for Parasmlogy. Published by Elsevier Science Ltd Printed in Great Britam 002&7519/97 $17.00+0.00
NOTE
Detection of Theileria sergenti Schizonts in Bovine Lymph Node KATSURO HAGIWARA,*§ SATOSHI KAWAMOTO,? *School
KOUJI TAKAHASHI,* TAKASHI KUROSAWA,* CHIAKI ISHIHARA*
HIROYUKI KAZUYOSHI
and
of Veterinary Medicine, Rakuno Gakuen University, Bunkyoudai, Ebetsu,
Hokkaido 069, Japan Shintoku Animal Husbandry Experimental Hokkaido 081, Japan $Section of Serology, Institute of Immunological Science, Hokkaido Hokkaido 060, Japan THokkaido
TANIYAMA,* IKUTAS
Prefectural
Station, Shintoku, University,
Sapporo,
(Received 4 March 1997; accepted 10 July 1997)
Abstract-Hagiwara K., Takahashi K., Taniyama H., Kawamoto S., Kurosawa T., Ikuta K. & Ishihara C. 1997. Detection of Theileria sergenti schizonts in bovine lymph node. International Journalfor Parasitology 27: 1375-1378. The growth of Theileria serge& schizonts in bovine lymph nodes was examined by experimental infection. Six naive Holstein calves were inoculated with a sporozoite stabilate into the superficial cervical and subiliac lymph nodes. Biopsy samples from the inoculated calves were collected every 24 h for lOdays. Histological examination demonstrated the schizont of T. sergenti on day 4 post-inoculation. The growth of schiionts in the lymph node was observed on days 4-g post-inoculation. Electron microscopic observation disclosed that the core of the schizont has a high density central core surrounded by numerous electrondense spherules. 0 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd.
Key words: Theileria sergenti; Schizonts; lymph node. Theileriosis is one of the more serious infectious diseases of cattle in Japan. The full life-cycle of TheiZerin sergenti remained obscure for a long time, since the intracellular parasites are very small and many of their morphological details are unrecognisable by light microscopy. By histological observation, the patterns of asexual reproduction within the salivary glands of the vector ticks (Higuchi, 1987; Kawai et al., 1993; Takahashi et al., 1993) and within the erythrocytes of cattle (Kawamoto et al., 1990a, 1990b) were identified with certainty. Histological study for T. sergenti schizonts in experimentally infected calves was reported (Sato et al., 1993, 1994); however, the development of schizont growth in cattle remained unknown. Some progress towards sub-unit vaccine develop-
§To whom correspondence should be addressed. Tel: +81-(11)-386-11ll;Fax: +81-(ll)-387-5890.
ment for Theileriaparva has been made (Musoke et al., 1992; Nene et al., 1995). These studies focused on the recombinant sporozoite vaccine antigen, ~67. Immunity against T. sergentiinfections is likely to be directed at both the sporozoite and the schizont lifecycle stages. Lack of an in vitro cultivation system for the schizont-stage parasite and the difficulty of detection of T. sergenti schizonts in lymphoid cells in infected cattle has hampered progress towards vaccine development. Here, we focus on the detection of schizont growth in calf lymph nodes, by inoculating with sporozoite stabilate. Sporozoite stabilate of T. sergenti (Chitose strain; Takahashi, 1976) was used for animal infection. The stabilate was derived from the nymphal ticks (Huemaphvsalis mageshimaensis) picked up from a calf infected with T. sergenti(parasitaemia of 15%). Four months after feeding, the ticks were fed on rabbits at 20°C for 3days. Thereafter, the salivary glands of
1315
1376
K. Hagiwara et al.
these ticks were removed and were stained with methyl green pyronin to examine the appearance of sporozoites, according to the method described by Takahashi et al. (1993). The sporozoite stabilate was suspended at approximately 300 infected salivary gland acini in 1 ml of phosphate buffered saline (PBS) pH 7.2. For inoculation of sporozoite stabilate to calves, six naive Holstein calves (3-5 months old) were inoculated with sporozoite stabilate (0.25 ml of sporozoite stabilate/one lymph node) into superficial cervical and subiliac lymph nodes, respectively. As a control, the same dose of uninfected tick salivary gland acini suspension was inoculated into a lymph node of control calves. For histological preparation, lymph nodes from those calves were collected by excision biopsies at 24-h intervals for lOdays. All the animals were treated according to the Laboratory Animal Control Guidelines of the Rakuno Gakuen University. Tissues fixed in 4% phosphate-buffered paraformaldehyde (pH 7.4) were prepared for histological or immunohistochemical studies. The tissues were dehydrated in ethanol and embedded in paraffin. Histological sections (&6-pm thick) were processed for H & E staining, or used for immunohistochemistry. None of the calves showed any clinical symptoms throughout the experiments. The development of T. sergenti merozoites was observed in the erythrocytes from all of the inoculated calves at approximately 15 days post-inoculation. The lymph nodes from these inoculated calves were collected by biopsy for 10 days post-inoculation. Histological observation revealed that there were no inflammatory reactions and no schizont-like structures in the lymph nodes from inoculated calves on days l-3, and from the control lymph nodes throughout the experiments. However, early stages of the schizonts, approximately 55 x 44pm, were observed in lymph nodes on day 4 postinoculation. The cells had round- or oval-shaped basophilic cores and poor cytoplasm (Fig. 1A). Significant growth of the schizont cells was observed on days 5-6 post-inoculation (Fig. 1B), approximately 166x 1lOpm on day 5 and 266x 166pm on day 6, respectively (Fig. IC). Along with the development of the schizont cells, the cells had enlarged and irregular shaped basophilic cores, and were rich in cytoplasm having basophilic granules. On day 8, the core disappeared from the enlarged schizont cells and the cells were filled with numerous basophilic granules (Fig. ID). By days 9 and 10, the granule cells had disappeared from the lymph node (data not shown). To verify the localisation of the theilerial antigens, the avidin-biotin complex method was used. Histological sections from lymph nodes fixed in 4% phosphate-buffered paraformaldehyde (pH 7.4) were incu-
Fig. 1. The development of T. sergenti schizonts. (A) Parasitic cell has a large basophilic core and a small amount of cytoplasm on day 4 after inoculation. Scale bar = 5 pm. (B) Developing schizont cell has an enlarged basophilic core with irregular shape and a large amount of cytoplasm containing many basophilic granules peripherally on day 5 after inoculation. Scale bar=5 pm. (C) An enlarged schizont has a segmental core and an abundant cytoplasm containing a large number of basophilic granules on day 6 after inoculation. Scale bar = 20 pm. (D) A most developed schizont has a large amount of cytoplasm filled with numerous basophilic granules without basophilic core on day 8 after inoculation. Scale bar = 20 pm.
bated initially in 2% normal goat serum in PBS pH 7.4. The primary antibodies, anti-T. sergenti bovine serum, which were collected from T. sergentiinfected cattle, were applied next; these included antiT. sergenti antibodies (IFA titer: 1: 10 000) to identify theilerial antigen. After washing, the appropriate secondary antibodies consisting of biotinylated antibovine immunoglobulin were applied. This was followed by application of a horseradish peroxidase (HRP)-linked avidin-biotin complex (Histofine ABC kit, Nichirei, Japan). Application of diaminobenzidine/hydrogen peroxide to the bound HRP enzyme caused a brown precipitate. Immunohistochemical examinations disclosed that the baso-
1377
Fig. 2. Photomicrograph of T. sergenfi schizont on day 6 after inoculation. The basophilic granules in the cytoplasm of schizont cells reacted positively with anti-T. sergenri bovine serum. Avidin-biotin-peroxidase complex stain with methyl green counterstain. Scale bar = 10 pm. Inset: all basophilic granules in the schizont show a positive immunoreactivity with anti-T. sergenti antibody. Scale bar =40pm.
philic granules in the cytoplasm of schizont cells reacted positively with anti-K sergenti bovine serum (Fig. 2). For electron microscopic observation, lymph node tissues from sporozoite stabilate-inoculated calves were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C. These tissues were washed with phosphate buffers, refixed in 2% osmium tetroxide in phosphate buffers at 4°C dehydrated in a graded series of ethanols followed by propylene oxide and embedded in Quetole 812. The ultra-thin sections were stained with uranyl acetate and subsequently lead citrate. The prepared specimens were examined with an electron microscope (JEM-l200EX, Nihon Denshi). Electron microscopic observation of the schizont cells in lymph node on day 6 post-inoculation showed that the core of the enlarged schizont cell had consisted of internal low electron-dense material and marginal high electron-dense granules, approximately 3310pm in diameter (data not shown). These spherules had a thin membrane and inner electrondense granules. Protrusions of the membranes of the spherules (approximately l-2 pm in diameter) suggested that they are budding (Fig. 3). Inoculation of a sporozoite stabilate into calf lymph nodes generated enlarged schizonts on days 48 postinoculation. Histological and immunohistochemical examination revealed that the schizonts consisted of central core structures and numerous basophilic granules in the cytoplasm. The granules reacted positively
with anti-T. sergenri antibodies. Electron microscopic observation showed that the enlarged schizonts have a central core and marginal granules which contain electron-dense granules. The granules are presumed to be the macroschizont or microschizont according to previous reports (Schein et al., 1978; Sato ef al., 1993). Although schizonts of T. parva and T. annduta are formed in lymphocytes or monocytes (DeMartini & Moulton. 1973), in the results of our experiment, schizonts of T. sergenti were observed in lymph nodes but not in the peripheral blood mononuclear cells obtained from all of the calves examined. Interestingly, infiltration of inflammatory cells was not observed in the inoculated lymph nodes from all the calves. As described, no schizonts were detected in the lymph node sections within 3 days post-inoculation. Thus, the sporozoites probably invade immediately into the host cells to escape from the host immunity as described in T. parl;a (Fawcett et al., 1982). Immunohistochemical examination showed that there is no T. sergenti antigen on the surface of the enlarged schizont cells. The T. sergenti sporozoite might invade the host cell during the early incubation period. Subsequently, schizont growth may occur in the lymph node coating with the host cell membrane. Therefore, investigation of the early stage of the schizont in the lymph node is important to identify the host cell. Although further study will be needed to clarify the schizont growth in the host cells, we could easily detect the schizont growth in a time-dependent manner.
K. Hagiwara
1378
Fig. 3.
Electron
er al.
micrograph of T. sergenti schizont on day 6 after inoculation. The spherule contains highly granules and has a protrusion of membrane suggesting budding (arrow). Scale bar = 1 pm.
Acknowledgements-The authors wish to thank the late Professor K. Takahashi, Dr M. Tsuji and MS M. Takahashi (Rakuno Gakuen University) for their helpful advice and encouragement. This work was supported by Research Fellowships of the Japan Society for the promotion of Science for Young Scientists.
REFERENCES DeMartini J. C. & Moulton J. E. 1973. Response of the bovine lymphatic system to infection by Theileriaparua. I. Histology and ultrastructure of lymph nodes in experPathimentally-infected calves. Journal of Comparative ology 83: 28 l-298. Fawcett D. W.. Doxsey S., Stagg D. A. & Young A. S. 1982. The entry of sporozoites of Theileria paraa into bovine lymphocytes in vitro. Electron microscopic observations. European Journal qf Cell Biologv 27: l&21. Higuchi S. 1987. Development of Theileria sergenti in the midgut of the tick, Haemaphvsalis longicornis. Japanese Journal of Veterinary Science 49: 341-347. Kawai S.. Takahashi K., Kurosawa T. & Sonoda M. 1993. Light and electron-microscopical observation of the early development stage of Theileria sergenti in the gut of Haemaphysalis longicornis. The Journal of Veterinary Medical Science 55: 837-839. Kawamoto S., Takahashi K., Kurosawa T.. Sonoda M. & Onuma M. 1990. Intraerythrocytic schizogony of Theileria sergenti in cattle. Japanese Journal of Veterinary Science 52: 1251-1259. Kawamoto S., Takahashi K., Onuma M., Kurosawa T. & Sonoda M. 1990. Invasion of bovine erythrocytes by
electron-dense
Theileria sergenti piroplasms in r’itro. Japanese Journal q/ Veterinary Science 5t: 126 1-l 263 Musoke A., Morzaria S., Nkonge C.. Jones E. & Nene V. 1992. A recombinant sporozoite surface antigen of Theileria parva induces protection in cattle. Proceedings of the National Academql of Sciences U.S.A. 89: 514518. Nene V.. Inumaru S.. McKeever D., Morzaria S.. Shaw M. & Musoke A. 1995. Characterization of an insect cellderived Theileria paroa sporozoite vaccine antigen and immunogenicity in cattle. Infection atzd Immunity 63: 503508. Sato M., Kamio T.. Kawazu S., Taniguchi T.. Minami T. & Fuiisaki K. 1993. Histological observations on the schizonts in cattle infected with Japanese Theileria sergenti. The Journal of Veterinarv Medical Science 55: 571L 574. Sato M., Kamio T.. Kawazu S.. Tanaka S., Taniguchi T. & Fujisaki K. 1994. Development of Theileriu sergenti schizonts in the lymph node of experimentally infected cattle. The Journal of Veterinary Medical Science 56: 715722. Schein E., Mehlhorn H. & Warnecke M. 1978. Electron microscopic studies on the schizogony of four Theileria species of cattle (T. naroa, T. lawrencei, T. annulata and T. &tans). Para&lo&a 14: 337-348. Takahashi K. 1976. Studies on the infection and immunitv of Theileria sergenti. Journal of the College of Dairying 8: 179-278. Takahashi K., Kawai S., Yaehata K.. Kawamoto S., Hagiwara K., Kurosawa T., Tajima M. & Sonoda M. 1993. Spologony of Theileria sergentiin the salivary glands of the tick vector Haemaphysalis longicornis. Parasito1og.v Research 79: 1-7.