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Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow
Veterinary Parasitology 90 (2000) 247–252
Rapid communication
Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow Masumi Sawada a,∗ , Hisayo Kondo b , Yukiko Tomioka a , Chun-Ho Park a , Takehito Morita b , Akinori Shimada b , Takashi Umemura a a b
Laboratory of Comparative Pathology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan Department of Veterinary Pathology, Faculty of Agriculture, Tottori University, Tottori 680-0945, Japan Received 5 January 2000; accepted 10 April 2000
Abstract Neospora caninum was isolated from the brain of a 2-year-old dairy cow that had aborted confirmed N. caninum-infected fetuses on two occasions. The cow had an indirect fluorescent antibody titer of 1:1600 to N. caninum. The cow was killed 24 days after its second abortion and the brain was bioassayed for N. caninum in nude mice. Multifocal areas of perivascular cuffing and glial nodules were observed in the cerebrum and mesencephalon of the cow, but N. caninum was not identified in histological sections of the brain. All three nude mice inoculated with brain homogenate of the cow, developed emaciation and paralysis. Microscopical examination of the nude mice revealed systemic N. caninum infection with demonstrable tachyzoites in various organs. The parasites isolated from fresh mouse brain were transferred successfully into Vero cell cultures. PCR procedure on the purified tachyzoites obtained from the Vero cell cultures amplified the specific DNA sequence for N. caninum. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Neospora caninum; Cow; Brain; Latent infection; Nude mice; Cell culture
1. Introduction Neospora caninum is a significant cause of abortions in cows and is distributed worldwide (Thilsted and Dubey, 1989; Dubey and Lindsay, 1996). The dog has been recognized as a definitive host of N. caninum (McAllister et al., 1998; Lindsay et al., 1999). In aborted fetuses and neonatal calves, N. caninum has predilection for ∗ Corresponding author. Tel.: +81-11-706-5193; fax: +81-11-717-3701. E-mail address: [email protected] (M. Sawada)
0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 2 4 8 - X
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the brain and spinal cord and has been isolated from the brain of aborted fetuses and newborn calves (Conrad et al., 1993; Yamane et al., 1997; Davison et al., 1999; Magnino et al., 1999). However, the parasite has never been isolated from the tissues of adult cattle. We report for the first time, the isolation of the parasite from the brain of a naturally infected cow. 2. Materials and methods 2.1. Animal A 2-year-old Holstein cow was obtained from a dairy herd in Okayama prefecture, Japan. Twenty-four days after the second abortion, the cow was killed and necropsied. Abortions due to neosporosis have been of frequent occurrence in this herd and this cow had not been pastured. 2.2. Serological analysis A serum sample of the cow was analyzed by the indirect fluorescent antibody test for antibody to N. caninum (Yamane et al., 1997). 2.3. Bioassay for N. caninum in nude mice and cell cultures The brain of the cow was removed aseptically and a small piece of the brain tissue was homogenized in sterile saline containing antibiotics and intraperitoneally inoculated into three nude mice as previously described (Sawada et al., 1997). The nude mice were necropsied on the 46th day post-inoculation because they had become emaciated and developed paralysis. Brain homogenate from the mice was prepared and inoculated into Vero cell cultures (Yamane et al., 1998). 2.4. Histological examination Bovine and mouse tissues collected from various organs were fixed in 10% neutral buffered formalin at necropsy and processed routinely for histologic examination. For immunohistochemical examination, anti-N. caninum goat serum (VMRD Inc., Pullman, WA, USA) and anti-Toxoplasma gondii goat serum (VMRD Inc., Pullman, WA, USA) were used at a dilution of 1:3200 and 1:6000, respectively, as the primary antibody. A routine avidin–biotin complex procedure was used to locate the primary antibody and 3,30 -diaminobenzidine was used as the chromogen. 2.5. PCR for N. caninum Parasite purification was performed by the Percoll gradient (Yamane et al., 1998) and DNA was extracted from purified tachyzoites. We used Np21 and Np6 for primer pairs. The PCR procedure was used to detect the N. caninum specific DNA sequence by the method of Yamage et al. (1996) with a minor modification. DNA of T. gondii (RH strain) was used as negative control.
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3. Results 3.1. History and serology of the cow and fetal examination The cow had aborted twice in the fourth month of gestation in two successive pregnancies, but otherwise remained clinically normal. The antibody titer of the cow to N. caninum was 1:1600 at necropsy. Histological examination revealed N. caninum tachyzoites in tissues of both aborted fetuses. 3.2. Pathological examination There were no visible gross lesions in the tissues of the cow. The main lesions observed were mild perivascular cuffing and glial nodules in the central nervous system (Fig. 1). Perivascular cuffs were diffusely observed in the cerebrum, mesencephalon and spinal cord. The cuffs were composed of lymphocytes, macrophages and plasma cells. Glial nodules were also located in the cerebrum and mesencephalon. Focal myositis was found in the heart and skeletal muscles. Small aggregates of lymphocytes and macrophages were seen in the liver and kidney. N. caninum was not identified in any bovine tissue sections examined. 3.3. Bioassays for N. caninum in nude mice and cell cultures The three nude mice inoculated with the brain homogenate had inflammatory lesions with tachyzoites of N. caninum in the brain, pancreas, heart, adrenal glands, stomach and skeletal muscles (Fig. 2). Tachyzoites of N. caninum (BT-3 strain) were detected in the Vero cell culture 39 days after inoculation with murine brain homogenate. These tachyzoites were
Fig. 1. Glial nodules (arrows) in the occipital lobe of the adult dairy cow naturally infected with Neospora caninum. HE, bar=40 m.
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M. Sawada et al. / Veterinary Parasitology 90 (2000) 247–252
Fig. 2. Aggregation of Neospora caninum tachyzoites (arrow) in the brain of an infected mouse. HE, bar=20 m.
demonstrated to be positive with anti-N. caninum sera, but negative with anti-T. gondii sera by immunohistochemical staining (Fig. 3). 3.4. Amplification of N. caninum DNA To distinguish the purified parasite from T. gondii, we used the PCR procedure to specifically amplify a N. caninum DNA fragment. A DNA fragment of expected size (328 bp) was amplified from the isolated parasite. On the other hand, no DNA fragments were amplified from a DNA extract of T. gondii.
Fig. 3. Isolated tachyzoites are positively stained by anti-Neospora caninum serum. Immunohistochemical method, bar=10 m.
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4. Discussion Although the histopathological lesions of adult cattle infected with N. caninum have not been described previously, the brain lesions observed in our cow resembled those described in aborted fetuses and reflected previously reported paucity of identifiable parasites in brain lesions (Barr et al., 1990; Dubey et al., 1992). Therefore, isolation of the parasites from the brain suggested that the brain lesions were caused by N. caninum infection. The correlation of the parasites with other visceral lesions of the cow, including focal myositis in the heart and skeletal muscles, and small aggregates of mononuclear cells in the liver and kidney, were inconclusive in this study. Several strains of bovine N. caninum have been isolated from aborted fetuses and newborn calves (Conrad et al., 1993; Yamane et al., 1997; Davison et al., 1999; Magnino et al., 1999), but this is the first report of isolation of N. caninum from a naturally infected cow. Neospora-specific DNA fragments were amplified from the brain of naturally infected cows (Ho et al., 1997). These results suggest that the brain is one of the possible sites for latent residence of the parasite in infected, but clinically normal cows, and that the brain is one organ from which reactivated tachyzoites may enter the bloodstream and infect a developing fetus. One of the factors triggering reactivation of the parasites may be pregnancy, but the precise mechanism remains to be determined.
Acknowledgements We thank Dr. K. Okuda for providing the seropositive cow. References Barr, B.C., Anderson, M.L., Blanchard, P.C., Daft, B.M., Kinde, H., Conrad, P.A., 1990. Bovine fetal encephalitis and myocarditis associated with protozoal infections. Vet. Pathol. 27, 354–361. Conrad, P.A., Barr, B.C., Sverlow, K.W., Anderson, M., Daft, B., Kinde, H., Dubey, J.P., Munson, L., Ardans, A., 1993. In vitro isolation and characterization of a Neospora sp. from aborted bovine foetuses. Parasitology 106, 239–249. Davison, H.C., Guy, F., Trees, A.J., Ryce, C., Ellis, J.T., Otter, A., Jeffrey, M., Simpson, V.R., Holt, J.J., 1999. In vitro isolation of Neospora caninum from a stillborn calf in the UK. Res. Vet. Sci. 67, 103–105. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Dubey, J.P., Lindsay, D.S., Anderson, M.L., Davis, S.W., Shen, S.K., 1992. Induced transplacental transmission of Neospora caninum in cattle. J. Am. Vet. Med. Assoc. 201, 709–713. Ho, M.S.Y., Barr, B.C., Rowe, J.D., Anderson, M.L., Sverlow, K.W., Packham, A., Marsh, A.E., Conrad, P.A., 1997. Detection of Neospora sp. from infected bovine tissues by PCR and probe hybridization. J. Parasitol. 83, 508–514. Lindsay, D.S., Dubey, J.P., Duncan, R.B., 1999. Confirmation that dogs are a definitive host for Neospora caninum. Vet. Parasitol. 82, 327–333. Magnino, S., Vigo, P.G., Fabbi, M., Colombo, M., Bandi, C., Genchi, C., 1999. Isolation of a bovine Neospora from a newborn calf in Italy. Vet. Rec. 144, 456. McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., McGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473–1478. Sawada, M., Park, C.H., Morita, T., Shimada, A., Umemura, T., Haritani, M., 1997. Pathological findings of nude mice inoculated with bovine Neospora. J. Vet. Med. Sci. 59, 947–948.
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Thilsted, J.P., Dubey, J.P., 1989. Neosporosis-like abortions in a herd of dairy cattle. J. Vet. Diagn. Invest. 1, 205–209. Yamage, M., Flechtner, O., Gottstein, B., 1996. Neospora caninum: specific oligonucleotide primers for the detection of brain ‘cyst’ DNA of experimentally infected nude mice by the polymerase chain reaction (PCR). J. Parasitol. 82, 272–279. Yamane, I., Kokuho, T., Shimura, K., Eto, M., Shibahara, T., Haritani, M., Ouchi, Y., Sverlow, K., Conrad, P.A., 1997. In vitro isolation and characterization of a bovine Neospora species in Japan. Res. Vet. Sci. 63, 77–80. Yamane, I., Shibahara, T., Kokuho, T., Shimura, K., Hamaoka, T., Haritani, M., Conrad, P.A., Park, C.H., Sawada, M., Umemura, T., 1998. An improved isolation technique for bovine Neospora species. J. Vet. Diagn. Invest. 10, 364–368.
Rapid communication
Isolation of Neospora caninum from the brain of a naturally infected adult dairy cow Masumi Sawada a,∗ , Hisayo Kondo b , Yukiko Tomioka a , Chun-Ho Park a , Takehito Morita b , Akinori Shimada b , Takashi Umemura a a b
Laboratory of Comparative Pathology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan Department of Veterinary Pathology, Faculty of Agriculture, Tottori University, Tottori 680-0945, Japan Received 5 January 2000; accepted 10 April 2000
Abstract Neospora caninum was isolated from the brain of a 2-year-old dairy cow that had aborted confirmed N. caninum-infected fetuses on two occasions. The cow had an indirect fluorescent antibody titer of 1:1600 to N. caninum. The cow was killed 24 days after its second abortion and the brain was bioassayed for N. caninum in nude mice. Multifocal areas of perivascular cuffing and glial nodules were observed in the cerebrum and mesencephalon of the cow, but N. caninum was not identified in histological sections of the brain. All three nude mice inoculated with brain homogenate of the cow, developed emaciation and paralysis. Microscopical examination of the nude mice revealed systemic N. caninum infection with demonstrable tachyzoites in various organs. The parasites isolated from fresh mouse brain were transferred successfully into Vero cell cultures. PCR procedure on the purified tachyzoites obtained from the Vero cell cultures amplified the specific DNA sequence for N. caninum. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Neospora caninum; Cow; Brain; Latent infection; Nude mice; Cell culture
1. Introduction Neospora caninum is a significant cause of abortions in cows and is distributed worldwide (Thilsted and Dubey, 1989; Dubey and Lindsay, 1996). The dog has been recognized as a definitive host of N. caninum (McAllister et al., 1998; Lindsay et al., 1999). In aborted fetuses and neonatal calves, N. caninum has predilection for ∗ Corresponding author. Tel.: +81-11-706-5193; fax: +81-11-717-3701. E-mail address: [email protected] (M. Sawada)
0304-4017/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 0 ) 0 0 2 4 8 - X
248
M. Sawada et al. / Veterinary Parasitology 90 (2000) 247–252
the brain and spinal cord and has been isolated from the brain of aborted fetuses and newborn calves (Conrad et al., 1993; Yamane et al., 1997; Davison et al., 1999; Magnino et al., 1999). However, the parasite has never been isolated from the tissues of adult cattle. We report for the first time, the isolation of the parasite from the brain of a naturally infected cow. 2. Materials and methods 2.1. Animal A 2-year-old Holstein cow was obtained from a dairy herd in Okayama prefecture, Japan. Twenty-four days after the second abortion, the cow was killed and necropsied. Abortions due to neosporosis have been of frequent occurrence in this herd and this cow had not been pastured. 2.2. Serological analysis A serum sample of the cow was analyzed by the indirect fluorescent antibody test for antibody to N. caninum (Yamane et al., 1997). 2.3. Bioassay for N. caninum in nude mice and cell cultures The brain of the cow was removed aseptically and a small piece of the brain tissue was homogenized in sterile saline containing antibiotics and intraperitoneally inoculated into three nude mice as previously described (Sawada et al., 1997). The nude mice were necropsied on the 46th day post-inoculation because they had become emaciated and developed paralysis. Brain homogenate from the mice was prepared and inoculated into Vero cell cultures (Yamane et al., 1998). 2.4. Histological examination Bovine and mouse tissues collected from various organs were fixed in 10% neutral buffered formalin at necropsy and processed routinely for histologic examination. For immunohistochemical examination, anti-N. caninum goat serum (VMRD Inc., Pullman, WA, USA) and anti-Toxoplasma gondii goat serum (VMRD Inc., Pullman, WA, USA) were used at a dilution of 1:3200 and 1:6000, respectively, as the primary antibody. A routine avidin–biotin complex procedure was used to locate the primary antibody and 3,30 -diaminobenzidine was used as the chromogen. 2.5. PCR for N. caninum Parasite purification was performed by the Percoll gradient (Yamane et al., 1998) and DNA was extracted from purified tachyzoites. We used Np21 and Np6 for primer pairs. The PCR procedure was used to detect the N. caninum specific DNA sequence by the method of Yamage et al. (1996) with a minor modification. DNA of T. gondii (RH strain) was used as negative control.
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3. Results 3.1. History and serology of the cow and fetal examination The cow had aborted twice in the fourth month of gestation in two successive pregnancies, but otherwise remained clinically normal. The antibody titer of the cow to N. caninum was 1:1600 at necropsy. Histological examination revealed N. caninum tachyzoites in tissues of both aborted fetuses. 3.2. Pathological examination There were no visible gross lesions in the tissues of the cow. The main lesions observed were mild perivascular cuffing and glial nodules in the central nervous system (Fig. 1). Perivascular cuffs were diffusely observed in the cerebrum, mesencephalon and spinal cord. The cuffs were composed of lymphocytes, macrophages and plasma cells. Glial nodules were also located in the cerebrum and mesencephalon. Focal myositis was found in the heart and skeletal muscles. Small aggregates of lymphocytes and macrophages were seen in the liver and kidney. N. caninum was not identified in any bovine tissue sections examined. 3.3. Bioassays for N. caninum in nude mice and cell cultures The three nude mice inoculated with the brain homogenate had inflammatory lesions with tachyzoites of N. caninum in the brain, pancreas, heart, adrenal glands, stomach and skeletal muscles (Fig. 2). Tachyzoites of N. caninum (BT-3 strain) were detected in the Vero cell culture 39 days after inoculation with murine brain homogenate. These tachyzoites were
Fig. 1. Glial nodules (arrows) in the occipital lobe of the adult dairy cow naturally infected with Neospora caninum. HE, bar=40 m.
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M. Sawada et al. / Veterinary Parasitology 90 (2000) 247–252
Fig. 2. Aggregation of Neospora caninum tachyzoites (arrow) in the brain of an infected mouse. HE, bar=20 m.
demonstrated to be positive with anti-N. caninum sera, but negative with anti-T. gondii sera by immunohistochemical staining (Fig. 3). 3.4. Amplification of N. caninum DNA To distinguish the purified parasite from T. gondii, we used the PCR procedure to specifically amplify a N. caninum DNA fragment. A DNA fragment of expected size (328 bp) was amplified from the isolated parasite. On the other hand, no DNA fragments were amplified from a DNA extract of T. gondii.
Fig. 3. Isolated tachyzoites are positively stained by anti-Neospora caninum serum. Immunohistochemical method, bar=10 m.
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4. Discussion Although the histopathological lesions of adult cattle infected with N. caninum have not been described previously, the brain lesions observed in our cow resembled those described in aborted fetuses and reflected previously reported paucity of identifiable parasites in brain lesions (Barr et al., 1990; Dubey et al., 1992). Therefore, isolation of the parasites from the brain suggested that the brain lesions were caused by N. caninum infection. The correlation of the parasites with other visceral lesions of the cow, including focal myositis in the heart and skeletal muscles, and small aggregates of mononuclear cells in the liver and kidney, were inconclusive in this study. Several strains of bovine N. caninum have been isolated from aborted fetuses and newborn calves (Conrad et al., 1993; Yamane et al., 1997; Davison et al., 1999; Magnino et al., 1999), but this is the first report of isolation of N. caninum from a naturally infected cow. Neospora-specific DNA fragments were amplified from the brain of naturally infected cows (Ho et al., 1997). These results suggest that the brain is one of the possible sites for latent residence of the parasite in infected, but clinically normal cows, and that the brain is one organ from which reactivated tachyzoites may enter the bloodstream and infect a developing fetus. One of the factors triggering reactivation of the parasites may be pregnancy, but the precise mechanism remains to be determined.
Acknowledgements We thank Dr. K. Okuda for providing the seropositive cow. References Barr, B.C., Anderson, M.L., Blanchard, P.C., Daft, B.M., Kinde, H., Conrad, P.A., 1990. Bovine fetal encephalitis and myocarditis associated with protozoal infections. Vet. Pathol. 27, 354–361. Conrad, P.A., Barr, B.C., Sverlow, K.W., Anderson, M., Daft, B., Kinde, H., Dubey, J.P., Munson, L., Ardans, A., 1993. In vitro isolation and characterization of a Neospora sp. from aborted bovine foetuses. Parasitology 106, 239–249. Davison, H.C., Guy, F., Trees, A.J., Ryce, C., Ellis, J.T., Otter, A., Jeffrey, M., Simpson, V.R., Holt, J.J., 1999. In vitro isolation of Neospora caninum from a stillborn calf in the UK. Res. Vet. Sci. 67, 103–105. Dubey, J.P., Lindsay, D.S., 1996. A review of Neospora caninum and neosporosis. Vet. Parasitol. 67, 1–59. Dubey, J.P., Lindsay, D.S., Anderson, M.L., Davis, S.W., Shen, S.K., 1992. Induced transplacental transmission of Neospora caninum in cattle. J. Am. Vet. Med. Assoc. 201, 709–713. Ho, M.S.Y., Barr, B.C., Rowe, J.D., Anderson, M.L., Sverlow, K.W., Packham, A., Marsh, A.E., Conrad, P.A., 1997. Detection of Neospora sp. from infected bovine tissues by PCR and probe hybridization. J. Parasitol. 83, 508–514. Lindsay, D.S., Dubey, J.P., Duncan, R.B., 1999. Confirmation that dogs are a definitive host for Neospora caninum. Vet. Parasitol. 82, 327–333. Magnino, S., Vigo, P.G., Fabbi, M., Colombo, M., Bandi, C., Genchi, C., 1999. Isolation of a bovine Neospora from a newborn calf in Italy. Vet. Rec. 144, 456. McAllister, M.M., Dubey, J.P., Lindsay, D.S., Jolley, W.R., Wills, R.A., McGuire, A.M., 1998. Dogs are definitive hosts of Neospora caninum. Int. J. Parasitol. 28, 1473–1478. Sawada, M., Park, C.H., Morita, T., Shimada, A., Umemura, T., Haritani, M., 1997. Pathological findings of nude mice inoculated with bovine Neospora. J. Vet. Med. Sci. 59, 947–948.
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M. Sawada et al. / Veterinary Parasitology 90 (2000) 247–252
Thilsted, J.P., Dubey, J.P., 1989. Neosporosis-like abortions in a herd of dairy cattle. J. Vet. Diagn. Invest. 1, 205–209. Yamage, M., Flechtner, O., Gottstein, B., 1996. Neospora caninum: specific oligonucleotide primers for the detection of brain ‘cyst’ DNA of experimentally infected nude mice by the polymerase chain reaction (PCR). J. Parasitol. 82, 272–279. Yamane, I., Kokuho, T., Shimura, K., Eto, M., Shibahara, T., Haritani, M., Ouchi, Y., Sverlow, K., Conrad, P.A., 1997. In vitro isolation and characterization of a bovine Neospora species in Japan. Res. Vet. Sci. 63, 77–80. Yamane, I., Shibahara, T., Kokuho, T., Shimura, K., Hamaoka, T., Haritani, M., Conrad, P.A., Park, C.H., Sawada, M., Umemura, T., 1998. An improved isolation technique for bovine Neospora species. J. Vet. Diagn. Invest. 10, 364–368.