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Histopathological features of canine distemper recently observed in Japan
J. Comp. Path. 1997 Vol. 116, 403-408
SHORT PAPER
Histopathological Features of Canine Distemper Recently Observed in Japan M. Okita*t, T. Yanai~, F. Ochikubow T. Gemma*, T. Mori*, T. Maseki++, K. Yamanouchi 82 T. Mikami* and C. Kai* *Department of VeterinaryMicrobiology, Faculty of Agriculture, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, ++Departmentof VeterinaryPathology, Gifu University, 1-1 Gifu 501-11, w Primate Center, Ibaraki 305, and 82 Institutefor Biological Sc#nce, Ome, Tokyo 198, Japan
Summary Eleven dogs with canine distemper (CD) from the Chubu region of Japan and the Tokyo area were examined. Clinically, respiratory and neurological signs were present in all animals. Histopathologically, all showed characteristic CD lesions of bronchopneumonia and demyelinating encephalitis. However, some differences in gastrointestinal abnormalities were observed. Three out of four dogs from the Chubu region had severe diarrhoea and gastroenteritis, associated with numerous eosinophilic inclusion bodies in the mucosal epithelia. The remaining dog from this area showed vomiting, but not diarrhoea, and also had a number of intraepithelial inclusion bodies in the gastric and intestinal mucosa. In contrast, the seven dogs from the Tokyo area showed neither g.astrointestinal symptoms nor intraepithelial inclusions in the stomach or intestine. Immunohistochemical examination for CD virus antigen, however, revealed that these seven dogs had immunoreactive products in the mucosal epithelia, suggesting that the epithelial cells had either a low level of infection with CD virus or were infected with a less cytopathogenic virus. These findings suggest that the dogs in this study were probably affected by two distinct types of CD, in terms of epitheliotropism and cytopathogenic effects on the gastrointestinal tissues. 9 1997 W.B.SaundersCompany Limited Canine distemper (CD) is an acute to subacute contagious systemic disease with a high mortality rate.in dogs and other carnivores. The disease has been reported throughout the world. The common histopathological lesions in dogs are depletion of lymphocytes in the lymphoid tissues, interstitial pneumonia, degenerative changes in the mucous membranes of the respiratory organs, urinary bladder and gastrointestinal tract, hyperkeratosis, and eosinophilic inclusion bodies in the epithelial cells of the gastrointestinal tract; the affected animals often exhibit degeneration of the central nervous system (CNS) (DeMonbreun, 1936; Appel, 1987; Blixenkrone-Moller, 1993). T h r o u g h extensive investigations of both natural and experimental CD in dogs, the Correspondence to: C. Kai. "~Present address: Ministry of Agriculture, Forestry and Fisheries, 1-2-1 Kasumigaseki, Chiyoda-ku, Tokyo 100, Japan. 0021 9975/97/040403+06 $12.00/0
9 1997 W.B. Saunders Company Limited
404
M. O k i t a e t a l . Table 1 Clinical details of the a n i m a l s
Dog no. Age
Sex
Breed
Major clinical signs
Vaccinated Region (and ci~)
1
2m
Female Golden retriever Diarrhoea, tic
No
2
6m
Female Pomeranian
No
3
3m
Male
4
3m
Female
5
5m
Male
6
3m
Male
7 8 9 10
5m 1m 2y 3m
Female Male Female Female
11
NK
Male
Coughing, vomiting, tic
Samoyed
Haemorrhagic diarrhoea, nasal and ocular discharge Samoyed Haemorrhagic diarrhoea, nasal and ocular discharge, hyperkeratosis of foot pads Husky Coughing, nasal discharge, seizure, dysstasia Schnauzer Coughing, nasal discharge, seizure, dysstasia Pomeranian Coughing, seizure Pug Coughing, nasal discharge, seizure Shiba Pyrexia, anorexia Cavalier King Coughing, nasal discharge, Charles spaniel anorexia Golden retriever Coughing, tic
Yes*
Chubu (Shizuoka) Chubu (Shizuoka) Chubu (Fukui)
Yes*
Chubu (Fukui)
Yes
TA (Kanagawa)
Yes
TA (Kanagawa)
Yes Yes* Yes NK
TA TA TA TA
Yes
TA (Tokyo)
(Kanagawa) (Kanagawa) (Tokyo) (Tokyo)
m, months; y, years; NK, not known; TA, Tokyo area. * Vaccine failure, probably due to the presence of maternal antibodies.
pathogenesis has been well defined (DeMonbreun, 1936; Appel, 1969, 1987). Recently we observed outbreaks of CD throughout Japan (Gemma et al., 1995, 1996a). Clinically, the affected dogs showed neurological, respiratory and gastrointestinal signs of varying degree (Gemma et al., 1995, 1996b). We were particularly interested, however, in outbreaks in the Tokyo area, in which the dogs showed severe CNS signs and mild respiratory signs but no gastrointestinal signs. Eleven animals were examined, seven from the Tokyo area and four from the Chubu region (central region of Japan), all of which were suspected to have CD and had either died or been humanely killed at the moribund stage a few days to 2 months after the first visit to the veterinarian. Brief clinical details are shown in Table 1. Of four animals from the Chubu region, three (nos 1, 3 and 4) showed typical clinical signs of CD, the first being either severe diarrhoea or haematochezia, followed by the gradual development of respiratory and neurological signs. However, the seven animals from the Tokyo area (nos 5-11) showed only respiratory and neurological signs. Clinically, one animal from the Chubu region (no. 2) showed respiratory and neurological signs and vomiting, but no diarrhoea. Specimens of the brain, lung, spleen, lymph nodes, intestine, urinary bladder and kidney, collected at necropsy, were fixed in 10% neutral-buffered formalin, dehydrated, and embedded in paraffin wax. They were processed routinely for haematoxylin and eosin (HE) staining and examined histopathologically. Immunohistochemical examination for canine distemper virus (CDV) antigens was performed on selected slides with a monoclonal antibody to CDV nucleoprotein (Hirayama et al., 1991) by the avidin-biotin peroxidase complex (ABC)
Histopathology of Canine Distemper
405
technique (Vector Laboratories, Burlingame, CA, USA) (Iwatsuki et al., 1995). CD was confirmed histopathologically in all 11 dogs on the basis of findings of bronchopneumonia, catarrhal or interstitial pneumonia, and changes in the CNS (encephalitis, demyelination, and the formation of cytoplasmic and intranuclear eosinophilic inclusion bodies in the glia). In the spleen and lymph nodes, degeneration and necrosis of the lymphoid follicles and eosinophilic intranuclear inclusion bodies were observed. The histopathological diagnosis was supported by the immunohistochemical detection of the CDV antigen in the intestine, spleen and CNS. The l 1 animals could be classified into two groups, according to the presence or absence of gastrointestinal lesions or inclusion bodies, or both. All four animals from the Chubu region (nos 1-4) showed such abnormalities and were designated the "enteritis group". Histopathologically, these four dogs exhibited numerous eosinophilic inclusion bodies in the epithelial cells of the lung, intestine (Fig. la), renal pelvis and urinary bladder. In three of these animals, which had shown severe diarrhoea or haematochezia, gastroenteritis (Fig. lb) with degeneration of epithelia, lymphocyte necrosis and neutrophil infiltration of the lamina propria was also observed. Although the remaining animal (no. 2) had shown no diarrhoea or gastrointestinal lesions, it was included in the enteritis group because it had exhibited vomiting and also had numerous inclusion bodies in the epithelial cells of the gastrointestinal mucosa. The histopathological lesions in its respiratory and neural organs were similar to those in clogs 1, 3 and 4. Animals in the second ("non-enteritis") group showed, histopathologically, bronchopneumonia and degenerative lesions in the CNS, but exhibited neither gastrointestinal lesions nor inclusion bodies in the intestinal or urinary epithelia. All the animals from the Tokyo area belonged to this group. However, immunohistochemical examination revealed viral antigens in the epithelial cells of the intestines, despite the absence of obvious histopathological lesions (Fig. lc,d). CDV is known to exhibit tropism to both lymphoid and epithelial cells (DeMonbreun, 1936; Yamanouchi, 1980; Appel, 1987; Blixenkrone-Moller, 1993). Recently, we succeeded in isolating several wild types of CDV (Kai et al., 1993; Iwatsuki et al., 1997); these isolates differed with regard to the electromobility of some viral proteins and exhibited substitutions of nucleotide sequences in relation to laboratory and vaccine strains (Iwatsuki et al., 1997). The non-enteritis type of CD might be the result of infection with wild types of CDV having reduced epitheliotropism or cytopathogenicity, or poor ability to replicate in the epithelial cells of the gastrointestinal tract. Further virological studies are necessary to clarify these points. It has been suggested that the range of clinical signs, as well as their severity, in CD are affected by factors such as host age, natural resistance to CDV, and possibly by secondary infections (Appel, 1987). The effects of vaccination and veterinary treatment should also be taken into consideration. In the present study, the four animals from the Chubu region with CD were assumed to have little or no vaccine-induced immunity. In fact, they had either not been vaccinated or had been vaccinated too soon after birth, when maternal
406
Fig. 1.
M . O k i t a et aL
Histopathological examination of dog 3 showed (a) eosinophilic inclusion bodies (arrowhead) in the gastric epithelia, and (b) an inflammatory lesion in the jejunum, with lymphocyte infiltration in the lamina propria, a: HE. x 400. b: HE. x 200. Histopathological and immunohistochemical examination of dog 11 showed (c) the absence of obvious histopathological lesions, but (d) viral antigens (arrows) were observed in the intestinal epithelia, c: HE. x 400. d: ABC, counter-stained with haematoxylin, x 400.
Histopathology of Canine Distemper
407
CDV antibodies may have been present; dogs 3 and 4 had been vaccinated once, 5 weeks after birth, and had shown CD symptoms at 8 weeks of age. In contrast, five of the seven animals (non-enteritis group) from the Tokyo area were known to have been vaccinated by approved procedures. It is therefore possible that the non-enteritis type of disease was induced in adequately vaccinated dogs by a CDV strain that differed antigenically from the vaccine strain. It does not seem that geographical area itself was responsible for the two patterns of disease, because some animals with the severe enteritic form of CD have been observed in Tokyo (data not shown). The antigenic and molecular biological properties of prevalent strains of CDV should be analysed to clarify their possible relevance to the different types of disease. Acknowledgments The authors thank Drs N. Goto and K. Uetsuka, Department of Veterinary Pathology, The University of Tokyo for technical advice. They also thank Drs T. Iinuma, T. Tanabe, A. Matsui and K. Akiyama, veterinary practitioners, for providing the dogs and relevant clinical information. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan, and a grant for a pioneering research project in biotechnology from the Ministry of Agriculture, Forestry, and Fisheries, Japan. References Appel, M . J . G . (1969). Pathogenesis of canine distemper. AmericanJournal of Veterinary Research, 30, 1167-1182. Appel, M . J . G . (1987). Canine distemper virus. In: l~rus Infections of Carnivores, Vol. 1. M. C. Horzinek, Ed., Elsevier, Amsterdam, pp.133-159. Blixenkrone-Moller, M. (1993). Biological properties of phocine distemper virus and canine distemper virus. Acta Pathologica, Microbiologica et Immunologica Scandinavica, 101 (Suppl. 36), 5-51. DeMonbreun, W. A. (1936). The histopathology of natural and experimental canine distemper. American Journal of Pathology, 13, 187-212. Gemma, T., Iwatsuki, K., Shin, Y.-S., Yoshida, E., Kai, C. and Mikami, T. (1996a). Serological analysis of canine distemper virus using an immunocapture enzymelinked immunosorbent assay. Journal of VeterinaryMedical Science, 58, 791-794. Gemma, T., Miyashita, N., Shin, Y.-S., Okita, M., Mori, T., Iwatsuki, K., Mikami, T. and Kai, C. (1995). Serological survey of canine distemper virus infection using enzyme-linked immunosorbent assay. Journal of VeterinaryMedical Science, 57, 761-763. Gemma, T., Watari, T., Akiyama, K., Miyashita, N., Shin, Y.-S., Iwatsuki, K., Kai, C. and Mikami, T. (1996b). Epidemiological observations on recent outbreaks of canine distemper in Tokyo area. Journal of VeterinaryMedical Science, 58, 547-550. Hirayama, N., Senda, M., Nakashima, N., Takagi, M., Sugiyama, M., Yoshikawa, Y. and Yamanouchi, K. (1991). Protective effects of monoclonal antibodies against lethal canine distemper virus infection in mice. Journal of General Nrology, 72, 2827-2830. Iwatsuki, K., Okita, M., Ochikubo, F., Gemma, T., Shin, Y.-S., Miyashita, N., Mikami, T. and Kai, C. (1995). Immunohistochemical analysis of the lymphoid organs of dogs naturally infected with canine distemper virus. Journal of Comparative Pathology, 113, 185-190. Iwatsuki, K., Miyashita, N., Yoshida, E., Gemma, T., Shin, Y.-S., Mori, T., Hirayama, N., Kai, C. and Mikami, T. (1997). Molecular and phylogenetic analyses of the
408
M. Okita et al.
haemagglutinin (H) proteins of field isolates of canine distemper virus from naturally infected dogs. Journal of General Nrology, 78, 373-380. Kai, C , Ochikubo, F., Okita, M., Iinuma, T., Mikami, T., Kobune, F. and Yamanouchi, K. (1993). Use of B95a cells for isolation of canine distemper virus from clinical cases. Journal of Veterinary Medical Science, 55, 1067 1070. Yamanouchi, K. (1980). Comparative aspects of pathogenicity of measles, canine distemper, and rinderpest virus. Japanese Journal of Medical Science and Biology, 33, 41-66.
Received, October 7 th, 1996 ] Accepted, January 13th, 1997J
SHORT PAPER
Histopathological Features of Canine Distemper Recently Observed in Japan M. Okita*t, T. Yanai~, F. Ochikubow T. Gemma*, T. Mori*, T. Maseki++, K. Yamanouchi 82 T. Mikami* and C. Kai* *Department of VeterinaryMicrobiology, Faculty of Agriculture, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, ++Departmentof VeterinaryPathology, Gifu University, 1-1 Gifu 501-11, w Primate Center, Ibaraki 305, and 82 Institutefor Biological Sc#nce, Ome, Tokyo 198, Japan
Summary Eleven dogs with canine distemper (CD) from the Chubu region of Japan and the Tokyo area were examined. Clinically, respiratory and neurological signs were present in all animals. Histopathologically, all showed characteristic CD lesions of bronchopneumonia and demyelinating encephalitis. However, some differences in gastrointestinal abnormalities were observed. Three out of four dogs from the Chubu region had severe diarrhoea and gastroenteritis, associated with numerous eosinophilic inclusion bodies in the mucosal epithelia. The remaining dog from this area showed vomiting, but not diarrhoea, and also had a number of intraepithelial inclusion bodies in the gastric and intestinal mucosa. In contrast, the seven dogs from the Tokyo area showed neither g.astrointestinal symptoms nor intraepithelial inclusions in the stomach or intestine. Immunohistochemical examination for CD virus antigen, however, revealed that these seven dogs had immunoreactive products in the mucosal epithelia, suggesting that the epithelial cells had either a low level of infection with CD virus or were infected with a less cytopathogenic virus. These findings suggest that the dogs in this study were probably affected by two distinct types of CD, in terms of epitheliotropism and cytopathogenic effects on the gastrointestinal tissues. 9 1997 W.B.SaundersCompany Limited Canine distemper (CD) is an acute to subacute contagious systemic disease with a high mortality rate.in dogs and other carnivores. The disease has been reported throughout the world. The common histopathological lesions in dogs are depletion of lymphocytes in the lymphoid tissues, interstitial pneumonia, degenerative changes in the mucous membranes of the respiratory organs, urinary bladder and gastrointestinal tract, hyperkeratosis, and eosinophilic inclusion bodies in the epithelial cells of the gastrointestinal tract; the affected animals often exhibit degeneration of the central nervous system (CNS) (DeMonbreun, 1936; Appel, 1987; Blixenkrone-Moller, 1993). T h r o u g h extensive investigations of both natural and experimental CD in dogs, the Correspondence to: C. Kai. "~Present address: Ministry of Agriculture, Forestry and Fisheries, 1-2-1 Kasumigaseki, Chiyoda-ku, Tokyo 100, Japan. 0021 9975/97/040403+06 $12.00/0
9 1997 W.B. Saunders Company Limited
404
M. O k i t a e t a l . Table 1 Clinical details of the a n i m a l s
Dog no. Age
Sex
Breed
Major clinical signs
Vaccinated Region (and ci~)
1
2m
Female Golden retriever Diarrhoea, tic
No
2
6m
Female Pomeranian
No
3
3m
Male
4
3m
Female
5
5m
Male
6
3m
Male
7 8 9 10
5m 1m 2y 3m
Female Male Female Female
11
NK
Male
Coughing, vomiting, tic
Samoyed
Haemorrhagic diarrhoea, nasal and ocular discharge Samoyed Haemorrhagic diarrhoea, nasal and ocular discharge, hyperkeratosis of foot pads Husky Coughing, nasal discharge, seizure, dysstasia Schnauzer Coughing, nasal discharge, seizure, dysstasia Pomeranian Coughing, seizure Pug Coughing, nasal discharge, seizure Shiba Pyrexia, anorexia Cavalier King Coughing, nasal discharge, Charles spaniel anorexia Golden retriever Coughing, tic
Yes*
Chubu (Shizuoka) Chubu (Shizuoka) Chubu (Fukui)
Yes*
Chubu (Fukui)
Yes
TA (Kanagawa)
Yes
TA (Kanagawa)
Yes Yes* Yes NK
TA TA TA TA
Yes
TA (Tokyo)
(Kanagawa) (Kanagawa) (Tokyo) (Tokyo)
m, months; y, years; NK, not known; TA, Tokyo area. * Vaccine failure, probably due to the presence of maternal antibodies.
pathogenesis has been well defined (DeMonbreun, 1936; Appel, 1969, 1987). Recently we observed outbreaks of CD throughout Japan (Gemma et al., 1995, 1996a). Clinically, the affected dogs showed neurological, respiratory and gastrointestinal signs of varying degree (Gemma et al., 1995, 1996b). We were particularly interested, however, in outbreaks in the Tokyo area, in which the dogs showed severe CNS signs and mild respiratory signs but no gastrointestinal signs. Eleven animals were examined, seven from the Tokyo area and four from the Chubu region (central region of Japan), all of which were suspected to have CD and had either died or been humanely killed at the moribund stage a few days to 2 months after the first visit to the veterinarian. Brief clinical details are shown in Table 1. Of four animals from the Chubu region, three (nos 1, 3 and 4) showed typical clinical signs of CD, the first being either severe diarrhoea or haematochezia, followed by the gradual development of respiratory and neurological signs. However, the seven animals from the Tokyo area (nos 5-11) showed only respiratory and neurological signs. Clinically, one animal from the Chubu region (no. 2) showed respiratory and neurological signs and vomiting, but no diarrhoea. Specimens of the brain, lung, spleen, lymph nodes, intestine, urinary bladder and kidney, collected at necropsy, were fixed in 10% neutral-buffered formalin, dehydrated, and embedded in paraffin wax. They were processed routinely for haematoxylin and eosin (HE) staining and examined histopathologically. Immunohistochemical examination for canine distemper virus (CDV) antigens was performed on selected slides with a monoclonal antibody to CDV nucleoprotein (Hirayama et al., 1991) by the avidin-biotin peroxidase complex (ABC)
Histopathology of Canine Distemper
405
technique (Vector Laboratories, Burlingame, CA, USA) (Iwatsuki et al., 1995). CD was confirmed histopathologically in all 11 dogs on the basis of findings of bronchopneumonia, catarrhal or interstitial pneumonia, and changes in the CNS (encephalitis, demyelination, and the formation of cytoplasmic and intranuclear eosinophilic inclusion bodies in the glia). In the spleen and lymph nodes, degeneration and necrosis of the lymphoid follicles and eosinophilic intranuclear inclusion bodies were observed. The histopathological diagnosis was supported by the immunohistochemical detection of the CDV antigen in the intestine, spleen and CNS. The l 1 animals could be classified into two groups, according to the presence or absence of gastrointestinal lesions or inclusion bodies, or both. All four animals from the Chubu region (nos 1-4) showed such abnormalities and were designated the "enteritis group". Histopathologically, these four dogs exhibited numerous eosinophilic inclusion bodies in the epithelial cells of the lung, intestine (Fig. la), renal pelvis and urinary bladder. In three of these animals, which had shown severe diarrhoea or haematochezia, gastroenteritis (Fig. lb) with degeneration of epithelia, lymphocyte necrosis and neutrophil infiltration of the lamina propria was also observed. Although the remaining animal (no. 2) had shown no diarrhoea or gastrointestinal lesions, it was included in the enteritis group because it had exhibited vomiting and also had numerous inclusion bodies in the epithelial cells of the gastrointestinal mucosa. The histopathological lesions in its respiratory and neural organs were similar to those in clogs 1, 3 and 4. Animals in the second ("non-enteritis") group showed, histopathologically, bronchopneumonia and degenerative lesions in the CNS, but exhibited neither gastrointestinal lesions nor inclusion bodies in the intestinal or urinary epithelia. All the animals from the Tokyo area belonged to this group. However, immunohistochemical examination revealed viral antigens in the epithelial cells of the intestines, despite the absence of obvious histopathological lesions (Fig. lc,d). CDV is known to exhibit tropism to both lymphoid and epithelial cells (DeMonbreun, 1936; Yamanouchi, 1980; Appel, 1987; Blixenkrone-Moller, 1993). Recently, we succeeded in isolating several wild types of CDV (Kai et al., 1993; Iwatsuki et al., 1997); these isolates differed with regard to the electromobility of some viral proteins and exhibited substitutions of nucleotide sequences in relation to laboratory and vaccine strains (Iwatsuki et al., 1997). The non-enteritis type of CD might be the result of infection with wild types of CDV having reduced epitheliotropism or cytopathogenicity, or poor ability to replicate in the epithelial cells of the gastrointestinal tract. Further virological studies are necessary to clarify these points. It has been suggested that the range of clinical signs, as well as their severity, in CD are affected by factors such as host age, natural resistance to CDV, and possibly by secondary infections (Appel, 1987). The effects of vaccination and veterinary treatment should also be taken into consideration. In the present study, the four animals from the Chubu region with CD were assumed to have little or no vaccine-induced immunity. In fact, they had either not been vaccinated or had been vaccinated too soon after birth, when maternal
406
Fig. 1.
M . O k i t a et aL
Histopathological examination of dog 3 showed (a) eosinophilic inclusion bodies (arrowhead) in the gastric epithelia, and (b) an inflammatory lesion in the jejunum, with lymphocyte infiltration in the lamina propria, a: HE. x 400. b: HE. x 200. Histopathological and immunohistochemical examination of dog 11 showed (c) the absence of obvious histopathological lesions, but (d) viral antigens (arrows) were observed in the intestinal epithelia, c: HE. x 400. d: ABC, counter-stained with haematoxylin, x 400.
Histopathology of Canine Distemper
407
CDV antibodies may have been present; dogs 3 and 4 had been vaccinated once, 5 weeks after birth, and had shown CD symptoms at 8 weeks of age. In contrast, five of the seven animals (non-enteritis group) from the Tokyo area were known to have been vaccinated by approved procedures. It is therefore possible that the non-enteritis type of disease was induced in adequately vaccinated dogs by a CDV strain that differed antigenically from the vaccine strain. It does not seem that geographical area itself was responsible for the two patterns of disease, because some animals with the severe enteritic form of CD have been observed in Tokyo (data not shown). The antigenic and molecular biological properties of prevalent strains of CDV should be analysed to clarify their possible relevance to the different types of disease. Acknowledgments The authors thank Drs N. Goto and K. Uetsuka, Department of Veterinary Pathology, The University of Tokyo for technical advice. They also thank Drs T. Iinuma, T. Tanabe, A. Matsui and K. Akiyama, veterinary practitioners, for providing the dogs and relevant clinical information. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan, and a grant for a pioneering research project in biotechnology from the Ministry of Agriculture, Forestry, and Fisheries, Japan. References Appel, M . J . G . (1969). Pathogenesis of canine distemper. AmericanJournal of Veterinary Research, 30, 1167-1182. Appel, M . J . G . (1987). Canine distemper virus. In: l~rus Infections of Carnivores, Vol. 1. M. C. Horzinek, Ed., Elsevier, Amsterdam, pp.133-159. Blixenkrone-Moller, M. (1993). Biological properties of phocine distemper virus and canine distemper virus. Acta Pathologica, Microbiologica et Immunologica Scandinavica, 101 (Suppl. 36), 5-51. DeMonbreun, W. A. (1936). The histopathology of natural and experimental canine distemper. American Journal of Pathology, 13, 187-212. Gemma, T., Iwatsuki, K., Shin, Y.-S., Yoshida, E., Kai, C. and Mikami, T. (1996a). Serological analysis of canine distemper virus using an immunocapture enzymelinked immunosorbent assay. Journal of VeterinaryMedical Science, 58, 791-794. Gemma, T., Miyashita, N., Shin, Y.-S., Okita, M., Mori, T., Iwatsuki, K., Mikami, T. and Kai, C. (1995). Serological survey of canine distemper virus infection using enzyme-linked immunosorbent assay. Journal of VeterinaryMedical Science, 57, 761-763. Gemma, T., Watari, T., Akiyama, K., Miyashita, N., Shin, Y.-S., Iwatsuki, K., Kai, C. and Mikami, T. (1996b). Epidemiological observations on recent outbreaks of canine distemper in Tokyo area. Journal of VeterinaryMedical Science, 58, 547-550. Hirayama, N., Senda, M., Nakashima, N., Takagi, M., Sugiyama, M., Yoshikawa, Y. and Yamanouchi, K. (1991). Protective effects of monoclonal antibodies against lethal canine distemper virus infection in mice. Journal of General Nrology, 72, 2827-2830. Iwatsuki, K., Okita, M., Ochikubo, F., Gemma, T., Shin, Y.-S., Miyashita, N., Mikami, T. and Kai, C. (1995). Immunohistochemical analysis of the lymphoid organs of dogs naturally infected with canine distemper virus. Journal of Comparative Pathology, 113, 185-190. Iwatsuki, K., Miyashita, N., Yoshida, E., Gemma, T., Shin, Y.-S., Mori, T., Hirayama, N., Kai, C. and Mikami, T. (1997). Molecular and phylogenetic analyses of the
408
M. Okita et al.
haemagglutinin (H) proteins of field isolates of canine distemper virus from naturally infected dogs. Journal of General Nrology, 78, 373-380. Kai, C , Ochikubo, F., Okita, M., Iinuma, T., Mikami, T., Kobune, F. and Yamanouchi, K. (1993). Use of B95a cells for isolation of canine distemper virus from clinical cases. Journal of Veterinary Medical Science, 55, 1067 1070. Yamanouchi, K. (1980). Comparative aspects of pathogenicity of measles, canine distemper, and rinderpest virus. Japanese Journal of Medical Science and Biology, 33, 41-66.
Received, October 7 th, 1996 ] Accepted, January 13th, 1997J