_Archives
Vi rology
Arch Virot (1991) 119:147-152
© SpringerWerlag 1991 Printed in Austria
Canine distemper virus infection and encephalitis in javelinas (collared peccaries) Brief R e p o r t M. J. G. Appel 1, C. Reggiardo 2, B. A. Summers 1, S. Pearce-Kelling 1, C. J. Mar~ 2, T. H. Noon2, R. E. Reed2, J. N. Shively2, and C. Orvell 3 l j. A. Baker Institute for Animal Health, Departments of Microbiology and Pathology, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 2 Department of Veterinary Science, College of Agriculture, University of Arizona, Tucson, Arizona, U.S.A. 3The National Bacteriological Laboratory, Department of Virology, Stockholm, Sweden
Accepted January 20, 1991
Summary. Canine distemper virus has been isolated in dog lymphocyte cultures from the brains of three javelinas that became moribund with signs of encephalitis. Canine distemper viral antigen was demonstrated predominantly in neurons and morbillivirus-like structures were seen by electron microscopy in brains of diseased animals. Serological studies suggest that CDV infection may be common in javelinas.
Attention has focused on morbilliviruses, family Paramyxoviridae, genus Morbiltivirus, during the last two years when agents resembling canine distemper virus (CDV) were isolated from harbour seals that had died in large numbers in the North Sea [12], the Baikal Lake [13] and from dolphins in the Mediterranean Sea [8]. By monoclonal antibody studies, virus isolated from seals in the North Sea was found to be closely related to CDV although differing in some epitopes [6, 11]. This virus was tentatively classified as phocine distemper virus (PDV). In contrast, virus isolated from seals in the Baikal Lake was almost identical to CDV [16]. The epizootiology of the disease outbreaks remains obscure. We now report the isolation of CDV from a further animal species. Javelinas (collared peccary, Tayassu tajacu [ 14]) are feral animals resembling small pigs which are common in the southwestern United States. A disease outbreak characterized by signs of encephalitis (blindness, myoclonus, reluc-
148
M.J.G. Appel et at.
Fig. 1. Electron micrograph of morbillivirus-like nucleocapsids in a neuron from a javelina with encephalitis, x 26,000 tance to move) and apparent high mortality in adult javelinas was observed by the Arizona Game and Fish Department in the deserts of southwest Arizona during 1989. Pathologic examination of nine affected animals revealed lesions confined almost exclusively to the central nervous system (CNS). Microscopic examination of the affected brains predominantly revealed neurons with prominent eosinophilic intracytoplasmic inclusions, variable neuronal necrosis, neuronophagia, mild perivascular cuffing by mononuclear cells and astrocytosis. Infection by rabies [9], pseudorabies [9], hog cholera [9], swine paramyxovirus (blue eye) [15], encephalomyocarditis [9], vomiting and wasting disease [9], swine vesicular disease [9] and Japanese encephalitis viruses [9] were ruled out on the basis of lesions or of specific laboratory tests. However, by electron microscopy, structures resembling morbillivirus nucleocapsids were demonstrated in the CNS (Fig. 1). By immunocytochemistry, CDV antigen could be demonstrated, predominantly in neurons of the cerebral cortex (Fig. 2). Control brains from unaffected javelinas did not show a reaction. Virus isolation attempts in a variety of cell lines and primary cells of pig, dog, cat, simian, and human origin failed. However, virus was isolated from the brains of three javelinas with signs of encephalitis in phytohemagglutinin (PHA) stimulated primary blood lymphocyte cultures prepared from specific pathogen free (SPF) dogs (Fig. 3). Uninoculated control cultures remained free of virus. The isolated virus produced syncytia in dog lung macrophage cultures
Canine distemper virus infection and encephalitis
149
Fig. 2. Javelina brain section (cerebrum) stained by the PAP technique using monoclonal mouse-anti-CDV-N antibody (4.772). x 350. Viral antigen is demonstrated within the dendrites, perikarya, and axons of pyramidal neurons [2] and did not readily grow in primary dog kidney cells, criteria previously associated with virulent CDV [1]. The virus was adapted to Vero cells by co-cultivation with infected lymphocytes. Infected Vero cells in the third passage were tested with mouse monoclonal antibodies to CDV [10] and to PDV [11]. The results indicated that the isolated virus was CDV that differed only with one CDV-H monoclonal antibody when compared with attenuated CDV (Table 1). For further testing virus virulence, three CDV susceptible young adult domestic ferrets (Mustelaputoriusfi~ro)were inoculated intraperitoneally with 1 ml each of a 10% suspension of brain tissue from a javelina with neurological signs. Close to 100% of domestic ferrets succumb to virulent CDV whereas seroconversion without signs of illness occurs after inoculation with attenuated live virus [3]. Only one of the three inoculated ferrets showed signs of distemper (anorexia, depression, conjunctivitis, dermatitis, ataxia) and was euthanized 16 days post inoculation. CDV was isolated in dog lung macrophage cultures [2] from lymphatic tissues and lungs. Microscopic lesions were typical of canine distemper: severe lymphoid depletion with syncytial formation in lymphatic tissues, moderate pneumonitis and bronchiolitis, mild gastritis, diffuse epidermitis with syncytia and intracytoplasmic inclusion bodies in urinary bladder, kidney pelvis, bronchial epithelium and skin. Conjunctivitis and anterior uveitis were also seen. The remaining two ferrets developed antibody to CDV but had no clinical
150
M.J.G. Appel et al.
Fig. 3. Blood tymphocytes from an SPF dog 5 days post inoculation with a t:50 dilution ofjavelina brain suspension. Lymphocytes were stained by direct FA with FITC labelled dog-anti-CDV serum. Lymphocytes were separated from blood in Phycoll-Hypaque and inoculated with brain suspension 1 day post stimulation with PHA (15 btg/ml)
signs nor virus in tissues when euthanized six weeks post inoculation, as usually seen in ferrets after inoculation with attenuated CDV. Sera from eight javelinas with signs of encephalitis did not have virus neutralizing antibody to CDV [4] with the exception of one with a low titer of 1:10. However, three of the same animals had CDV antibody when tested by ELISA. This corresponds with observations in dogs with acute distemper encephalitis. A preliminary survey of clinically normal, hunter-killed, javelinas from an enzootic focus of the disease revealed that 20 of 33 javelinas had CDV neutralizing antibody titers ranging from 1:10 to 1:2000. In an earlier study [7], javetinas, which belong to the Tayassuidae and not to the family Suidae [14], showed clinical signs when inoculated with rinderpest virus (RPV), another morbillivirus. In contrast, domestic pigs (Suidae) were previously found to be susceptible to infection with CDV [5] and RPV [7] without the development of disease. Both viruses replicate in lymphatic tissues of pigs without causing clinical signs and virus shedding. We now report the isolation of CDV from javelinas with clinical signs of encephalitis and CNS lesions as seen in dogs with canine distemper. These wild animals may be endemically infected with CD as are many wildlife carnivore species. The seal and dolphin epidemic and this episode highlight our incomplete knowledge of both the susceptibility of animal species to morbilliviruses and the epidemiology of these diseases.
Canine distemper virus infection and encephalitis
151
Table 1. Reactivity of mAbs with javelina virus adapted to Vero cells (indirect FA) Anti CDV mAbs
JV
CDV-Ond
PDV
Anti PDV mAbs
JV
CDV-Ond
PDV
N3.721 N3.805 N3.991
+ + +
+ + +
+ -
P P P P P
3.630 3.695 3.698 3.768 4.088
+ + + + +
+ + + + +
+ + +
F F F F F
1.062 E2 1.067D2 1.068 B2 1.073G10 1.092 C3
+ + + + +
+ + + + +
+ + + + +
F F F F
3.551 3.584 3.633 5.148
+ + + +
+ + + +
+ + + +
H 1.122Dll H 1.062G5 H 1 . 0 6 3 E9 H 1 . 0 6 7 E5 H 1.068 F2 H 1.070 B5
+ -
+ -
+ + + + + +
H2.267 H3.734 H3.775 H4.043
+ + +
+ + + +
+
J V Virus isolated from javelina brain tissue in dog blood lymphocytes and adapted to Vero cells CD V-Ond Onderstepoort strain of CDV isolated in chick embryos and adapted to Vero cells PD V Phocine distemper virus isolated from a seal in Denmark and adapted to Vero cells
References 1. Appet M (1978) Reversion to virulence of attenuated canine distemper virus in vivo and in vitro. J Gen Virol 41:385-393 2. Appet M, Jones O R (1967) Use of alveolar macrophages for cultivation of canine distemper virus. Proc Soc Exp Bio Med 126:571-574 3. Appel M, Gillespie JE (1972) Canine distemper monograph. In: Gard S, Hallauer C, Meyer K F (eds) Handbook of virus research. Springer, Wien New York, pp 34-63 4. Appel M, Robson DA (1973) A microneutralization test for canine distemper virus. Am J Vet Res 34:1459-1463 5. Appel M, Sheffy BE, Percy DH, Gaskin JM (1974) Canine distemper virus in domesticated cats and pigs. Am J Vet Res 35:803-806 6. Cosby SL, McQuaid S, Duffy N, Lyons C, Rima BK, Allan GM, McCuUough SJ, Kennedy S, Smyth JA, McNeilly F, Craig C, ~}rvell C (1988) Characterization of seal morbillivirus. Nature 336:115-116 7. Dardiri AH, Yedloutschnig RJ, Taylor W D (1969) Clinical and serologic response of American white-collored peccaries to African swine fever, foot-and-mouth disease, vesicular stomatitis, vesicular exanthema of swine, hog cholera, and rinderpest viruses. In: Proceedings 73rd Annu Meet US Anim Health Assoc, pp 437-452
152
M . J . G . Appel et al. : Canine distemper virus infection and encephalitis
8. Domingo M, Ferrer L, Pumarola M, Marco A, Plana J, Kennedy S, McAliskey M, Rima BK (1990) Morbillivirus in dolphins. Nature 348:21 9. Fenner F, Bachmann PA, Gibbs EPJ, Murphy FA, Studdert MJ, White DO (1987) Veterinary virology. Academic Press, Orlando 10. Orvell C, Sheshberadaran H, Norrby E (1985) Preparation and characterization of monoclonal antibodies directed against four structural components of canine distemper virus. J Gen Virol 66:443-456 11. Orvell C, Blixenkrone-M611er M, Svansson V, Have P (1990) Immunological relationships between phocid and canine distemper virus studied with monoclonal antibodies. J Gen Virol 71:2085-2092 12. Osterhaus ADME, Groen J, de Vries P, UytdeHaag FGCM, Klingeborn B, Zarnke R (1988) Canine distemper virus in seals. Nature 335:403-404 13. Osterhaus ADME, Groen J, UytdeHaag FGCM, Visser IKG, van de Bildt MWG, Bergman A, Klingeborn B (1989) Distemper virus in Baikal seals. Nature 338: 209210 14. Sowls LK (1984) The peccaries. University of Arizona Press, Tucson, AZ 15. Stephano HA, Gay GM, Ramirez TC (1988) Encephalomyelitis, reproductive failure and corneal opacity (blue eye) in pigs, associated with a paramyxovirus infection. Vet Rec 122:6-10 16. Visser IKG, Kumarev VP, Orvell C, de Vries P, Broeders WJ, van de Bildt MWG, Groen J, Teppema JS, Burger MC, UytdeHaag FGCM, Osterhaus ADME (1990) Comparison of two morbilliviruses isolated from seals during outbreaks of distemper in North West Europe and Siberia. Arch Virol 111:149-164 Authors' address: M.J.G. Appel, James A. Baker Institute for Animal Health, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, U.S.A. Received January 2, 1991
Verleger: Springer-Verlag KG, Saehsenplatz 4-6, A-1201 Wien. - Herausgeber: Dr. F. A. Murphy, Center for Infectious Diseases, Centers for Disease Control, Atlanta, G A 30333, U . S . A . - Redaktion: Sachsenplatz 4-6, A-1201 Wien. - Hersteller: Adolf Holzhausens Nachfolger, Kandlgasse 19-21, A-1070 Wien. - Verlagsort: Wien. - Herstetlungsort: Wien. - Printed in Austria
Vi rology
Arch Virot (1991) 119:147-152
© SpringerWerlag 1991 Printed in Austria
Canine distemper virus infection and encephalitis in javelinas (collared peccaries) Brief R e p o r t M. J. G. Appel 1, C. Reggiardo 2, B. A. Summers 1, S. Pearce-Kelling 1, C. J. Mar~ 2, T. H. Noon2, R. E. Reed2, J. N. Shively2, and C. Orvell 3 l j. A. Baker Institute for Animal Health, Departments of Microbiology and Pathology, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 2 Department of Veterinary Science, College of Agriculture, University of Arizona, Tucson, Arizona, U.S.A. 3The National Bacteriological Laboratory, Department of Virology, Stockholm, Sweden
Accepted January 20, 1991
Summary. Canine distemper virus has been isolated in dog lymphocyte cultures from the brains of three javelinas that became moribund with signs of encephalitis. Canine distemper viral antigen was demonstrated predominantly in neurons and morbillivirus-like structures were seen by electron microscopy in brains of diseased animals. Serological studies suggest that CDV infection may be common in javelinas.
Attention has focused on morbilliviruses, family Paramyxoviridae, genus Morbiltivirus, during the last two years when agents resembling canine distemper virus (CDV) were isolated from harbour seals that had died in large numbers in the North Sea [12], the Baikal Lake [13] and from dolphins in the Mediterranean Sea [8]. By monoclonal antibody studies, virus isolated from seals in the North Sea was found to be closely related to CDV although differing in some epitopes [6, 11]. This virus was tentatively classified as phocine distemper virus (PDV). In contrast, virus isolated from seals in the Baikal Lake was almost identical to CDV [16]. The epizootiology of the disease outbreaks remains obscure. We now report the isolation of CDV from a further animal species. Javelinas (collared peccary, Tayassu tajacu [ 14]) are feral animals resembling small pigs which are common in the southwestern United States. A disease outbreak characterized by signs of encephalitis (blindness, myoclonus, reluc-
148
M.J.G. Appel et at.
Fig. 1. Electron micrograph of morbillivirus-like nucleocapsids in a neuron from a javelina with encephalitis, x 26,000 tance to move) and apparent high mortality in adult javelinas was observed by the Arizona Game and Fish Department in the deserts of southwest Arizona during 1989. Pathologic examination of nine affected animals revealed lesions confined almost exclusively to the central nervous system (CNS). Microscopic examination of the affected brains predominantly revealed neurons with prominent eosinophilic intracytoplasmic inclusions, variable neuronal necrosis, neuronophagia, mild perivascular cuffing by mononuclear cells and astrocytosis. Infection by rabies [9], pseudorabies [9], hog cholera [9], swine paramyxovirus (blue eye) [15], encephalomyocarditis [9], vomiting and wasting disease [9], swine vesicular disease [9] and Japanese encephalitis viruses [9] were ruled out on the basis of lesions or of specific laboratory tests. However, by electron microscopy, structures resembling morbillivirus nucleocapsids were demonstrated in the CNS (Fig. 1). By immunocytochemistry, CDV antigen could be demonstrated, predominantly in neurons of the cerebral cortex (Fig. 2). Control brains from unaffected javelinas did not show a reaction. Virus isolation attempts in a variety of cell lines and primary cells of pig, dog, cat, simian, and human origin failed. However, virus was isolated from the brains of three javelinas with signs of encephalitis in phytohemagglutinin (PHA) stimulated primary blood lymphocyte cultures prepared from specific pathogen free (SPF) dogs (Fig. 3). Uninoculated control cultures remained free of virus. The isolated virus produced syncytia in dog lung macrophage cultures
Canine distemper virus infection and encephalitis
149
Fig. 2. Javelina brain section (cerebrum) stained by the PAP technique using monoclonal mouse-anti-CDV-N antibody (4.772). x 350. Viral antigen is demonstrated within the dendrites, perikarya, and axons of pyramidal neurons [2] and did not readily grow in primary dog kidney cells, criteria previously associated with virulent CDV [1]. The virus was adapted to Vero cells by co-cultivation with infected lymphocytes. Infected Vero cells in the third passage were tested with mouse monoclonal antibodies to CDV [10] and to PDV [11]. The results indicated that the isolated virus was CDV that differed only with one CDV-H monoclonal antibody when compared with attenuated CDV (Table 1). For further testing virus virulence, three CDV susceptible young adult domestic ferrets (Mustelaputoriusfi~ro)were inoculated intraperitoneally with 1 ml each of a 10% suspension of brain tissue from a javelina with neurological signs. Close to 100% of domestic ferrets succumb to virulent CDV whereas seroconversion without signs of illness occurs after inoculation with attenuated live virus [3]. Only one of the three inoculated ferrets showed signs of distemper (anorexia, depression, conjunctivitis, dermatitis, ataxia) and was euthanized 16 days post inoculation. CDV was isolated in dog lung macrophage cultures [2] from lymphatic tissues and lungs. Microscopic lesions were typical of canine distemper: severe lymphoid depletion with syncytial formation in lymphatic tissues, moderate pneumonitis and bronchiolitis, mild gastritis, diffuse epidermitis with syncytia and intracytoplasmic inclusion bodies in urinary bladder, kidney pelvis, bronchial epithelium and skin. Conjunctivitis and anterior uveitis were also seen. The remaining two ferrets developed antibody to CDV but had no clinical
150
M.J.G. Appel et al.
Fig. 3. Blood tymphocytes from an SPF dog 5 days post inoculation with a t:50 dilution ofjavelina brain suspension. Lymphocytes were stained by direct FA with FITC labelled dog-anti-CDV serum. Lymphocytes were separated from blood in Phycoll-Hypaque and inoculated with brain suspension 1 day post stimulation with PHA (15 btg/ml)
signs nor virus in tissues when euthanized six weeks post inoculation, as usually seen in ferrets after inoculation with attenuated CDV. Sera from eight javelinas with signs of encephalitis did not have virus neutralizing antibody to CDV [4] with the exception of one with a low titer of 1:10. However, three of the same animals had CDV antibody when tested by ELISA. This corresponds with observations in dogs with acute distemper encephalitis. A preliminary survey of clinically normal, hunter-killed, javelinas from an enzootic focus of the disease revealed that 20 of 33 javelinas had CDV neutralizing antibody titers ranging from 1:10 to 1:2000. In an earlier study [7], javetinas, which belong to the Tayassuidae and not to the family Suidae [14], showed clinical signs when inoculated with rinderpest virus (RPV), another morbillivirus. In contrast, domestic pigs (Suidae) were previously found to be susceptible to infection with CDV [5] and RPV [7] without the development of disease. Both viruses replicate in lymphatic tissues of pigs without causing clinical signs and virus shedding. We now report the isolation of CDV from javelinas with clinical signs of encephalitis and CNS lesions as seen in dogs with canine distemper. These wild animals may be endemically infected with CD as are many wildlife carnivore species. The seal and dolphin epidemic and this episode highlight our incomplete knowledge of both the susceptibility of animal species to morbilliviruses and the epidemiology of these diseases.
Canine distemper virus infection and encephalitis
151
Table 1. Reactivity of mAbs with javelina virus adapted to Vero cells (indirect FA) Anti CDV mAbs
JV
CDV-Ond
PDV
Anti PDV mAbs
JV
CDV-Ond
PDV
N3.721 N3.805 N3.991
+ + +
+ + +
+ -
P P P P P
3.630 3.695 3.698 3.768 4.088
+ + + + +
+ + + + +
+ + +
F F F F F
1.062 E2 1.067D2 1.068 B2 1.073G10 1.092 C3
+ + + + +
+ + + + +
+ + + + +
F F F F
3.551 3.584 3.633 5.148
+ + + +
+ + + +
+ + + +
H 1.122Dll H 1.062G5 H 1 . 0 6 3 E9 H 1 . 0 6 7 E5 H 1.068 F2 H 1.070 B5
+ -
+ -
+ + + + + +
H2.267 H3.734 H3.775 H4.043
+ + +
+ + + +
+
J V Virus isolated from javelina brain tissue in dog blood lymphocytes and adapted to Vero cells CD V-Ond Onderstepoort strain of CDV isolated in chick embryos and adapted to Vero cells PD V Phocine distemper virus isolated from a seal in Denmark and adapted to Vero cells
References 1. Appet M (1978) Reversion to virulence of attenuated canine distemper virus in vivo and in vitro. J Gen Virol 41:385-393 2. Appet M, Jones O R (1967) Use of alveolar macrophages for cultivation of canine distemper virus. Proc Soc Exp Bio Med 126:571-574 3. Appel M, Gillespie JE (1972) Canine distemper monograph. In: Gard S, Hallauer C, Meyer K F (eds) Handbook of virus research. Springer, Wien New York, pp 34-63 4. Appel M, Robson DA (1973) A microneutralization test for canine distemper virus. Am J Vet Res 34:1459-1463 5. Appel M, Sheffy BE, Percy DH, Gaskin JM (1974) Canine distemper virus in domesticated cats and pigs. Am J Vet Res 35:803-806 6. Cosby SL, McQuaid S, Duffy N, Lyons C, Rima BK, Allan GM, McCuUough SJ, Kennedy S, Smyth JA, McNeilly F, Craig C, ~}rvell C (1988) Characterization of seal morbillivirus. Nature 336:115-116 7. Dardiri AH, Yedloutschnig RJ, Taylor W D (1969) Clinical and serologic response of American white-collored peccaries to African swine fever, foot-and-mouth disease, vesicular stomatitis, vesicular exanthema of swine, hog cholera, and rinderpest viruses. In: Proceedings 73rd Annu Meet US Anim Health Assoc, pp 437-452
152
M . J . G . Appel et al. : Canine distemper virus infection and encephalitis
8. Domingo M, Ferrer L, Pumarola M, Marco A, Plana J, Kennedy S, McAliskey M, Rima BK (1990) Morbillivirus in dolphins. Nature 348:21 9. Fenner F, Bachmann PA, Gibbs EPJ, Murphy FA, Studdert MJ, White DO (1987) Veterinary virology. Academic Press, Orlando 10. Orvell C, Sheshberadaran H, Norrby E (1985) Preparation and characterization of monoclonal antibodies directed against four structural components of canine distemper virus. J Gen Virol 66:443-456 11. Orvell C, Blixenkrone-M611er M, Svansson V, Have P (1990) Immunological relationships between phocid and canine distemper virus studied with monoclonal antibodies. J Gen Virol 71:2085-2092 12. Osterhaus ADME, Groen J, de Vries P, UytdeHaag FGCM, Klingeborn B, Zarnke R (1988) Canine distemper virus in seals. Nature 335:403-404 13. Osterhaus ADME, Groen J, UytdeHaag FGCM, Visser IKG, van de Bildt MWG, Bergman A, Klingeborn B (1989) Distemper virus in Baikal seals. Nature 338: 209210 14. Sowls LK (1984) The peccaries. University of Arizona Press, Tucson, AZ 15. Stephano HA, Gay GM, Ramirez TC (1988) Encephalomyelitis, reproductive failure and corneal opacity (blue eye) in pigs, associated with a paramyxovirus infection. Vet Rec 122:6-10 16. Visser IKG, Kumarev VP, Orvell C, de Vries P, Broeders WJ, van de Bildt MWG, Groen J, Teppema JS, Burger MC, UytdeHaag FGCM, Osterhaus ADME (1990) Comparison of two morbilliviruses isolated from seals during outbreaks of distemper in North West Europe and Siberia. Arch Virol 111:149-164 Authors' address: M.J.G. Appel, James A. Baker Institute for Animal Health, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, U.S.A. Received January 2, 1991
Verleger: Springer-Verlag KG, Saehsenplatz 4-6, A-1201 Wien. - Herausgeber: Dr. F. A. Murphy, Center for Infectious Diseases, Centers for Disease Control, Atlanta, G A 30333, U . S . A . - Redaktion: Sachsenplatz 4-6, A-1201 Wien. - Hersteller: Adolf Holzhausens Nachfolger, Kandlgasse 19-21, A-1070 Wien. - Verlagsort: Wien. - Herstetlungsort: Wien. - Printed in Austria
