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Specific diagnosis of parvovirus enteritis in dogs and cats by in situ hybridization
J. Comp.
Path.
1992 Vol.
Specific
107, 141-146
Diagnosis of Parvovirus Enteritis and Cats by In Situ Hybridization
in Dogs
A. S. Waldvogel, S. Hassam *, N. Stoerckle, R. Weilenmann, J. D. Tratschint, G. Siegl$ and A. Pospischil Institut fir Veterintirpathologie und *Institut ftir klinische Pathologie der Universitiit
Summary In a retrospective study, the fixed intestines of 10 dogs and 10 cats with intestinal lesionscharacteristic of parvovirus infection were assayedfor the presenceof parvovirus by in situ hybridization and immunohistochemistry. Parvoviral nucleic acid was localized by in situ hybridization in intestinal tissuein all 10 dogs and in nine of the 10 cats, whereasantigen was detected only in seven of 10 canine and eight of 10 feline intestines by immunohistdchemistry. We conclude that an aetiological diagnosiscan be establishedwith a high degree of certainty by routine histology. Demonstration of the infectious agent by in situ hybridization, however, proves to be a valuable specific tool which allows an exact cellular localization of parvovirus in formalin-fixed, paraffin wax-embedded tissuesections. Introduction Fatal enteritis due to infection with parvovirus is still a fairly common disease among young cats and dogs despite the widespread use of vaccines. Eighteen and 14 per cent, respectively, of cats and dogs lessthan 1 year old, submitted to our institute show lesions characteristic of parvovirus infection. Diagnosis is based on the presence of classical lesions, such as loss of intestinal epithelium beginning in the crypts, necrosis of lymphoid tissue and depletion of lymphocytes (Appel and Parrish, 1987; Pedersen, 1987). Although these lesions are generally considered to be pathognomonic, Barker and vanDreume1 (1985) mention several conditions of dogs requiring differential diagnoses from parvovirus enteritis and, in cats, feline leukemia virus (FeLV) is a likely cause of similar lesions (Reinacher, 1987). Definite confirmation of the aetiology can only be achieved by demonstrating the virus within the lesions, e.g. by detecting specific viral nucleic acids or antigen. In our study we proposed to evaluate and compare the detection of viral nucleic acid and viral protein by in situ hybridization and immunohistochemistry, respectively, in canine and feline intestines with lesions characteristic of parvovirus enteritis. We also aimed to determine the diagnostic value of morphological criteria of parvovirus enteritis. Address for correspondence: Institut 268, 8057 Zurich, Switzerland. 0021-9975/92/060141+06
$08.00/O
fk
VeterGrpathologie
der UniversitPt
0
Ziirich,
Winterthurerstrasse
1992 Academic
Press Limited
142
A. S. Waldvogel Materials
and
et al.
Methods
The tissuesused were retrieved from the archives of the Department of Veterinary Pathology of the University of Zurich. They originated from 10 dogs and 10 cats with intestinal lesionssuggestiveof parvovirus enteritis. Small intestine was available from all animals, lymph nodes from two dogs and two cats, and spleenfrom one dog. All thesetissueshad been fixed in formalin and embeddedin paraffin wax a few days later. Tissue sections were mounted on organosilan treated glassslides (Rentrop, Knapp, Winter and Schweizer, 1986). Sections from myocardium, previously shown to contain canine parvovirus nucleic acid by in situ hybridization (Waldvogel, Hassam, Weilenmann, Tratschin, Siegl, Hanichen, Briner and Pospischil, 1991), served as a positive control. To generate the probe, we obtained a cloned genomic DNA of canine parvovirus inserted in pBR322, designated pNCPV2, and consisting of the entire VPl- and VPP-coding sequences(Reed, Jones and Miller, 1988). This plasmid wasdigested with PvuII and HindIII, resulting in four fragments: a 660bp and a 1300bp DNA fragment both containing CPV genome sequencesonly, a 2620bp fragment, containing both pBR322 and CPV sequences,and a 2320bp fragment containing only pBR322 DNA. The restriction products were separated by gel electrophoresis and the 660bp and 1300bp fragments isolated from the gel with the Geneclean II Kit (BlO 101, La Jolla, CA, U.S.A.) and utilized together as a probe for in situ hybridization. Labelling with 35S-dATP (Amersham, U.K.) or with Biotin-1 I-dUTP was performed with a random primed labelling kit (Boehringer Mannheim GmbH, Germany). Becausethere is a homology of at least 98 per cent between the nucleotide sequencesof canine and feline parvoviruses in the genome region coding for VP-l and VP-2 (Parrish, Aquadro and Carmichael, 1988; Reed et al., 1988), we decided to use this probe for in situ hybridization both in canine and feline tissues. Our protocol for in situ hybridization with biotinylated probes has been described previously (Waldvogel et al., 1991). Approximately 6 ng of the biotinylated probe was usedin 20 p1 hybridization solution per slide. For the 35S-labelledprobe, pretreatment and hybridization was also carried out by the above mentioned protocol, with approximately 300 000 cpm per section. Post-hybridization washing was more stringent and included a brief rinse in 1 x PBS, washing on a shaker platform in freshly prepared 10mM dithiotreitol (DTT) in 2 x SSC at room temperature for 10 min, 2 x SSC at 37°C three times for 20 min, 1 x SSC at 37°C for 30 min and 0.1 x SSC at 37°C for 30 min. For autoradiography, the slideswere dehydrated in graded ethanol and 0.3 M NH,Ac, air-dried, dipped in Ilford nuclear track emulsion (diluted 1 in 1 with 0.6 M NH.+Ac), air-dried and exposedfor 5 to 6 days at 4°C in a light-proof box. The slideswere then developed in Kodak D19 developer (diluted 1 in 1 with water), fixed in Kodak fixer, all at 14°C and stained with haemalum and eosin. Biotinylated pBR322 and 35S-labelledh DNA were usedasnegative control probes. For immunohistochemistry by the peroxidase-antiperoxidase (PAP) method, the sectionswere dewaxed and digested in 0.1 per cent pronase XXVII (Sigma Chemie, 9470 Buchs, Switzerland) for 1 min at 37°C. Parvovirus antigen was demonstrated with a polyclonal goat antibody against feline parvovirus (generousgift from Prof. Dr H. Lutz, Clinic for Veterinary Internal Medicine, University of Zurich, Switzerland). This primary antibody was applied at a dilution of 1 in 100in an overnight incubation at room temperature. Visualization was achieved with the DAK0 PAP KITTM goat (DAK0 A/S, Glostrup, Denmark). Counterstaining wasdone with haematoxylin for 1 min. Results
The intestines showed various degrees of autolysis ranging from loss of the villous epithelium (but otherwise preserved mucosa) to the disappearance of the nucleus of the cells in the lamina propria mucosae. Nevertheless, parvovir-
Diagnosis
of parvovirus
by hybridization
143
us-specific DNA reacting with the CPV probe could be demonstrated in the intestines of all dogs and nine of 10 cats by in situ hybridization (Table 1, Fig. 1). Although viral DNA could be demonstrated in as many tissue samples using the biotinylated probe as by using the 35S-labelled probe, the latter yielded a much higher signal strength per cell and detected a higher number of infected cells per specimen. This was most conspicuous in the centre of lymph follicles in Peyer’s patches or lymph nodes, where an intense signal was often generated by the 35S-labelled probe (Fig. 2), although no viral nucleic acid could be demonstrated by in situ hybridization with non-radioactively labelled probes. On the other hand, the biotinylated probe had a much better signal resolution and allowed the more accurate cellular localization of the signal: only with this probe was it possible to recognize that not only the nucleus, but also the cytoplasm of enterocytes and mononuclear leucocytes in Peyer’s patches contained parvoviral nucleic acid (Fig. 3). Parvoviral antigen was not always detected in intestines where nucleic acid was present (Table 1). Minimal non-specific staining, limited to cell membranes in adipose tissue and to intestinal content, was observed with the biotinylated parvovirus as well as the biotinylated pBR322 probe. Hybridization with 35S-labelled h DNA remained negative. Discussion
Using a CPV-specific DNA probe, parvoviral nucleic acid could be demonstrated in all dogs and nine of 10 cats with lesions considered to be typical of parvovirus enteritis. The morphological changes of the intestines therefore allow an aetiological diagnosis with a high degree of reliability. As anticipated, the CPV probe also hybridized to feline parvovirus. Intestinal lesions, indistinguishable from parvovirus enteritis, might be associated with FeLV and in our study in one cat, neither viral nucleic acid nor viral antigen could ..be demonstrated. However, the average age of cats affected by FeLV enteritis is 2.5 years (Reinacher, 1987). The age of this cat was not known, but based on records of its body weight and crown rump length, it was judged to be juvenile. Furthermore, there were no lesions indicative of a FeLV infection in other available organs (lymph nodes, lung). FeLV, therefore, was an unlikely explanation for the intestinal lesions. The inability to detect parvovirus might, therefore, have been due to the virus having already been eliminated or at least reduced below detection level by the time the animal died. An involvement of some other, as yet unknown, agent cannot be ruled out. Although parvoviral DNA replication takes place within the nucleus (Berns, 1990) and the staining was strictly limited to the nucleus of myocardial cells in parvovirus myocarditis (Waldvogel et al., 1991), not only viral antigen but also viral nucleic acid was found in the cytoplasm of intestinal cells and mononuclear leucocytes of Peyer’s patches and in lymph nodes. This may be due to hybridization to viral mRNA present in the cytoplasm or may indicate the diffusion of virus and viral components into cytoplasm after the breakdown of
144
Fig.
A. S. Waldvogel
I.
In situ hybridization with the biotinylated acid within cells of an autolysed intestine.
et
al.
probe demonstrates the presence of parvovirus Haemalum counterstain. Bar = 50 bm.
nucleic
Fig. 2.
In situ hybridization with the 35S-labelled probe reveals a strong signal in the centre of lymph follicles in Peyer’s patches, indicating the presence of virus. With the biotinylated probe it was not possible to localize CPV in this tissue. HE counterstain. Bar= 300 pm.
Fig.
In situ hybridization with the biotinylated probe shows parvoviral nucleic nucleus, but also in the cytoplasm of infected cells (arrow). No counterstain.
3.
acid, not only Bar= 30 pm.
in the
Diagnosis Sensitivity
of parvovirus
Table of immunohistochemistry
1 and
in situ
hybridization
Assay
SHUitiUity*
Immunohistochemistry In situ hybridization
“3 probe
In situ hybridization
biotinylated
*Sensitivity: number intestines tested.
of intestines
145
by hybridization
probe where
parvovirus
Canine intestines
Feline intestines
7/10
S/l0
lo/lo
S/IO
lo/lo
s/10
was identified
divided
by the number
of suspect
the nuclear membrane in dead cells, particularly in the enterocytes, or may indicate phagocytosis of viruses in lymphoid organs. In situ hybridization with biotinylated probes for the detection of parvovirus proved to be somewhat more sensitive, but not more time-consuming or labour-intensive than immunohistochemistry. Furthermore, we have a welldefined range of CPV probes with known specificity and a good control over the stringency of the hybridization reaction. Because of the higher sensitivity of in situ hybridization with the radioactive probe, this probe is more suitable for studying the extent of viral distribution within an animal. The biotinylated probe, however, proved to be sensitive enough to confirm the aetiology in the intestines we examined. Our experiments were performed only in animals with strongly suspected parvovirus enteritis. To evaluate further the efficacy of this assay, it will be necessary to examine a larger series of enteritis specimens and correlate the findings with those obtained by routine histological diagnosis. References
Appel, M. and Parrish, C. R. (1987). Canine parvovirus type 2. In Viws Infections of Carnivores. M. J. Appel, Ed., Elsevier Science PublishersB. V., Amsterdam, pp. 69-92. Berns, K. I. (1990). Parvoviridae and their replication. In Fields Virology, Vol. 2, 2nd Edit. B. N. Fields and D. M. Knipe, Eds, Raven Press,New York, p . 1743-1763. Barker, I. K. and vanDreume1, A. A. (1985). The alimentary system. Pn Pathology of Domestic Animals, Vol. 2, 3rd Edit. K. V. F. Jubb, P. C. Kennedy and N. Palmer, Eds, Academic Press,Inc., Orlando, pp. l-237. Parrish, C. R., Aquadro, C. F. and Carmichael, L. E. (1988). Canine host range and a specific epitope map along with variant sequencesin the capsid protein gene of canine parvovirus and related feline, mink, and raccoon parvoviruses. Virology, 166,293-307.
Pedersen,N. C. (1987). Feline panleukopenia virus. In Virus Znfections of Carnivores. M. J. Appel, Ed., Elsevier Science Publishers B. V., Amsterdam, pp. 247-254. Reed, A. P., Jones, E. V. and Miller, T. J. (1988). Nucleotide sequenceand genome organization of canine parvovirus. Journal of Virology, 62, 26G276. Reinacher, M. (1987). Feline leukemia virus-associated enteritis-a condition with features of feline panleukopenia. Veterinary Pathology, 24, I+. Rentrop, M., Knapp, B., Winter, H. and Schweizer, J. (1986). Aminoalkylsilanetreated glassslidesas support for in situ hybridization of keratin cDNAs to frozen tissuesections under varying fixation and pretreatment conditions. Histochemical Journal,
18, 271-276.
A. S. Waldvogel
146
et al.
Waldvogel, A. S., Hassam,S., Weilenmann, R., Tratschin, J. D., Siegl, G., Hgnichen, T., Briner, J. and Pospischil,A. (1991). Retrospective study of myocardial canine parvovirus infection by in situ hybridization. Journal of Veterinary Medicine Series B, 38, 353-357.
1
Received, February 13th, 1992 Accepted, March 3Oth, 1992
Path.
1992 Vol.
Specific
107, 141-146
Diagnosis of Parvovirus Enteritis and Cats by In Situ Hybridization
in Dogs
A. S. Waldvogel, S. Hassam *, N. Stoerckle, R. Weilenmann, J. D. Tratschint, G. Siegl$ and A. Pospischil Institut fir Veterintirpathologie und *Institut ftir klinische Pathologie der Universitiit
Summary In a retrospective study, the fixed intestines of 10 dogs and 10 cats with intestinal lesionscharacteristic of parvovirus infection were assayedfor the presenceof parvovirus by in situ hybridization and immunohistochemistry. Parvoviral nucleic acid was localized by in situ hybridization in intestinal tissuein all 10 dogs and in nine of the 10 cats, whereasantigen was detected only in seven of 10 canine and eight of 10 feline intestines by immunohistdchemistry. We conclude that an aetiological diagnosiscan be establishedwith a high degree of certainty by routine histology. Demonstration of the infectious agent by in situ hybridization, however, proves to be a valuable specific tool which allows an exact cellular localization of parvovirus in formalin-fixed, paraffin wax-embedded tissuesections. Introduction Fatal enteritis due to infection with parvovirus is still a fairly common disease among young cats and dogs despite the widespread use of vaccines. Eighteen and 14 per cent, respectively, of cats and dogs lessthan 1 year old, submitted to our institute show lesions characteristic of parvovirus infection. Diagnosis is based on the presence of classical lesions, such as loss of intestinal epithelium beginning in the crypts, necrosis of lymphoid tissue and depletion of lymphocytes (Appel and Parrish, 1987; Pedersen, 1987). Although these lesions are generally considered to be pathognomonic, Barker and vanDreume1 (1985) mention several conditions of dogs requiring differential diagnoses from parvovirus enteritis and, in cats, feline leukemia virus (FeLV) is a likely cause of similar lesions (Reinacher, 1987). Definite confirmation of the aetiology can only be achieved by demonstrating the virus within the lesions, e.g. by detecting specific viral nucleic acids or antigen. In our study we proposed to evaluate and compare the detection of viral nucleic acid and viral protein by in situ hybridization and immunohistochemistry, respectively, in canine and feline intestines with lesions characteristic of parvovirus enteritis. We also aimed to determine the diagnostic value of morphological criteria of parvovirus enteritis. Address for correspondence: Institut 268, 8057 Zurich, Switzerland. 0021-9975/92/060141+06
$08.00/O
fk
VeterGrpathologie
der UniversitPt
0
Ziirich,
Winterthurerstrasse
1992 Academic
Press Limited
142
A. S. Waldvogel Materials
and
et al.
Methods
The tissuesused were retrieved from the archives of the Department of Veterinary Pathology of the University of Zurich. They originated from 10 dogs and 10 cats with intestinal lesionssuggestiveof parvovirus enteritis. Small intestine was available from all animals, lymph nodes from two dogs and two cats, and spleenfrom one dog. All thesetissueshad been fixed in formalin and embeddedin paraffin wax a few days later. Tissue sections were mounted on organosilan treated glassslides (Rentrop, Knapp, Winter and Schweizer, 1986). Sections from myocardium, previously shown to contain canine parvovirus nucleic acid by in situ hybridization (Waldvogel, Hassam, Weilenmann, Tratschin, Siegl, Hanichen, Briner and Pospischil, 1991), served as a positive control. To generate the probe, we obtained a cloned genomic DNA of canine parvovirus inserted in pBR322, designated pNCPV2, and consisting of the entire VPl- and VPP-coding sequences(Reed, Jones and Miller, 1988). This plasmid wasdigested with PvuII and HindIII, resulting in four fragments: a 660bp and a 1300bp DNA fragment both containing CPV genome sequencesonly, a 2620bp fragment, containing both pBR322 and CPV sequences,and a 2320bp fragment containing only pBR322 DNA. The restriction products were separated by gel electrophoresis and the 660bp and 1300bp fragments isolated from the gel with the Geneclean II Kit (BlO 101, La Jolla, CA, U.S.A.) and utilized together as a probe for in situ hybridization. Labelling with 35S-dATP (Amersham, U.K.) or with Biotin-1 I-dUTP was performed with a random primed labelling kit (Boehringer Mannheim GmbH, Germany). Becausethere is a homology of at least 98 per cent between the nucleotide sequencesof canine and feline parvoviruses in the genome region coding for VP-l and VP-2 (Parrish, Aquadro and Carmichael, 1988; Reed et al., 1988), we decided to use this probe for in situ hybridization both in canine and feline tissues. Our protocol for in situ hybridization with biotinylated probes has been described previously (Waldvogel et al., 1991). Approximately 6 ng of the biotinylated probe was usedin 20 p1 hybridization solution per slide. For the 35S-labelledprobe, pretreatment and hybridization was also carried out by the above mentioned protocol, with approximately 300 000 cpm per section. Post-hybridization washing was more stringent and included a brief rinse in 1 x PBS, washing on a shaker platform in freshly prepared 10mM dithiotreitol (DTT) in 2 x SSC at room temperature for 10 min, 2 x SSC at 37°C three times for 20 min, 1 x SSC at 37°C for 30 min and 0.1 x SSC at 37°C for 30 min. For autoradiography, the slideswere dehydrated in graded ethanol and 0.3 M NH,Ac, air-dried, dipped in Ilford nuclear track emulsion (diluted 1 in 1 with 0.6 M NH.+Ac), air-dried and exposedfor 5 to 6 days at 4°C in a light-proof box. The slideswere then developed in Kodak D19 developer (diluted 1 in 1 with water), fixed in Kodak fixer, all at 14°C and stained with haemalum and eosin. Biotinylated pBR322 and 35S-labelledh DNA were usedasnegative control probes. For immunohistochemistry by the peroxidase-antiperoxidase (PAP) method, the sectionswere dewaxed and digested in 0.1 per cent pronase XXVII (Sigma Chemie, 9470 Buchs, Switzerland) for 1 min at 37°C. Parvovirus antigen was demonstrated with a polyclonal goat antibody against feline parvovirus (generousgift from Prof. Dr H. Lutz, Clinic for Veterinary Internal Medicine, University of Zurich, Switzerland). This primary antibody was applied at a dilution of 1 in 100in an overnight incubation at room temperature. Visualization was achieved with the DAK0 PAP KITTM goat (DAK0 A/S, Glostrup, Denmark). Counterstaining wasdone with haematoxylin for 1 min. Results
The intestines showed various degrees of autolysis ranging from loss of the villous epithelium (but otherwise preserved mucosa) to the disappearance of the nucleus of the cells in the lamina propria mucosae. Nevertheless, parvovir-
Diagnosis
of parvovirus
by hybridization
143
us-specific DNA reacting with the CPV probe could be demonstrated in the intestines of all dogs and nine of 10 cats by in situ hybridization (Table 1, Fig. 1). Although viral DNA could be demonstrated in as many tissue samples using the biotinylated probe as by using the 35S-labelled probe, the latter yielded a much higher signal strength per cell and detected a higher number of infected cells per specimen. This was most conspicuous in the centre of lymph follicles in Peyer’s patches or lymph nodes, where an intense signal was often generated by the 35S-labelled probe (Fig. 2), although no viral nucleic acid could be demonstrated by in situ hybridization with non-radioactively labelled probes. On the other hand, the biotinylated probe had a much better signal resolution and allowed the more accurate cellular localization of the signal: only with this probe was it possible to recognize that not only the nucleus, but also the cytoplasm of enterocytes and mononuclear leucocytes in Peyer’s patches contained parvoviral nucleic acid (Fig. 3). Parvoviral antigen was not always detected in intestines where nucleic acid was present (Table 1). Minimal non-specific staining, limited to cell membranes in adipose tissue and to intestinal content, was observed with the biotinylated parvovirus as well as the biotinylated pBR322 probe. Hybridization with 35S-labelled h DNA remained negative. Discussion
Using a CPV-specific DNA probe, parvoviral nucleic acid could be demonstrated in all dogs and nine of 10 cats with lesions considered to be typical of parvovirus enteritis. The morphological changes of the intestines therefore allow an aetiological diagnosis with a high degree of reliability. As anticipated, the CPV probe also hybridized to feline parvovirus. Intestinal lesions, indistinguishable from parvovirus enteritis, might be associated with FeLV and in our study in one cat, neither viral nucleic acid nor viral antigen could ..be demonstrated. However, the average age of cats affected by FeLV enteritis is 2.5 years (Reinacher, 1987). The age of this cat was not known, but based on records of its body weight and crown rump length, it was judged to be juvenile. Furthermore, there were no lesions indicative of a FeLV infection in other available organs (lymph nodes, lung). FeLV, therefore, was an unlikely explanation for the intestinal lesions. The inability to detect parvovirus might, therefore, have been due to the virus having already been eliminated or at least reduced below detection level by the time the animal died. An involvement of some other, as yet unknown, agent cannot be ruled out. Although parvoviral DNA replication takes place within the nucleus (Berns, 1990) and the staining was strictly limited to the nucleus of myocardial cells in parvovirus myocarditis (Waldvogel et al., 1991), not only viral antigen but also viral nucleic acid was found in the cytoplasm of intestinal cells and mononuclear leucocytes of Peyer’s patches and in lymph nodes. This may be due to hybridization to viral mRNA present in the cytoplasm or may indicate the diffusion of virus and viral components into cytoplasm after the breakdown of
144
Fig.
A. S. Waldvogel
I.
In situ hybridization with the biotinylated acid within cells of an autolysed intestine.
et
al.
probe demonstrates the presence of parvovirus Haemalum counterstain. Bar = 50 bm.
nucleic
Fig. 2.
In situ hybridization with the 35S-labelled probe reveals a strong signal in the centre of lymph follicles in Peyer’s patches, indicating the presence of virus. With the biotinylated probe it was not possible to localize CPV in this tissue. HE counterstain. Bar= 300 pm.
Fig.
In situ hybridization with the biotinylated probe shows parvoviral nucleic nucleus, but also in the cytoplasm of infected cells (arrow). No counterstain.
3.
acid, not only Bar= 30 pm.
in the
Diagnosis Sensitivity
of parvovirus
Table of immunohistochemistry
1 and
in situ
hybridization
Assay
SHUitiUity*
Immunohistochemistry In situ hybridization
“3 probe
In situ hybridization
biotinylated
*Sensitivity: number intestines tested.
of intestines
145
by hybridization
probe where
parvovirus
Canine intestines
Feline intestines
7/10
S/l0
lo/lo
S/IO
lo/lo
s/10
was identified
divided
by the number
of suspect
the nuclear membrane in dead cells, particularly in the enterocytes, or may indicate phagocytosis of viruses in lymphoid organs. In situ hybridization with biotinylated probes for the detection of parvovirus proved to be somewhat more sensitive, but not more time-consuming or labour-intensive than immunohistochemistry. Furthermore, we have a welldefined range of CPV probes with known specificity and a good control over the stringency of the hybridization reaction. Because of the higher sensitivity of in situ hybridization with the radioactive probe, this probe is more suitable for studying the extent of viral distribution within an animal. The biotinylated probe, however, proved to be sensitive enough to confirm the aetiology in the intestines we examined. Our experiments were performed only in animals with strongly suspected parvovirus enteritis. To evaluate further the efficacy of this assay, it will be necessary to examine a larger series of enteritis specimens and correlate the findings with those obtained by routine histological diagnosis. References
Appel, M. and Parrish, C. R. (1987). Canine parvovirus type 2. In Viws Infections of Carnivores. M. J. Appel, Ed., Elsevier Science PublishersB. V., Amsterdam, pp. 69-92. Berns, K. I. (1990). Parvoviridae and their replication. In Fields Virology, Vol. 2, 2nd Edit. B. N. Fields and D. M. Knipe, Eds, Raven Press,New York, p . 1743-1763. Barker, I. K. and vanDreume1, A. A. (1985). The alimentary system. Pn Pathology of Domestic Animals, Vol. 2, 3rd Edit. K. V. F. Jubb, P. C. Kennedy and N. Palmer, Eds, Academic Press,Inc., Orlando, pp. l-237. Parrish, C. R., Aquadro, C. F. and Carmichael, L. E. (1988). Canine host range and a specific epitope map along with variant sequencesin the capsid protein gene of canine parvovirus and related feline, mink, and raccoon parvoviruses. Virology, 166,293-307.
Pedersen,N. C. (1987). Feline panleukopenia virus. In Virus Znfections of Carnivores. M. J. Appel, Ed., Elsevier Science Publishers B. V., Amsterdam, pp. 247-254. Reed, A. P., Jones, E. V. and Miller, T. J. (1988). Nucleotide sequenceand genome organization of canine parvovirus. Journal of Virology, 62, 26G276. Reinacher, M. (1987). Feline leukemia virus-associated enteritis-a condition with features of feline panleukopenia. Veterinary Pathology, 24, I+. Rentrop, M., Knapp, B., Winter, H. and Schweizer, J. (1986). Aminoalkylsilanetreated glassslidesas support for in situ hybridization of keratin cDNAs to frozen tissuesections under varying fixation and pretreatment conditions. Histochemical Journal,
18, 271-276.
A. S. Waldvogel
146
et al.
Waldvogel, A. S., Hassam,S., Weilenmann, R., Tratschin, J. D., Siegl, G., Hgnichen, T., Briner, J. and Pospischil,A. (1991). Retrospective study of myocardial canine parvovirus infection by in situ hybridization. Journal of Veterinary Medicine Series B, 38, 353-357.
1
Received, February 13th, 1992 Accepted, March 3Oth, 1992