J Vet Diagn Invest 4: 19-22 (1992)
Fatal disease in nursing puppies associated with minute virus of canines Lenn R. Harrison, Eloise L. Styer, Alfred R. Pursell, Leland E. Carmichael, Jerome C. Nietfeld Abstract. Thirteen cases of a previously undescribed parvoviral infection affecting puppies ranging in age from 5 to 21 days is described. The cases were originally thought to represent an unusual pathologic manifestation of canine parvovirus-2 (CPV-2) infection. However, failure to confirm CPV-2 infection in any of the cases suggested a different parvovirus was involved. Minute virus of canines (MVC) was subsequently isolated from a case by using the Walter Reed Canine Cell Line, the only cell line which will support the growth of MVC. The pathologic and virologic findings for these 13 cases are described in this report.
Two distinct parvoviruses are known to infect dogs: minute virus of canines (MVC, canine parvovirus-l), first isolated in 1967 from the feces of normal dogs,1 and canine parvovirus-2 (CPV-2), which has caused a worldwide pandemic of enteritis and myocarditis since 1978.7 The two viruses differ in host cell range, spectra of hemagglutination, genomic properties, and antigenicity 1,2,9 Disease caused by CPV-2 is now well documented, although infection with MVC has received minimal study. MVC can replicate and cause pathologic changes in oronasally exposed neonatal pups9 and can cause transplacental infections with embryo resorptions, especially in dams given inoculations prior to gestation day 30.3 Mild diarrhea in dogs that previously suffered confirmed CPV-2 infection9 and a nonfatal transient diarrhea in young pups has been attributed to MVC.4 Over the past 7 years, 13 cases of a parvoviral infection in neonatal puppies that were negative for CPV-2 infection were studied. MVC was isolated from 1 case using the only continuous cell line known to support growth of this virus. Pathologic, virologic, and other laboratory results obtained from these 13 cases are reported.
mitted as fixed tissues taken at necropsy by the referring veterinarian. The signalment, clinical signs and initial reported morbidity and mortality for each case are listed in Table 1. The two litters represented by cases 3-6 were caesarean derived. Cases 1-6, 11, and 13 were single litter house pets, and cases 7-10 and 12 were from kennels. All of the bitches had received routine immunizations against the common canine viral pathogens: canine distemper virus, canine adenovirus, and CPV-2. Pathologic studies. A complete necropsy was done on all twelve cadavers, and specimens collected included brain, lung, liver, kidney, spleen, pancreas, stomach, urinary bladder, small intestine, and colon. Formalin-fixed 5-µm paraffinembedded sections were stained with hematoxylin and eosin (HE) for histologic examination. Electron microscopic examinations. Negative-contrast electron microscope (NCEM) studies were done on intestinal content samples of cases 2-13. The samples were prepared for NCEM by a method routinely used for feces. The samples were diluted approximately 9:1 in distilled water and briefly centrifuged to remove the large and heavy debris. The supernatant was poured off and concentrated by centrifugation for 60 min at 12,200 x g. The resultant pellet was resuspended in water and negatively stained with an equal volume of 1.5% solution of phosphate tungstic acid at pH 6.5. Transmission EM studies were done on formalin-fixed sections of the jejunum of cases 1, 2, 12, and 13 and of the myocardium of case 2. The selected tissue areas were cut in 1-2-mm cubes, secondarily fixed in osmium tetroxide, dehydrated, and embedded in Spurr’s epoxy resin. The negatively stained samples and the ultrathin tissue sections were examined with an RCA EMU-4 electron microscope. Bacteriologic examinations. Lung, liver, kidney, spleen, and intestinal contents were inoculated onto 5% bovine blood agar. Standard methods were used for isolation attempts and identification. Immunofluorescence assays (FAT). Sections of small intestine from cases 2-13 were blocked with OCT compound, sectioned at 5 µm in a cryostat, stained with fluoresceinconjugated hyperimmune CPV-2 antisera, and examined by
Materials and methods Case histories. Thirteen cases of a canine parvoviral infection that had similar pathologic intestinal features not previously associated with CPV-2 were studied. Twelve of these cases were submitted as cadavers, and 1 case was subFrom the University of Georgia, Veterinary Diagnostic and Investigational Laboratory, Tifton, GA 31793 (Harrison, Styer, Pursell), the Baker Institute for Animal Health, New York State College of Veterinary Medicine, Ithaca, NY 14853 (Carmichael), and the University of Georgia, Veterinary Diagnostic Assistance Laboratory, College of Veterinary Medicine, Athens, GA 30602 (Nietfeld). Received for publication October 8, 1991. 19
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Harrison et al.
20 Table 1.
Suspected cases of minute virus of canines.
epifluorescence. Crystal-cut sections of the small intestine of case 13 were subjected to indirect fluorescent antibody (IFA) staining with hyperimmune rabbit anti-MVC serum and examined by epifluorescence. Virus isolation. Virus isolation attempts included inoculation of intestinal contents from cases 2-9 on Maden Darby canine kidney cultures, cases 4-9 on Crandell feline kidney cell line cultures and cases 6-9 on A-72 cell line cultures, and cases 10 and 13 on Walter Reed canine (WRC) cell line cultures. IFA staining was done on tissue cultures of case 10 using previously prepared hyperimmune rabbit anti-MVC serum.
with exudate in airways of two littermates and mineralized focal to diffuse areas of myocardial necrosis with giant cells in case 2. No lesions were seen in brain, liver, kidney, spleen, pancreas, colon, or urinary bladder of any of the puppies.
Results Gross findings
A small amount of fecal staining was noted on the tail and the hairs around the anus of most of the pups. The contents of the small intestine were liquid, and the colonic contents varied from liquid to pasty soft and pale to bright yellow. The myocardium of case 2 had large tan to pale gray streaks and irregular areas deep within the myocardium. One pup had a mild Ancylostoma caninum infection. Histologic findings
All 13 puppies had large intranuclear epithelial inclusions at the tips of the villi in the jejunum (Figs. 1, 2). These inclusions were eosinophilic and often appeared to fill the nuclei. Other intestinal changes noted included crypt epithelial hyperplasia and single-cell necrosis of crypt epithelial cells. These changes also appeared to be limited to the duodenum and jejunum. The other pathologic feature common to all of the specimens was moderate to marked depletion and/or Figure 1. Photomicrograph of section of the jejunum of a puppy necrosis of the lymphoid cells of Peyer’s patches. Le- with MVC. Note tip area of villi showing numerous intranuclear sions seen in other tissues included severe pneumonitis inclusions characteristic of all cases. HE.
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MVC infection of puppies
Figure 2. Enlarged area of Fig. 1 demonstrating the swollen nuclei with the large intranuclear inclusions. HE.
Electron microscopy
The epithelial cells at the tips of the villi contained aggregates of electron-dense material that contained numerous small virus particles approximately 20 nm in size (Figs. 3, 4). The negatively stained intestinal contents also had viral particles approximately 20 nm in size that are remarkably similar to the viral particles in the nuclei (Fig. 5). No viral particles were seen in the myocytes of case 2. Bacteriologic findings
A pure growth of Escherchia coli was recovered from intestinal cultures of cases 3 and 7 and from the lung of case 13. Intestinal cultures from the other cases yielded a variety of bacteria. Culture of the other internal organs of all 12 cadavers yielded no significant growth. Virologic results
The FAT for CPV-2 was negative in all 12 cases. Selective attempts to isolate a virus from the intestinal contents and other tissues of cases 2-9 and 13 were unsuccessful. Cytopathic changes were observed in the WRC cells inoculated with intestinal contents from case 10. The presence of MVC in the WRC cell culture was confirmed by IFA staining using the hyperimmune
21
Figure 3. Electronmicrograph of the intranuclear inclusions in a section of jejunum from a puppy with MVC. The electron-dense material is typical of the inclusions. Bar equals 10 µm.
rabbit anti-MVC serum. The MVC IFA done on the sections of the small intestine of case 13 was positive. Discussion These cases were originally thought to represent an unusual pathologic manifestation of CPV-2 infection. However, the consistently negative FAT results and the failure to isolate CPV-2 in appropriate tissue culture systems suggested that another parvoviral infection was involved. The isolation of MVC from case 10 suggests that the prior failures to identify the parvovirus were not due the failure of the test procedures but due to infection with an antigenically unrelated virus. Failure to isolate MVC from the earlier cases was probably due to the use of tissue culture lines that will not support the growth of MVC. Only the WRC cell line is known to support growth of MCV,1 and isolation of this virus is rare. The positive IFA for MVC on case 13 also confirms that MVC was the virus affecting these puppies. The pathologic changes seen in these cases are different from those described for CPV-2 infection in neonatal pups. 2,8,10 Enteritis due to CPV-2 in puppies less than 3 weeks of age results in loss of normal ar-
Downloaded from vdi.sagepub.com at University of Aberdeen on March 21, 2015
Harrison et al.
Figure 4. Electronmicrograph of the intranuclear inclusions in a section of jejunum from a puppy with MVC. Numerous viral particles are present. Bar equals 100 nm.
chitecture, collapse of the mucosa, blunting and fusion of villi, and necrosis of villus epithelium. By comparison, the 13 cases in the present study retained normal intestinal architecture. Crypt epithelial cell necrosis was minimal, generally affecting only a few cells located together. The pattern of hyperplasia was different from that observed in CPV-2 enteritis in older pups. The villus epithelial hyperplasia was generalized and consistently uniform from villus to villus. In experimental and confirmed fatal natural CPV-2 cases, there are few crypts that are lined by hyperplastic epithelium generally surrounded by collapsed mucosa. The only experimental pathogenicity study done on neonatal puppies with MVC demonstrated pathologic changes in lymphoid tissue and duodenal crypts.9 The intestinal lesions in experimental MVC consisted of crypt epithelial single-cell necrosis and large pale intranuclear inclusion bodies. The inclusion bodies, although seen only in crypts, resembled those in the tips of the villi in the natural cases. Lymphoid depletion and necrosis observed in the experimental cases were similar to those in the natural cases. Although thymic lesions were seen in the experimentally infected pups, no comparison can be made with the natural cases because thymus tissue was not taken from any of them.
Figure 5. Electronmicrograph of the negatively stained intestinal contents of a puppy with MVC. The viral particles are 20 nm in size and have morphologic features typical of parvoviruses. Bar equals 200 nm.
MVC has been considered a nonpathogenic parvovirus of dogs; however, it should be considered a pathogen for puppies, although infection is generally mild and rarely fatal. References 1. Binn LN, Lazar EC, Eddy GA, et al.: 1970, Recovery and characterization of a minute virus of canines. Infect Immunity 1:503508. 2. Carmichael LE, Joubert JC, Pollock RVH: 1980, Hemagglutination by canine parvovirus: serologic studies and diagnostic applications. Am J Vet Res 41:784-791. 3. Carmichael LE, Schlafer DH, Hashimoto A: 1991, Pathogenicity of minute virus of canines (MVC) for the canine fetus. Cornell Vet 81:151-171. 4. Eugster AK, Nairn C: 1977, Diarrhea in puppies: parvoviruslike particles demonstrated in their feces. Southwest Vet 30:5960. 5. Lenghaus C, Studdert MJ: 1982, Generalized parvovirus disease in neonatal pups. J Am Vet Med Assoc 181:41-45. 6. Macartney L, Parrish CR, Binn LN, et al.: 1988, Characterization of minute virus of canines (MVC) and its pathogenicity for pups. Cornell Vet 78:131-145. 7. McArdaragh JP, Eustis SL, Nelson DT, et al.: 1982, Experimental infection of conventional dogs with canine parvovirus. Am J Vet Res 43:693-696. 8. McCandlish IAP, Thompson H, Fisher EW, et al.: 1981, Canine parvovirus infection. In Pract (May):5-14. 9. Pollock RVH: 1982, Experimental canine parvovirus infection in dogs. Compend Contin Ed 72: 103-119.
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Fatal disease in nursing puppies associated with minute virus of canines Lenn R. Harrison, Eloise L. Styer, Alfred R. Pursell, Leland E. Carmichael, Jerome C. Nietfeld Abstract. Thirteen cases of a previously undescribed parvoviral infection affecting puppies ranging in age from 5 to 21 days is described. The cases were originally thought to represent an unusual pathologic manifestation of canine parvovirus-2 (CPV-2) infection. However, failure to confirm CPV-2 infection in any of the cases suggested a different parvovirus was involved. Minute virus of canines (MVC) was subsequently isolated from a case by using the Walter Reed Canine Cell Line, the only cell line which will support the growth of MVC. The pathologic and virologic findings for these 13 cases are described in this report.
Two distinct parvoviruses are known to infect dogs: minute virus of canines (MVC, canine parvovirus-l), first isolated in 1967 from the feces of normal dogs,1 and canine parvovirus-2 (CPV-2), which has caused a worldwide pandemic of enteritis and myocarditis since 1978.7 The two viruses differ in host cell range, spectra of hemagglutination, genomic properties, and antigenicity 1,2,9 Disease caused by CPV-2 is now well documented, although infection with MVC has received minimal study. MVC can replicate and cause pathologic changes in oronasally exposed neonatal pups9 and can cause transplacental infections with embryo resorptions, especially in dams given inoculations prior to gestation day 30.3 Mild diarrhea in dogs that previously suffered confirmed CPV-2 infection9 and a nonfatal transient diarrhea in young pups has been attributed to MVC.4 Over the past 7 years, 13 cases of a parvoviral infection in neonatal puppies that were negative for CPV-2 infection were studied. MVC was isolated from 1 case using the only continuous cell line known to support growth of this virus. Pathologic, virologic, and other laboratory results obtained from these 13 cases are reported.
mitted as fixed tissues taken at necropsy by the referring veterinarian. The signalment, clinical signs and initial reported morbidity and mortality for each case are listed in Table 1. The two litters represented by cases 3-6 were caesarean derived. Cases 1-6, 11, and 13 were single litter house pets, and cases 7-10 and 12 were from kennels. All of the bitches had received routine immunizations against the common canine viral pathogens: canine distemper virus, canine adenovirus, and CPV-2. Pathologic studies. A complete necropsy was done on all twelve cadavers, and specimens collected included brain, lung, liver, kidney, spleen, pancreas, stomach, urinary bladder, small intestine, and colon. Formalin-fixed 5-µm paraffinembedded sections were stained with hematoxylin and eosin (HE) for histologic examination. Electron microscopic examinations. Negative-contrast electron microscope (NCEM) studies were done on intestinal content samples of cases 2-13. The samples were prepared for NCEM by a method routinely used for feces. The samples were diluted approximately 9:1 in distilled water and briefly centrifuged to remove the large and heavy debris. The supernatant was poured off and concentrated by centrifugation for 60 min at 12,200 x g. The resultant pellet was resuspended in water and negatively stained with an equal volume of 1.5% solution of phosphate tungstic acid at pH 6.5. Transmission EM studies were done on formalin-fixed sections of the jejunum of cases 1, 2, 12, and 13 and of the myocardium of case 2. The selected tissue areas were cut in 1-2-mm cubes, secondarily fixed in osmium tetroxide, dehydrated, and embedded in Spurr’s epoxy resin. The negatively stained samples and the ultrathin tissue sections were examined with an RCA EMU-4 electron microscope. Bacteriologic examinations. Lung, liver, kidney, spleen, and intestinal contents were inoculated onto 5% bovine blood agar. Standard methods were used for isolation attempts and identification. Immunofluorescence assays (FAT). Sections of small intestine from cases 2-13 were blocked with OCT compound, sectioned at 5 µm in a cryostat, stained with fluoresceinconjugated hyperimmune CPV-2 antisera, and examined by
Materials and methods Case histories. Thirteen cases of a canine parvoviral infection that had similar pathologic intestinal features not previously associated with CPV-2 were studied. Twelve of these cases were submitted as cadavers, and 1 case was subFrom the University of Georgia, Veterinary Diagnostic and Investigational Laboratory, Tifton, GA 31793 (Harrison, Styer, Pursell), the Baker Institute for Animal Health, New York State College of Veterinary Medicine, Ithaca, NY 14853 (Carmichael), and the University of Georgia, Veterinary Diagnostic Assistance Laboratory, College of Veterinary Medicine, Athens, GA 30602 (Nietfeld). Received for publication October 8, 1991. 19
Downloaded from vdi.sagepub.com at University of Aberdeen on March 21, 2015
Harrison et al.
20 Table 1.
Suspected cases of minute virus of canines.
epifluorescence. Crystal-cut sections of the small intestine of case 13 were subjected to indirect fluorescent antibody (IFA) staining with hyperimmune rabbit anti-MVC serum and examined by epifluorescence. Virus isolation. Virus isolation attempts included inoculation of intestinal contents from cases 2-9 on Maden Darby canine kidney cultures, cases 4-9 on Crandell feline kidney cell line cultures and cases 6-9 on A-72 cell line cultures, and cases 10 and 13 on Walter Reed canine (WRC) cell line cultures. IFA staining was done on tissue cultures of case 10 using previously prepared hyperimmune rabbit anti-MVC serum.
with exudate in airways of two littermates and mineralized focal to diffuse areas of myocardial necrosis with giant cells in case 2. No lesions were seen in brain, liver, kidney, spleen, pancreas, colon, or urinary bladder of any of the puppies.
Results Gross findings
A small amount of fecal staining was noted on the tail and the hairs around the anus of most of the pups. The contents of the small intestine were liquid, and the colonic contents varied from liquid to pasty soft and pale to bright yellow. The myocardium of case 2 had large tan to pale gray streaks and irregular areas deep within the myocardium. One pup had a mild Ancylostoma caninum infection. Histologic findings
All 13 puppies had large intranuclear epithelial inclusions at the tips of the villi in the jejunum (Figs. 1, 2). These inclusions were eosinophilic and often appeared to fill the nuclei. Other intestinal changes noted included crypt epithelial hyperplasia and single-cell necrosis of crypt epithelial cells. These changes also appeared to be limited to the duodenum and jejunum. The other pathologic feature common to all of the specimens was moderate to marked depletion and/or Figure 1. Photomicrograph of section of the jejunum of a puppy necrosis of the lymphoid cells of Peyer’s patches. Le- with MVC. Note tip area of villi showing numerous intranuclear sions seen in other tissues included severe pneumonitis inclusions characteristic of all cases. HE.
Downloaded from vdi.sagepub.com at University of Aberdeen on March 21, 2015
MVC infection of puppies
Figure 2. Enlarged area of Fig. 1 demonstrating the swollen nuclei with the large intranuclear inclusions. HE.
Electron microscopy
The epithelial cells at the tips of the villi contained aggregates of electron-dense material that contained numerous small virus particles approximately 20 nm in size (Figs. 3, 4). The negatively stained intestinal contents also had viral particles approximately 20 nm in size that are remarkably similar to the viral particles in the nuclei (Fig. 5). No viral particles were seen in the myocytes of case 2. Bacteriologic findings
A pure growth of Escherchia coli was recovered from intestinal cultures of cases 3 and 7 and from the lung of case 13. Intestinal cultures from the other cases yielded a variety of bacteria. Culture of the other internal organs of all 12 cadavers yielded no significant growth. Virologic results
The FAT for CPV-2 was negative in all 12 cases. Selective attempts to isolate a virus from the intestinal contents and other tissues of cases 2-9 and 13 were unsuccessful. Cytopathic changes were observed in the WRC cells inoculated with intestinal contents from case 10. The presence of MVC in the WRC cell culture was confirmed by IFA staining using the hyperimmune
21
Figure 3. Electronmicrograph of the intranuclear inclusions in a section of jejunum from a puppy with MVC. The electron-dense material is typical of the inclusions. Bar equals 10 µm.
rabbit anti-MVC serum. The MVC IFA done on the sections of the small intestine of case 13 was positive. Discussion These cases were originally thought to represent an unusual pathologic manifestation of CPV-2 infection. However, the consistently negative FAT results and the failure to isolate CPV-2 in appropriate tissue culture systems suggested that another parvoviral infection was involved. The isolation of MVC from case 10 suggests that the prior failures to identify the parvovirus were not due the failure of the test procedures but due to infection with an antigenically unrelated virus. Failure to isolate MVC from the earlier cases was probably due to the use of tissue culture lines that will not support the growth of MVC. Only the WRC cell line is known to support growth of MCV,1 and isolation of this virus is rare. The positive IFA for MVC on case 13 also confirms that MVC was the virus affecting these puppies. The pathologic changes seen in these cases are different from those described for CPV-2 infection in neonatal pups. 2,8,10 Enteritis due to CPV-2 in puppies less than 3 weeks of age results in loss of normal ar-
Downloaded from vdi.sagepub.com at University of Aberdeen on March 21, 2015
Harrison et al.
Figure 4. Electronmicrograph of the intranuclear inclusions in a section of jejunum from a puppy with MVC. Numerous viral particles are present. Bar equals 100 nm.
chitecture, collapse of the mucosa, blunting and fusion of villi, and necrosis of villus epithelium. By comparison, the 13 cases in the present study retained normal intestinal architecture. Crypt epithelial cell necrosis was minimal, generally affecting only a few cells located together. The pattern of hyperplasia was different from that observed in CPV-2 enteritis in older pups. The villus epithelial hyperplasia was generalized and consistently uniform from villus to villus. In experimental and confirmed fatal natural CPV-2 cases, there are few crypts that are lined by hyperplastic epithelium generally surrounded by collapsed mucosa. The only experimental pathogenicity study done on neonatal puppies with MVC demonstrated pathologic changes in lymphoid tissue and duodenal crypts.9 The intestinal lesions in experimental MVC consisted of crypt epithelial single-cell necrosis and large pale intranuclear inclusion bodies. The inclusion bodies, although seen only in crypts, resembled those in the tips of the villi in the natural cases. Lymphoid depletion and necrosis observed in the experimental cases were similar to those in the natural cases. Although thymic lesions were seen in the experimentally infected pups, no comparison can be made with the natural cases because thymus tissue was not taken from any of them.
Figure 5. Electronmicrograph of the negatively stained intestinal contents of a puppy with MVC. The viral particles are 20 nm in size and have morphologic features typical of parvoviruses. Bar equals 200 nm.
MVC has been considered a nonpathogenic parvovirus of dogs; however, it should be considered a pathogen for puppies, although infection is generally mild and rarely fatal. References 1. Binn LN, Lazar EC, Eddy GA, et al.: 1970, Recovery and characterization of a minute virus of canines. Infect Immunity 1:503508. 2. Carmichael LE, Joubert JC, Pollock RVH: 1980, Hemagglutination by canine parvovirus: serologic studies and diagnostic applications. Am J Vet Res 41:784-791. 3. Carmichael LE, Schlafer DH, Hashimoto A: 1991, Pathogenicity of minute virus of canines (MVC) for the canine fetus. Cornell Vet 81:151-171. 4. Eugster AK, Nairn C: 1977, Diarrhea in puppies: parvoviruslike particles demonstrated in their feces. Southwest Vet 30:5960. 5. Lenghaus C, Studdert MJ: 1982, Generalized parvovirus disease in neonatal pups. J Am Vet Med Assoc 181:41-45. 6. Macartney L, Parrish CR, Binn LN, et al.: 1988, Characterization of minute virus of canines (MVC) and its pathogenicity for pups. Cornell Vet 78:131-145. 7. McArdaragh JP, Eustis SL, Nelson DT, et al.: 1982, Experimental infection of conventional dogs with canine parvovirus. Am J Vet Res 43:693-696. 8. McCandlish IAP, Thompson H, Fisher EW, et al.: 1981, Canine parvovirus infection. In Pract (May):5-14. 9. Pollock RVH: 1982, Experimental canine parvovirus infection in dogs. Compend Contin Ed 72: 103-119.
Downloaded from vdi.sagepub.com at University of Aberdeen on March 21, 2015
