Vet. Pathol. 17: 589-599 (1980) Canine Parvoviral Disease: Experimental Reproduction of the Enteric Form with a Parvovirus Isolated from a Case of Myocarditis W. F. ROBINSON, G. E. WILCOX and R. L. P. FLOWER Division of Veterinary Biology, School of Veterinary Studies, Murdoch University, Murdoch, Western Australia, Australia Abstract. Five 7-week-old pups and four 4-week-old pups, all seronegative to canine parvovirus, were inoculated intravenously with 1000 haemagglutinating units of canine parvovirus originally isolated from the myocardium of a dog with naturally occurring myocarditis. After three days, pups in both litters became pyrexic, anorectic and depressed, with vomiting and diarrhoea. The 4-week-old pups were killed on day 4, and the 7-week-old pups died or were killed on day 5 post-inoculation. Histological examination showed degeneration and necrosis of intestinal crypt epithelial cells and villous atrophy. All pups had thymic atrophy caused by lymphoid depletion. Peyer s patches, mesenteric lymph node and spleen also had lymphoid depletion. Lymphoid necrosis was present occasionally in these tissues. In the bone marrow, granulocytes and granulocyte and erythroid precursors were depleted. Amphophilic intranuclear inclusion bodies were abundant in crypt epithelial nuclei, less so in myocardial nuclei. Canine parvovirus was isolated from intestinal contents, thymus, spleen, mesenteric lymph node and liver in most pups, but not from kidney or myocardium. Two new viral diseases of dogs, parvoviral enteritis [5, 9, 12, 171 and parvoviral myocarditis [7, 8,9, 10, 131 have been described. Clinical signs in dogs with parvoviral enteritis include anorexia, pyrexia, vomiting and diarrhoea. Panleucopenia often is present in the early phase. The characteristic lesion is necrosis of rapidly dividing cells in bone marrow, intestinal crypt epithelium and lymphoid tissues. Intranuclear inclusion bodies are found in intestinal crypt epithelial cells. Canine parvoviral enteritis mimics the classical form of feline panleucopenia in many respects [ 151. Canine parvoviral myocarditis occurs only in pups 3 to 8 weeks old. Clinical signs are severe ventricular arrhythmias [lo, 13, 191, resulting in sudden death or death following a brief period of restlessness and dyspnoea. Subclinical ventricular arrhythmias may be detected by electrocardiography in affected animals [ 191. The primary lesion is multifocal myocardial necrosis, with an inflammatory reaction of variable intensity. Large intranuclear inclusion bodies are found in myocardial cells and are pathognomic for the disease. Although an apparently identical parvovirus has been isolated from naturally occurring cases of both diseases [5, 6, 11, 17, 181, clinicopathologic overlap between 589
590 Robinson, Wilcox and Flower them has not been reported. It has been suggested that the form the disease takes is a function of the age of the puppy at the time of infection [7, 201, but pathogenesis remains to be defined. We report the reproduction of the enteric form of the disease with a parvovirus isolated from the myocardium of a dog with naturally occurring myocarditis. Virus Materials and Methods Canine parvovirus isolate MV54 isolated from a 4-week-old puppy with histologically confirmed myocarditis [ 181 was inoculated onto 50% to 60% confluent Crandell feline kidney cells with Eagle s minimal essential medium containing 10% foetal calf serum with 100 international units/ml penicillin, neomycin (50 pg/ml) and amphotericin B (2.5 pg/ml). The medium was replaced 72 hours later with Eagle s minimal essential medium containing 2% foetal calf serum and antibiotics. After another 72 hours incubation, the cultures were frozen and thawed, sonicated and centrifuged at 1000 X gravity for 20 minutes at room temperature. The supernatant was aspirated and tested for haemagglutinating activity as described [ 181. An uninoculated culture of Crandell feline kidney cells was treated identically. Experimental animals A 7-week-old litter of seven crossbred puppies and a 4-week-old litter of six were housed in separate rooms for one week of acclimatization and observation prior to inoculation. Serum samples were taken and tested for haemagglutinating antibodies against canine parvovirus as described [MI. Following the period of acclimatization and observation, two puppies from each litter were removed and housed in a separate building as controls. Experimental design Nine puppies, five from the 7-week-old group (litter 1) and four from the 4-week-old group (litter 2) were inoculated intravenously with 5 ml of the supernatant containing canine parvovirus with a haemagglutination titre of 1000. The two remaining puppies from each litter were inoculated intravenously with 5 ml of the supernatant from the control culture. All were observed daily and rectal temperatures were taken. A complete blood count was done on days 3 and 4 post-inoculation. Serum samples were taken on day 4 post-inoculation or at death. Puppies either died or were killed by an intravenous injection of pentobarbitone sodium. Necropsy procedure Complete necropsies were done on all puppies. Samples for histologic examinations were taken from the following tissues and fixed in 10% neutral buffered formalin: tongue, oesophagus, stomach, duodenum, jejunum, ileum, colon, thymus, retropharyngeal and mesenteric lymph nodes, tonsil, spleen, adrenal, thyroid and parathyroid, costochondral junction, brain, pituitary, eyes, heart, lung, pancreas, parotid salivary gland and bone marrow. After routine processing, tissue samples embedded in paraffin were sectioned at 6 pm and stained with haematoxylin and eosin (HE). Samples of heart, thymus, spleen, mesenteric lymph node, liver, kidney and intestinal contents were taken aseptically and stored at -70 C. In addition, l-mm3 pieces of duodenum, jejunum and ileum from all puppies were fixed in 3% glutaraldehyde in 0.1 mol/liter phosphate buffer ph 7.4. These blocks were then fmed in 1% osmium tetroxide in the same buffer and processed for epon embedding. Sections for light microscopy were cut 1 pm thick and stained with toluidine blue. Thin sections were cut from selected areas, stained
Canine Parvoviral Disease 591 T "4 0 CONTROL (L) A LITER1 (5) 37 LmER 2 [LI 0 1 2 3 L DAYS-POST INOCULATION Fig. 1: Mean +- standard deviation of rectal temperature of two inoculated litters and 4 control pups. Rise in rectal temperature in two inoculated groups on days 3 and 4 after infection. Number of pups per group in brackets. with lead citrate and uranyl acetate and examined in an electron microscope. Bone marrow smears were made for cytologic examination. Viral isolation Tissues collected at necropsy were minced and homogenized in tissue grinders with Eagle's minimal essential medium to approximately a 10% suspension, centrifuged at 1000 X gravity for 15 minutes, and the supernatant collected. Approximately 0.1 ml of each tissue supernatant was inoculated onto 50% to 60% confluent Crandell feline kidney cells and allowed to adsorb for two hours at 37 C. Eagle's minimal essential medium containing 2% foetal calf serum with antibiotics was added and changed 24 hours later to Eagle's minimal essential medium containing 2% foetal calf serum with antibiotics. Cell cultures were incubated for four days. The culture media negative for haemagglutination were passaged. This procedure was repeated for a third passage. Samples were considered negative if haemagglutination was not evident after three passages. Clinical findings Results Preinoculation serum samples from all puppies had haemagglutination inhibition titres of -= 10. This indicated lack of previous exposure to canine parvovirus. From day 0 to day 2 post-inoculation, puppies in both experimental litters remained clinically normal. On day 3, they were pyrexic (fig. 1) and depressed. Mild diarrhoea was present in litter 2. Four puppies in litter 1 were lymphopenic. On day 4, both
592 Robinson, Wilcox and Flower Table I. Total and differential white-cell counts from dogs with experimental canine Darvovirus infection (mean f standard deviation) Days Litter 1 Litter 2 postinocu- Inocu- Inocu- Controls (2) lation lated (5) lated (4) Controls (2j Total white cell 3 15,849 14,350 15,900 16,050 count per pl +4,125 f 1,060 f5,500 f3,606 4 3,699 12,500 14,350 15,605 +3,581 k141 f4,5 11 +4,392 Total neutrophil 3 count per pl 4 Total lymphocyte 3 count per pl 4 14,531 +3,569 2,565 f2.904 983 f5 17 749 +616 6,573 +270 6,807 f895 6,888 +509 4,879 k762 12,080 +3,332 10,929 +3,775 1,926 +783 1,095 f502 10,146 +3,275 10,737 22,456 3,598 f698 2,765 f1.435 litters were pyrexic, anorexic, depressed and vomiting with litter 1 more severely affected. A pink-white diarrhoea containing red flecks of blood also was present. Four puppies in litter 1 were panleucopenic and three puppies in litter 2 were lymphopenic (table I). Litter 2 was killed on the morning of day 4. By the afternoon of day 4, puppies in litter 1 had deteriorated rapidly and were vomiting, anorexic, markedly depressed and diarrhoeic. The diarrhoea was dark red. On day 5, three puppies in litter 2 were found dead and one was moribund and soon died. The remaining puppy was depressed, dehydrated and vomited frequently, and was killed. Gross post-mortem fmdings Lesions were similar in all puppies in litter 1. The puppies were dehydrated and thin with little subcutaneous fat. Serosal congestion and a moderate number of petechiae were present from the duodenum to the ileocaecal junction, most severely in the jejunum. The small intestinal was dilated and atonic, and contained much clear yellow fluid. In some cases, the fluid was reddish in the jejunum, becoming darker brown in the ileum. The colon was usually empty. The intestinal mucosa usually appeared normal, but in some, thin strands of fibrin adhered to the surface. The thymus was always very small and consisted of a thin light brown band of tissue. The bone marrow was macroscopically normal. The pups in litter 2 were thin and dehydrated, and showed muscle wasting. One puppy had numerous serosal petechiae of the ileum. The mucosa in this pup was covered with fibrin strands. The remaining puppies had semi-fluid whitish-pink intestinal contents. The thymus in all puppies was small.
Histology and electron microscopy Canine Parvoviral Disease 593 Lesions in both litters were similar, consisting of degeneration, necrosis and hyperplasia of the epithelium of the crypts from the pylorus to the rectum; the duodenum and jejunum were affected most severely. In many crypts the lesion was focal. Affected crypts were dilated and contained cellular debris in the lumen. A varied proportion of surviving crypt epithelial cells were flattened. Many epithelial cells contained large intranuclear basophilic inclusion bodies, often surrounded by eosinophilic material (fig. 2). Villous atrophy and fusion were prominent in many pups, particularly those with the more severe crypt lesions. Goblet cell metaplasia occasionally was present in the duodenum and there was involution of Peyer's patches with loss of lymphocytes from germinal centres (fig. 3). Coccidia were present in sections of ileum from litter 2. Electron microscopic examination of affected crypts showed desquamated cells in the lumen. The cells lining the crypt had moderately dilated rough endoplasmic reticulum; some nuclei contained inclusion bodies (fig. 4) characterized by chromatin margination and abundant particles 20 nm in diameter, which were interpreted as parvovirus virions. There was loss of cortical lymphocytes in the thymus, but small lymphocytes were still present, although reduced in number, in the medulla. The number of thymic epithelial cells appeared greater because of this loss. Hassal's corpuscles also were larger. Some thymuses were diffusely congested. Periarteriolar lymphoid depletion and lymphocyte necrosis were present in the spleen. The paracortical and medullary areas of mesenteric and retropharyngeal lymph nodes were depleted of lymphocytes; there was also an increase in the number of fixed macrophages lining the sinusoids (fig. 5). Committed myeloid series and early erythroid series were lost from the bone marrow (fig. 6, 7). Megakaryocytes were normal, as were the later stages of the erythroid series. Large basophilic rectangular intranuclear inclusion bodies were found in a few myocardial cells. No fibre degeneration or inflammatory cells were present. Virology The results of virus isolation are shown in table 11. Canine parvovirus was isolated consistently from intestinal contents, thymus, spleen, liver and mesenteric lymph node from litter 2, but only from intestinal contents in litter 1. Serology All puppies were sero-negative prior to inoculation. Terminal serum samples from puppies inoculated with canine parvovirus taken on day 4 post-inoculation from litter 2 were negative, whereas litter 1 had haemagglutination titres from 320 to 1280. The four control puppies remained negative.
594 Robinson, Wilcox and Flower Fig. 2 Dilated duodenal crypt (bottom). Inclusion bodies correspond to eosinophilic inclusions seen with HE (broad arrow). More basophilic inclusion bodies (narrow arrow) associated with marginated chromatin. One-pm section. Toluidine blue. Fig. 3 Terminal ileum: crypt dilation, villous atrophy and fusion. Depletion of lymphocytes in Peyer s patches (arrow) and lymphatic diluation. HE.
Canine Parvoviral Disease 595 Discussion The original object of this study was to reproduce experimentally canine parvoviral myocarditis, but the most interesting result was the induction instead of enteric parvoviral disease. The critical factor in the pathogenesis of parvoviral infections is actively-replicating DNA, and thus a population of rapidly dividing cells [21]. The vulnerability of any particular tissue could be expected to be different in the prenatal, neonatal and adult animal, with periods of transition occurring at different times for different tissues. This is well demonstrated by the differing clinicopathologic features of parvoviral disease in neonatal kittens and adults [3,4, 151. In neonatal kittens, the bone marrow and external granular layer of the cerebellum are most affected, while intestinal lesions are minimal [4]. It has been shown that the parvoviral isolate used in this experiment (MV54) has a close serologic relationship to an isolate recovered from a case of canine parvoviral enteritis [18]. It was suggested that the two isolates were identical. The findings in our study substantiate those virologic findings and emphasize the possibility that the clinicopathologic manifestations of canine parvoviral infection are related to age at infection, myocarditis occurring when pups are infected at a younger age than that at which enteritis occurs. Further indications are seen by comparing the difference in severity of the disease between the two litters. The younger litter (4 weeks) had neither as marked clinical signs nor as severe gross pathological changes as the older litter (7 weeks). Indeed, by the fourth day postinoculation, the 4-week-old litter was improving clinically whereas the older pups (7 weeks old) were deteriorating rapidly. The younger litter was lymphopenic whereas the older litter was panleucopenic. The implication is that the intestinal crypt epithelium and bone marrow contained a larger number of vulnerable cells in the older animals. The severity of clinical signs in conventional dogs in this study is in contrast to the mild enteric disease in specific pathogen free dogs given canine parvovirus [ 11. The reasons for the difference remain undefined. Factors influencing the expression of the disease may include the route of infection, the amount of virus administered and the composition of the microbial flora in the intestinal tract. Data are not available on the normal replacement time of intestinal crypt epithelial cells in the dog, but it is known that in the pig the transition from the slower neonatal replacement time to the faster adult replacement time occurs at about three weeks of age [14, 161. Canine parvovims was isolated consistently from the intestinal contents, thymus, spleen and liver in litter 2, and cell destruction had occurred in all but the liver. However, unequivocal inclusion bodies were found only in the nuclei of intestinal crypt epithelium and myocardium. Post-mortem findings were similar in pups in litter 1 which died or were killed 24 hours later than litter 2, with virus consistently isolated only from intestinal contents. These findings, coupled with the extensive lymphoid depletion with little remaining evidence of necrosis in the thymus, spleen, mesenteric lymph node and Peyer s patches and acute crypt epithelial necrosis, suggest that the virus initially replicated in lymphoid tissue and subsequently invaded,
596 Robinson, Wilcox and Flower Fig. 4 Electron micrograph of affected jejunal crypt. Nuclei containing inclusion bodies (arrows): electron dense outer nuclear membrane, marginated chromatin and many small round particles fill nucleus. Fig. 5 Mesenteric lymph node. Extensive reticuloendothelial hyperplasia in medullary sinusoids. HE.
Canine Parvoviral Disease 591 Fig. 6 Bone marrow from control pup. Fig. 7: Bone marrow from inoculated pup: extensive depletion of myeloid and erythroid precursors. Committed erythroid series cells and megakaryocytes still present. Severe congestion.
598 Robinson, Wilcox and Flower Table 11. Recovery of canine parvovirus from inoculated pups Litter 1 Litter 2 Inoculated Controls Inoculated Controls Intestinal contents 3/4 0/2 4/4 0/2 Spleen 0/4 0/2 3/4 0/2 Thymus 2/4 o/ 1 3/4 0/2 Mesenteric lymph node 2/4 0/2 2/4 0/2 Liver 1 /4 0/2 4/4 0/2 Kidney 0/4 0/2 0/4 0/2 Mvocardium 0/4 0/2 0/4 0/2 replicated in and destroyed intestinal crypt epithelial cells. This is analogous to findings in experimental feline panleucopenia in which lymphoid, reticulo-endothelial and bone marrow precursor cells are infected initially, followed by a second phase with invasion and destruction of crypt epithelial cells [3]. Inclusion bodies in the myocardium have not been reported in natural cases of canine parvoviral enteritis. However, myocardial inclusion bodies without myocardial necrosis have been described in experimental panleucopenia in neonatal kittens [3]. The lack of myocardial cell necrosis and inflammation could be explained by the pups succumbing to the extracardiac effects of the virus before myocardial damage could occur. This seems unlikely, however, as extra-cardiac effects of the virus have not been seen in naturally occurring cases of myocarditis. It is known that myocardial cell division is maximal in puppies during the first three weeks of life [2]. It is likely that in both groups of experimental pups, the period of greatest myocardial vulnerability had passed at the time of inoculation. Acknowledgements We gratefully acknowledge the excellent technical assistance of Messrs. H. Findlay, P. Fallon and P. Hinchliffe and Ms. J. Robertson. References APPEL, M.J.G.; SCOTT, F.W.; CARMICHAEL, L.E.: Isolation and immunisation studies of a canine parvo-like virus from dogs with haemorrhagic enteritis. Vet Rec 105: 156-159, 1979 BISHOP, S.P.: Effect of aortic stenosis on myocardial cell growth, hyperplasia and ultrastructure in neonatal dogs. Recent Advances in Studies on Cardiac Structure and Metabolism 3637-655, 1972 CARLSON, J.H.; SCOTT, F.W.: Feline panleucopenia. 11. The relationship of intestinal mucosal cell proliferation rates to viral infection and development of lesion. Vet Pathol 14 173-181, 1977 CSIZA, C.K.; DE LAHUNTA, A.; SCOTT, F.W.; GILLESPIE, J.H.: Pathogenesis of feline panleucopenia in susceptible newborn kittens. 11. Pathology and immunofluorescence. Infect Immun 3: 838-846, 1971 EUGSTER, A.K.; BENDELE, R.A.; JONES, C.P.: Parvovirus infection in dogs. J Am Vet Med ASSOC 173: 1340-1341, 1978
Canine Parvoviral Disease 599 6 GAGNON, A.N.; POVEY, R.C.: A possible parvovirus associated with an epidemic gastroenteritis of dogs in Canada. Vet Rec 104.263-264, 1979 7 HAYES, M.A.; RUSSEL, R.G.; BABUIK, L.A.: Sudden death in young dogs with myocarditis caused by a parvovirus. J Am Vet Med Assoc 174 1197-1203, 1979 8 HUXTABLE, C.R.; HOWELL, J. McC.; ROBINSON, W.F.; WILCOX, G.E.; PASS, D.A.: Sudden death in puppies associated with a suspected viral myocarditis. Aust Vet J 5537-38, 1979 9 JEFFERIES, A.R.; BLAKEMORE, W.F.: Myocarditis and enteritis in puppies associated with parvovirus. Vet Rec 104:221, 1979 10 JEZYK, P.F.; HASKINS, M.E.; JONES, C.L.: Myocarditis of probable viral origin in pups of weaning age. J Am Vet Med Assoc 1741204-1207, 1979 11 JOHNSON, R.H.; SPRADBROW, P.B.: Isolation from dogs with severe enteritis of a parvovirus related to feline panleucopenia virus. Aust Vet J 55151, 1979 12 KELLY, W.R.: An enteric disease of dogs resembling feline panleucopenia. Aust Vet J 54: 593, 1978 13 KELLY, W.R.; ATWELL, R.B.: Diffuse subacute myocarditis of possibie viral aetiology-a cause of sudden death in pups. Aust Vet J 5536, 1979 14 KENT, T. W.; MOON, H.W.: The comparative pathogenesis of some enteric diseases. Vet Pathol 10414-469, 1973 15 LARSEN, S.; FLAGSTAD, A.; AALBROOK, B.: Experimental feline panleucopenia in the conventional cat. Vet Path0113 216-240, 1976 16 MOON, H.W.: Epithelial cell migration in the alimentary tract mucosa in the suckling pig. Proc SOC Exp Biol Med 1371651, 1971 17 POLLOCK, R.V.H.; CARMICHAEL, L.E.: Canine viral enteritis: Recent developments. Mod Vet Pract 60:375-380, 1979 18 ROBINSON, W.F.; WILCOX, G.E.; FLOWER, R.L.P.; SMITH, J.: Evidence for a parvovirus as the aetiologic agent in myocarditis of puppies. Aust Vet J 55:294295, 1979 19 ROBINSON, W.F.; HUXTABLE, C.R.; HOWELL, J. McC.; PASS, D.A.: Clinical and electrocardiographic findings in suspected viral myocarditis of puppies. Aust Vet J 55351-355, 1979 20 ROBINSON, W.F.; HUXTABLE, C.R.; PASS, D.A.: Canine parvoviral myocarditis: A morphologic description of the natural disease. Vet Pathol 1R282-293, 1980 21 SIEGL, G.: The Parvoviruses, pp. 4-6. Springer-Verlag, Wien, 1976 Request reprints from W. F. Robinson, Division of Veterinary Biology, School of Veterinary Studies, Murdoch University, Murdoch, Western Australia, 6 153 (Australia).