Canine Distemper Virus-Associated Cardiac Necrosis In The Dog

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1 Vet. Pathol. 18: ( 198 I j Canine Distemper Virus-Associated Cardiac Necrosis In The Dog R. J. HIGGINS, S. KRAKOWKA, A. E. METZLER. and A. KOESTNER Department of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, Columbus. Ohio Abstract. An age-related canine distemper virus-associated cardiomyopathy characterized by multifocal myocardial degeneration. necrosis and mineralization with minimal inflammatory cell response was found in gnotobiotic Beagle pups. Of the 30 dogs infected experimentally at 5 to 7 days of age with virulent R252 strain of canine distemper virus. I 1 had gross or microscopic cardiac involvement as early as 16 days post-infection. The 25 dogs similarly infected at 10 to 21 days of age. the uninfected age-matched controls. and pups infected at 5 to 7 days of age with avirulent R252 canine distemper virus had no cardiac lesions. Although the lesions are attributed to a direct viral effect. they occur against a background of other canine distemper virus-related changes including immunosuppression. anemia and encephalomyelitis. All these factors may have a modifying role in the development of this agedependent susceptibility to virus-associated myocardial necrosis. There is now convincing evidence that several viral infections in man are associated with an often fatal myocarditis [33]. Viruses most commonly implicated include those of the picornavirus group, e.g. polio virus [35], Coxsackie [6], measles virus [I], and several others A greater susceptibility to myocarditis in neonates and young children than in adults is a common feature of these infections 1331, particularly with the Coxsackie B group [6]. This age-dependent susceptibility to virus-associated cardiomyopathies has been demonstrated experimentally in a wide range of laboratory animals [16, 33,451. In these models, modulation of the classic lesions can be produced by such exogenous factors as hypoxia [43], exercise [52], and environmental temperature 1431, and by endogenous host-related factors such as age, immunity and immunoresponsiveness [27]. In domestic animals, only a few viruses have been associated with cardiomyopathy. Of these, picornaviruses in pigs and cattle had similar age-dependent effects on myocardium [25]. Recently, parvoviruses have been linked to epizootic outbreaks of viral myocarditis in young dogs [20, 241. Experimental reproduction of the myocardial lesions in susceptible dogs with these parvovirus isolates has not yet been successful [20, 241. Canine distemper virus is a pantropic viral pathogen of dogs. Following initial replication in lymphoid tissues, widespread dissemination to other tissues occurs in 41 2

2 CDV and Myocardial Necrosis 413 dogs that fail to develop an effective immunity to the virus. Although myocardial lesions have been seen in suckling pups infected naturally with canine distemper virus [25], more obvious manifestations of the infection, mainly encephalitis, systemic lymphoid depletion and interstitial pneumonia, have received the most attention. The occurrence, incidence and morphology of canine distemper virus-related cardiomyopathy assumes differential diagnostic importance in the light of the sudden appearance of parvovirus-related myocarditis in dogs. This retrospective study was done to examine myocardial tissues from gnotobiotic dogs infected with canine distemper virus for evidence of direct viral involvement, to describe these lesions in detail, and to determine whether they can be differentiated from parvovirus myocarditis. Materials and Methods A total of 71 gnotobiotic pure-bred Beagle dogs from 17 litters were derived by Cesarian section [32] from date-mated bitches and then maintained in sterile flexible plastic isolation units for the duration of the experiments, as previously described [ 17, 32). All the dogs were examined clinically every day and fed up to five times a day. Rectal temperatures were monitored daily. Fifty-seven dogs (Groups I and 2) received intraperitoneally 0.2 ml of virulent R252 canine distemper virus containing pulmonary macrophage culture infective doses. Thirty dogs (Group I) were infected when 5 to 7 days old, while 27 dogs (Group 2) were infected when 10 to 21 days old. Fourteen dogs (Group 3) were given either 0.2 ml of an R252 canine distemper virus bovine macrophage-adapted neurovirulent strain containing pulmonary macrophage culture infective doses intracerebrally or intraperitoneally, or 0.05 to 0.2 ml of an R252 canine distemper virus Vero cell-adapted nonvirulent strain containing pulmonary macrophage culture infective doses either intracerebrally or intraperitoneally. Six dogs (Group 4) were isolated as age-matched uninoculated controls. At various intervals. whole blood was collected from all dogs for complete blood counts and serum determinations. Serial serum calcium, magnesium and phosphate determinations and serum activities of creatine phosphokinase were done on selected dogs with an SMA-I2 analyzer. For serum vitamin E levels, heparinized blood samples were centrifuged immediately at 4"C, and the plasma was decanted and stored at -7OOC until analysis [14]. Whole blood collected for serum selenium determination and selenium-dependent glutathione peroxidase activity was allowed to clot at 37OC in a glass tube for one hour. Tubes were then centrifuged at 4OC, and the serum was decanted and stored at -70 C prior to determination Evaluations of leukocyte-associated [29] and plasma-phase viremia [3 I] were made by direct immunofluorescence techniques as previously described. At various intervals post-inoculation, dogs were anesthetized and fresh central nervous system tissue quickly removed from selected sites before perfusion fixation of the remaining tissues [37]. Tissues selected for light microscopic examination were embedded in paraffin, sectioned at 6 pm, and stained with hematoxylin and eosin (HE). Selected sections of heart muscle also were stained with von Kossa, alizarin red S, Perk' Prussian blue, Masson's trichrome. phosphotungstic acid and Lendrum's phloxine-tartrazine stains [36]. For electron microscopic examination, selected blocks of heart muscle were post-fixed for two hours in 2.5% glutaraldehyde in Millonig's buffer at ph 7.2, processed routinely and embedded in epon. Semi-thin 1-pm sections were stained with alkaline toiuidine blue. Ultra-thin sections were cut from selected areas, mounted on copper grids, stained with uranyl acetate and lead citrate and examined with an electron microscope [40].

3 414 Higgins er a1 Hematology and clinical pathology Results Group I dogs had a sustained leukopenia, primarily because of an absolute lymphopenia [32]; and regenerative anemia with 8% to 15% nucleated red blood cells and polychromasia. For nine affected dogs, the red blood cell range was 2.1 to 3.4 (mean 2.6) x 106/pl; packed cell volume 18% to 30% (mean 25.4% f standard error of mean 1.4%); hemoglobin 4.3 to 9.0 g/dl (mean 6.7 f standard error 0.4) terminally; and thrombocytopenia. Six uninfected dogs had corresponding values of red blood cell range 6.1 to 6.5 (mean 6.3) X 106/pl; packed cell volume 37% to 41% (mean 38.8% f standard error 0.5%); hemoglobin range 11.1 to 11.6 g/dl (mean 11.3). A cell-associated and plasma-phase viremia persisted in all dogs in groups I and 2 after day 7 post-inoculation, with some dogs still viremic 28 days post-inoculation [30]. Group 2 dogs had a similar canine distemper virus-induced leukopenia with absolute lymphopenia but with less severe anemia within this group; detailed examination of the anemia was performed in nine dogs. Values obtained were: total erythrocytes range of 3.7 to 4.3 (mean 4.1) x 106/p1; packed cell volume range of 28% to 36% (mean 31% f standard error of 1.0%); hemoglobin range of 7.0 to 9.9 g/ dl (mean 8.9); and regenerative anemia with 6% to 10% nucleated red blood cells and polychromasia, up to 39 days post-inoculation. Their viremic pattern was similar to that of Group 1. There was no apparent correlation between the severity of the anemia, lymphopenia, thrombocytopenia or the duration of virernia and the presence of cardiac lesions in the neonatally infected group. Serum canine distemper virus-neutralizing antibody production was not seen in sera from dogs in Group 1, but a low titer (I I 16) was found 27 days post-inoculation in one dog in Group 2. All other dogs remained seronegative. No serum hemagglutinin-inhibiting antibody titers to Toxoplasma gondii were found in sera of six of the tested dogs with lesions, or from age-matched dogs in Group 2. Serum selenium concentrations in 10 dogs varied from to parts per million (mean.i55 f standard error 0.018). The milk diet contained parts per million selenium and the solid diet parts per million selenium. These concentrations are within adequate parenteral and dietary intake levels for dogs [53]. Selenium-dependent glutathione peroxidase activity in the same 10 dogs was 0.82 to 4.65 IU/ml/minute (mean 2.24 f standard error 0.39), within accepted normal limits for conventionally raised dogs. Serum vitamin E concentrations in seven dogs ranged from 1.08 to 2.27 mg/dl (mean standard error 0.14), considered to be well above deficient levels [ 191. Serum aspartate transaminase activity (3 to 21 IU/I) remained within accepted normal limits (5 to 80 IU/ I). Creatine phosphokinase activity in dogs without cardiac lesions ranged from 90 to 155 IU/l (normal = 10 to 62 IU/l), while approximately weekly determinations post-inoculation in two dogs with gross lesions were 54, 40 and 115 IU/I and 90, 80 and 250 IU/l, respectively. Increased creatine phosphoki-

4 CDV and Myocardial Necrosis 415 nase activity was not found in the eight dogs with only microscopic lesions or in one other dog with gross lesions. Both weekly and terminal serum Ca++, Mg++ and phosphate levels were determined in 13 dogs from Group 1. Ranges of Ca++ from 9.4 to 11.9 (mean 10.1 & standard error 0.3) mg/dl; Mg++ from 1.9 to 3.9 (mean 2.8 k standard error 0.2) mg/ dl; and phosphate from 3.6 to 7.4 (mean 5.5 k standard error 0.4) mg/dl did not differ significantly from the normal laboratory reference values for dogs of Ca to 11.2 mg/dl, Mg" 1.8 to 2.4 mg/dl, and phosphate 3.6 to 6.0 mg/dl. Clinical findings Four of the 11 dogs in Group 1 with gross or microscopic cardiac involvement had clinical signs. No cardiac lesions were found in any of the dogs of Group 2 or Group 3 (table I). In the three dogs with cardiac and pulmonary lesions grossly visible at necropsy, the first clinical sign was an increased respiratory rate (60/min) starting between days 14 and 18 post-inoculation. After one to four days of depression and anorexia there was a sudden rapid progression to deep labored abdominal breathing with collapse and prostration in the last 24 hours before the animals died or were killed. Dogs were killed or died between days 16 and 23 post-inoculation. The only exception to this pattern was one dog that showed only variable depression and abdominal breathing starting on day 24 and lasting until the dog was killed on day 39, when it also had mild ataxia. Rectal temperatures remained subnormal or normal throughout the course of infection with a drop to between 34.5"C and 35.5"C when moribund. In the eight dogs with only microscopic cardiac lesions, one or more neurologic abnormalities developed after day 22 post-inoculation. These neurologic signs included depression, various degrees of truncal ataxia and incoordination, myoclonus, fine intention head tremors, intermittent clonic masticatory muscle activity with salivation (chewing gum seizures), and episodic grand-ma1 seizures. These dogs also had normal or subnormal temperatures, subnormal when moribund. At necropsy at Table I. Incidence of myocardial lesions in gnotobiotic beagle dogs infected with R252 strains of canine distemper virus. Group No. of dogs Viral inocula 1 30 V-R252-CDV' 5-1 I I (36%) 2 21 V-R252-CDV AV-R252-CDV2 7 0 BC-R252-CDV Control NA 0 V-R252-CDV = virulent strain. ' AV-R252-CDV = avirulent Vero-cell-adapted strain. BC-R252-CDV = avirulent bovine-cell-adapted strain. NA = Not applicable.

5 476 Higgins er a/ 20 to 36 days post-inoculation, four dogs in this group showed gross pulmonary edema and hemorrhage. The only other significant clinical findings were a mildly elevated respiratory rate with anorexia terminally. Gross lesions Three dogs had multiple, linear, sometimes confluent, well-demarcated whitish streaks approximately 1 X 3 mm distributed randomly on endocardia1 and epicardial surfaces of both left and right ventricles (fig. I ), including papillary muscles. On transverse sections of fixed tissue, these foci were present throughout ventricular and interventricular myocardium. No lesions were found in the atrial musculature. In one dog killed 3 I days post-inoculation, right ventricular dilation and apparent hypertrophy was associated with excess clear unclotted pericardial fluid. The lungs were examined in situ via thoracotomy immediately before cardiac perfusion. In eight of the dogs with gross or microscopic cardiac involvement, most lung lobes were Fig. 1: Section of left ventricle and left atrium following perfusion. Multiple linear white foci of necrosis in myocardium (arrows). Fig. 2 Perfused myocardium. Coagulation necrosis and degeneration. No inflammatory cell infiltrate. Day 16. Masson s trichrome. Fig. 3: Myocardium. Focus of lysis and necrosis with minimal inflammatory cell infiltration. Day 21. HE. Fig. 4 Myocardium. Focus of advanced necrosis with mineralization. Day 24. Von Kossa. HE.

6 CDV and Myocardial Necrosis 411 enlarged, greyish pink with red mottling, and edematous. All lobes contained bright red irregular petechial and ecchymotic hemorrhages up to 1 cm in diameter, most prominent in the diaphragmatic lobes. Other findings common to dogs in all groups receiving virulent R252 canine distemper virus were systemic lymphoid depletion and, in the lungs, prominent greyish well-demarcated solid areas of consolidation in the hilar regions around the bronchial bifurcations. Microscopic lesions In the heart, the most striking and consistent microscopic lesion was multifocal myocardial degeneration and necrosis with mineralization. It had a similar topographic distribution in all affected dogs. Two different types of histologic lesions, presumably arising at different times, involved random groups of myofibrils in a nodular, irregular or linear pattern. Microscopic lesions, first found 16 days post-inoculation, consisted of multiple foci of coagulative necrosis of groups of myofibers with some interstitial mononuclear cells (fig. 2). The sarcoplasm of swollen fibers was granular, clumped and deeply eosinophilic with HE. These areas were more accentuated with trichrome (fig. 3) or phosphotungstic acid-hematoxylin stains. Some muscle nuclei were pyknotic or karyolytic. Single or multiple, irregular, canine distemper virus inclusion-like, intensely eosinophilic structures were found occasionally in the sarcoplasm. Lendrum s stain for ribonucleic acid on these structures was not definitive. Some myocytes in and immediately adjacent to the lesions had prominent eosinophilic nucleoli with a granular sarcoplasm. No mineralization was found in any of these fibers. The myocardium of dogs with gross lesions 18 to 24 days post-inoculation contained more and larger areas of degeneration and necrosis. These foci consisted of myocytes in various stages of necrosis mixed with elongated spindle-shaped fibroblast-like cells, a few macrophages and deeply basophilic granules (fig. 4), positive with both von Kossa and alizarin red S stains. This prominent intracellular mineral was deposited irregularly within or at the margin of these lesions (fig. 5). Slight collagen production was found in some of these lesions. Giant cells bordering areas of intense mineral-

7 478 Higgins er 01. ization were seen occasionally, usually with developing fibrosis. No lesions were found in any cardiac or aortic valve leaflets or atrial musculature. Blood vessels were unremarkable. There was no obvious vascular orientation of these lesions or attempts at regeneration of myofibers. The histologic findings in lymphoid organs, including the thymus, were essentially as described elsewhere [3 11. In the lungs of some affected dogs in Group 1 there was generalized intra-alveolar, peribronchial and perinuclear edema and hemorrhage. Additional findings in all groups of dogs receiving virulent virus included patchy necrotizing bronchiolitis, mononuclear cell accumulation within alveoli and their walls, and occasional syncytium formation by intra-alveolar macrophages. Another common feature was a disseminated canine distemper virus-associated meningoencephalomyelitis with widespread viral infection of neurons and glial cells, including the ependyma and choroid plexis, as seen by immunofluorescence and electron microscopy. There was associated neuronal necrosis, neuronophagia, local microgliosis, and minimal or absent perivascular inflammatory cell response. No lesions were found in the tongue or in other skeletal musculature. Ultrastructural lesions Ultrastructural changes in myocytes prior to complete necrosis involved mitochondria, sarcoplasmic reticulum, and lipid deposits and were restricted to myofibers within or adjacent to degenerating foci. An increase in the number and size of randomly dispersed lipid droplets was found before mitochondria1 changes (fig. 6). The swollen mitochondria had closely applied inner and outer membranes, and the fragmented, disoriented cristae were dispersed throughout a granular, dense matrix. There were also some focal. electron-lucent areas in this matrix. Mitochondria of various sizes aggregated in clusters throughovt the sarcoplasm. The nucleus at this stage was unremarkable. Randomly distributed within the sarcoplasm of altered myofibers were single or less often multiple, non-membrane-bound intracytoplasrnic aggregates of compactly arranged filaments of varied size and shape. These filaments were comprised of a core with an inner tubular diameter of 18 to 20 nm and an outer fuzzy coat 35 to 40 nm in diameter (fig. 6, 7); they were of various lengths, and were readily identified as paramyxovirus-like nucleocapsids [4 I] identical with R252 canine distemper virus strain nucleocapsids occurring both in vitro [ 1 11 and in vivo [40]. Intracytoplasmic canine distemper virus nucleocapsids also were identified in mononuclear cells found interstitially throughout the myocardium (fig. 8), but there was no evidence of mature virion budding or incorporation of the characteristic nucleocapsid protein spikes in the cytoplasmic membrane of either cell. In other fibers, more advanced degenerative changes in mitochondria consisted of electron-dense clusters of curved or linear spicules (fig. 9), or of circular granules with an inner dense core and central electron-lucent ring. Compact or loose membrane-bound myelin-like figures and dilated single membrane-bound structures interpreted as arising from sarcoplasmic reticulum or T tubules developed in most myofibers (fig. 6). Focal discontinuous gaps in the sarcolemmal membrane occurred

8 CDV and Myocardial Necrosis 479 Fig. 6: Myocardial fiber. Increased lipid droplets, myelin-like membranous profiles and multiple intracytoplasmic canine distemper virus nucleocapsid aggregates (arrowheads). Correlated tubuloreticular crystalline structures in normal endothelial cell (arrow). Inset: Tubular nucleocapsids with inner core 18 to 20 nm (arrow), outer fuzzy margin 45 nm diameter. with widespread mineralization of mitochondria and sarcoplasm, granular compaction of myofibril structure, loss of nuclear chromatin patterns, and separation of intercalated disc junctions. Widened interstitial gaps were partially filled with fibrin and granular debris (fig.7). In these areas, macrophages contained membrane-bound cell organelle debris. Blood vessels were morphologically intact (fig. 7) and no changes were found in the Purkinje s fibers examined. Discussion This study demonstrates the occurrence of canine distemper virus-associated, often fatal, cardiac necrosis in experimentally infected neonatal gnotobiotic dogs. The distinctive gross or microscopic lesions included acute multifocal ventricular myocardial degeneration, necrosis, and minimal inflammatory cell response with later mineralization and fibrosis. Ultrastructurally, the characteristic intracytoplasmic filamentous aggregates of canine distemper virus nucleocapsids were found consistently in degenerating myocytes in and adjacent to affected foci and also in regional

9 480 Higgins ei al. Fig. 7: Myocardial fiber at periphery of lesion with canine distemper virus intracytoplasmic inclusion bodies (arrowheads). Intact capillary with interstitial debris and macrophage cell processes with nucleocapsids (arrow). Fig. 8: Myocardium. Normal myocardial fibers with interstitial macrophage containing nucleocapsids (arrowhead).

10 CDV and Myocardial Necrosis 48 I macrophages. The lesions were found during morphologic and virologic studies on the pathogenesis of canine distemper virus-induced encephalomyelitis, and the observations reported here represent a retrospective analysis. Although conclusive proof that canine distemper virus was directly responsible for the lesions would require re-isolation from myocardium, the presence of viral nucleocapsids in lesions and not in normal myocardium strongly suggests that direct infection of myocytes by blood-borne virus initiates the myocardial changes seen here. Further, the absence of these lesions in age-matched uninfected controls and in dogs infected with an avirulent R252 canine distemper virus indicates that these changes occur only in dogs exposed to virulent canine distemper virus. Finally, others have noted that multifocal cardiac necrosis and mineralization can occur in young dogs naturally infected with canine distemper virus [2,25]. In spite of this evidence, other factors influencing either the initiation of the lesion or its progression through mineralization must be considered in the interpretation and delineation of causes of myocardial damage in dogs. These influences fall into two general categories: exogenous factors, including nutritional deficiencies, altered electrolyte levels and concurrent infection with other infectious agents such as Toxoplasma gondii and canine parvovirus; and endogenous factors related chiefly to the concurrent effects of canine distemper virus upon hematopoiesis, lymphoid structure and function [3 I], and the neurologic status of experimentally infected puppies. A persistent and profound virulent canine distemper virus strain-induced bone marrow suppression, reflected in the marked anemia with evidence of regeneration and thrombocytopenia, occurred in the neonatally infected dogs. Severe anemia induced by controlled hemorrhage will predispose the canine myocardium to hypoxic injury [9]. Myocardial ischemia can produce disseminated myocytic necrosis, intense inflammatory cell response, and mineralization [54]. In rabbits, experimental myocardial ischemia has increased both the number and size of virus-induced myocardial lesions [42]. Thus, a regional ischemia could potentiate both virus infection and subsequent myocardial necrosis in such compromised areas. Anemia as the initiating cause of the lesions seen in our study is unlikely because not all of the severely

11 482 Higgins er a/. anemic dogs had myocardial involvement, and virus nucleocapsids were not seen in normal myocytes. Unlike the only other documented, naturally occurring viral myocarditis caused by parvovirus infection, dogs with canine distemper virus-associated myocardial necrosis did not show any remarkable degree of interstitial inflammatory cell infiltration. The lack of a pronounced inflammatory cell response visible by light microscopy in and around myocardial lesions in early cases in our series is not surprising, as this is a common finding in mammalian fetal and neonatal cardiac lesions of both metabolic [ 131 and infectious [ 14,211 origin. Delayed resorption of necrotic fibers may account for the dystrophic mineralization that develops in these lesions [3]. Supporting these observations and correlating with our findings are data collected from a paramyxovirus-induced myocarditis in the young chicken. In that study, Newcastle disease virus, also a paramyxovirus, was associated with direct myocyte infection, resulting in focal myocardial necrosis without concurrent anemia. This necrosis preceded any inflammatory cell response by several days [ 101. A similar sequence of events has been documented for experimentally-induced picornavirus myocarditis in neonatal and immature mice [7,32,45]. Also, coronavirus infection in both immature and adult rabbits recently was reported to cause a fatal cardiomyopathy [49] characterized by focal myocardial degeneration, necrosis and mineralization essentially similar to the lesions in the Group I dogs. Although the rabbits were not anemic, the concomitant depletion of lymphocyte populations in lymph nodes and the thymus, similar to the profound canine distemper virus-related immunosuppression [3 I], suggests that this common immunosuppression may modify the inflammatory cell response to the virus-induced myocardial necrosis. The apparent age-related difference in susceptibility to virus-induced myocarditis is a feature of a number of other viral infections. Increased neonatal susceptibility to viral myocarditis is well known in spontaneous [ 161 and experimental [22,44] picornaviral infections in neonatal and adult mice. Inherent host strain-related differences in susceptibility to myocardial damage occur in neonatal mice with Coxsackie B virus infection [ 181. Also, adverse ambient temperatures are known to influence the pathogenesis of several viral diseases [ 151. Reduced virulence of Herpes canis has been demonstrated in experimentally infected neonatal pups with artificially raised body temperatures [8]. Since newborn pups have only partially competent homeothermic regulatory mechanisms until the second week of life, the subnormal temperatures of Group 1 could lead to enhanced infectious virus production as demonstrated for a paramyxovirus in vifro [50], or to more virulent temperaturesensitive mutants [43]. Various patterns and degrees of mineralization were a feature of the lesions examined ultrastructurally. The ultrastructural granular and crystalline forms of mitochondria1 mineralization, with swelling and focal loss of matrix and cristae, are nonspecific changes associated with a wide variety of toxic, metabolic, infectious and degenerative conditions in cardiac muscle [4], including hypercalcemia and hypo-

12 CDV and Myocardial Necrosis 483 magnesemia [22]. Mineralization is considered one of the earliest morphological expressions of myocardial cell injury [9], and reflects alterations in cell membrane permeability [4,47]. Resulting calcium influx overloads the cytosol and mitochondria1 calcium binding sites [9]. Although one report presents evidence for disturbed calcium homeostasis in a group of moribund gnotobiotic Beagle dogs infected neonatally with canine distemper virus [55], this was not confirmed in our dogs. Parallel serum phosphate and magnesium levels remained within normal limits, also suggesting that disturbed mineral homeostasis is not a contributing factor to the process of mineralization. Experimental dietary deficiency of potassium produced myocardial necrosis in six of 16 Beagle dogs [51], but the lesions were not histologically similar to those described here. Dietary levels of potassium in this study were adequate [5 11. It is well established that central nervous system lesions can lead to myocardial damage in man [ 121 and laboratory animals [5,23], including dogs. Focal myocardial lesions can be found in about 8% of people with terminal primary neurologic conditions, predominantly intracranial hemorrhage, but also including trauma, bacterial and viral infections, and tumors. The lesions in man are described as fuscinophilia of groups of myocytes proceeding to myocytolysis with intact sarcolemmal sheaths and muscle nuclei. In these cases, loss of sarcoplasm does not result in mineralization or an inflammatory cell response [ 121, although interstitial fibrosis does occur [48]. In rodents, elevated circulating catecholamines, and direct sympathetic stimulation of the heart secondary to brain damage [6], lead to myocardial hypoxia. In dogs, cardiac lesions including focal necrosis with hemorrhage and interstitial polymorphonuclear leukocyte and mononuclear cell infiltration have been produced by stellate ganglion [28] or vagal nerve stimulation [39]. These lesions occur most frequently in the papillary muscles and subendocardial myocardium. Mineralization is not a feature of these lesions. While the pattern and distribution of encephalomyelitis in the Group 1 dogs was more severe than in Group 2, there are marked histologic differences between the brain lesions in man [ 121 and experimental animals [28], and those in our dogs. Myocardial necrosis occurs frequently with several dietary deficiencies. Extensive but topographically restricted myocardial necrosis in lambs, calves and pigs [26] is a consistent feature of white muscle disease, a syndrome of vitamin E and selenium deficiencies [34]. Typically, focal cardiac lesions appear sequentially as acute hyaline degeneration, intense macrophage infiltration, mineralization and fibrosis [26]. One poorly documented report speculates on the possibility of a diet-induced selenium deficiency in bitches contributing to sudden death from cardiac failure in two groups of neonatal pups [38]. No biochemical analyses were done and the canine distemper virus status of the bitches was not determined. Beagles have been subjected experimentally to combined vitamin E/selenium deficiency (531. Lesions in the heart consisted of necrosis and mineralization of the subendocardial Purkinje s fiber

13 484 Higgins era/. network and adjacent myofibers, which also occur in ruminants with white muscle disease [26]. Other changes in these dogs were intestinal lipofuscinosis and widespread skeletal muscle degeneration. To exclude subclinical dietary deficiency in our dogs, particularly since these animals were gnotobiotic, dietary and parenteral concentrations of these nutrients and of selenium-dependent glutathione peroxidase were determined. All values were within the normal reported range for dogs [19, 531. The gross histologic and ultrastructural findings were not compatible with lesions in the experimentally induced syndrome. Recently, a fatal parvovirus-related myocarditis of young dogs has been described [22, 241. Parvovirus infection is emerging as an important viral pathogen of dogs. Myocardial lesions in parvovirus-infected dogs include diffuse interstitial edema, lymphoplasmacytic cellular infiltration, and basophilic intranuclear viral inclusion bodies in myocytes. Mineralization is rare. Lesions of canine distemper virus-related myocardial necrosis described in this study are not compatible with those reported for parvovirus infection. Acknowledgements Supported by The State of Ohio, Canine Research Funds and Grant No. A , Public Health Service, NIH. The excellent technical assistance of David Long and Nancy Austin is gratefully acknowledged. References I ATTAL, C.: MOZZICONAZZI, P.: Measles: clinical features. In: Clinical Virology: The Evaluation and Management of Human Viral Infections. ed. Debre and Celers, pp W. B. Saunders and Co.. Philadelphia AYERS, K.M.; JONES, S.R.: The cardiovascular system. In: Pathology of Laboratory Animals, p. 13. Springer-Verlag. New York, BOLANDE, R.: Cellular Aspects of Developmental Pathology. Lea & Febiger, Philadelphia, BONUCCI, E.: SADUN, R.: Experimental calcification of the myocardium. Am J Pathol71: , BOYKO, W.J.; GALABRU. C.K.: MCGEER, E.G.; MCGEER, P.L.: Thalamic injections of kainic acid produce myocardial necrosis. Life Sci , BURCH, G.E.; SUN, S-C.: CHU, K.C.: Interstitial and Coxsackie virus B myocarditis in infants and children. A comparative histologic and immunofluorescent study of 50 autopsied hearts. JAMA , BURCH, G.E.: Ultrastructural myocardial changes produced by viruses. Recent Adv Stud Cardiac Struct Metab CARMICHAEL, L.E.; BARNES. F.D.: PERCY. D.H.: Temperature as a factor in resistance of young puppies to canine herpes virus. J Infect Dis , CHEVILLE, N.F.: Cell Pathology. p Iowa State University Press, Ames, CHEVILLE, N.F.: BEARD, C.W.: Cytopathology of Newcastle disease. Lab Invest 27: CONFER, A.W.; KHAN. D.E.: KOESTNER. A,: KRAKOWKA, S.: Comparison of canine distemper viral strains: An electron microscopic study. Am J Vet Res 38: , 1975

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15 486 Higgins er a/. system tissue for immunofluorescence, light and electron microscopic evaluation. Am J Vet Res 38: , MANKETLOW, B.W.: Myopathy resembling white muscle disease of sheep. NZ Vet J 11: 52-55, MANNING, G.W.; HALL, G.E.; BANTING, F.G.: Vagal stimulation and the production of myocardial damage. Can Med Assoc J 37: , MCCULLOUGH, B.; KRAKOWKA. S.; KOESTNER, A.: Experimental canine distemper virusinduced demyelination. Lab Invest 31: , NORRBY, E.; FRIDING, B.; ROCKBORN, G.; GARD, S.: The ultrastructure of canine distemper virus. Arch Gesamte Virusforsch , PEARCE. J.M.; LANGE, G.: Cardiac anoxia as the factor determining the occurrence of experimental virus carditis. Arch Pathol44: , PREBLE, O.T.; YOUNGER, J.S.: Temperature-sensitive viruses and the etiology of chronic and inapparent infections. J Infect Dis 131: , RABIN, E.R.; HASSAN, S.A.; JENSON, A.B.; MELNIK, J.L.: Coxsackie virus B:, myocarditis in mice. Am J Pathol44: , RABIN, E.; MELNIK, J.: Experimental acute myocarditis. Prog Cardiovasc Dis 7: 65-72, I ROTRUCK, J.T.; POPE. A.L.; GANTHER, H.E.; SWANSON. A.D.; HAFEMAN, D.G.; HOEKSTRA, W.G.: Selenium: Biochemical role as a component of glutathione peroxidase. Science , SCHANNE. F.A.X.; KANE, A.B.; YOUNG, E.E.; FARBER, J.L.: Calcium dependence of toxic cell death: A final common pathway. Science 206: , SCHLESINGER, M.J.; REINER, L.: Focal myocytolysis of the heart. Am J Pathol31: , SMALL, J.D.; AURELIAN, L.; SQUIRE, R.A.: STRANDBERG. J.D.; MELBY, E.C.; TURNER, T.B.; NEWMAN, B.: Rabbit cardiomyopathy. Am J Pathol , STALLCUP, K.C.; WECHSLER, S.L.: FIELDS, B.N.: Purification of measles virus and characterization of subviral components. J Virol30: , I TATE, C.L.; BAGDON, W. J.; BOKELMAN, D.L.: Morphologic abnormalities in potassiumdeficient dogs. Am J Path , TILLES, J.G.; ELSON, S. H.; SHAKA, J.A.; ABELMANN, W.H.; FINLAND, M.: Effects of exercise on Coxsackie virus A9 myocarditis in adult mice. Proc SOC Exp Biol Med 117: , VAN VLEET, J.F.: Experimentally induced vitamin E-selenium deficiency in the growing dog. J Am Vet Med Assoc 166: , WEAVER, D.Q.; HENSON, E.C.; CROWELL, J.W.; ARELGER, R.B.; BRUNSON, J.G.: Structural alterations produced in dogs in sublethal haemorrhagic shock. Arch Pathol , I WEISBRODE, S.E.; KRAKOWKA. S.: Canine distemper virus-associated hypocalcemia. Am J Vet Res 40: , 1979 Request reprints from Dr. Steven Krakowka. Department of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, Columbus, OH (USA).