Biomedical subjects
Publications and source records attributed to M J Appel.
Antibody titers were measured in domestic or European ferret (Mustela putorius furo) jills vaccinated with modified-live canine distemper virus (CDV) vaccine and in their kits. The half-life of maternal antibody to CDV in ferrets was 9.43 days. Ferret kits should be vaccinated against CDV at 6, 10, and 14 weeks of age.
In September and October 1985, six black-footed ferrets (Mustela nigripes) were captured from the only known population, located near Meeteetse, Wyoming for captive propagation. Two days following capture an adult male showed signs of canine distemper and an adult female displayed similar signs 7 days postcapture; these infections were undoubtedly acquired prior to capture. Subsequently the four remaining captive black-footed ferrets also developed canine distemper and all eventually died. Clinical signs included severe pruritus, hyperkeratosis and progressive loss of body condition. A few animals had intermittent diarrhea and respiratory disease. Intranuclear and intracytoplasmic inclusion bodies were numerous in epithelial tissues and two black-footed ferrets had a mild to moderate meningoencephalitis. Canine distemper virus was isolated from four animals and paramyxovirus nucleocapsids were observed by electron microscopy of feces from all affected black-footed ferrets. Antibodies to canine distemper virus were not detected in sera of sick black-footed ferrets. Antibodies to canine distemper virus were found in sera of badgers (Taxidea taxus) and coyotes (Canis latrans) collected in the Meeteetse area in 1986. Most free-ranging black-footed ferrets in the colony apparently died of canine distemper during the summer and fall of 1985. An attempt was made to capture all surviving animals in the affected area in order to abort the epizootic and provide black-footed ferrets for captive propagation.
A study was undertaken to determine the safety and suitability of vaccinia virus as an eukaryotic expression vector in dogs. Clinical signs were not seen in inoculated dogs, with the exception of small nodules at the site of inoculation. Vaccinia virus did not spread from dogs that were inoculated by SC (n = 5), intradermal (ID; n = 13), or intranasal (n = 3) routes to noninoculated dogs maintained in close contact. Replication of vaccinia virus appeared to be restricted in dogs because greater than or equal to 10(5) plaque-forming units of virus were required to induce an immune response by ID inoculation. Results were better with ID inoculation than with SC or intranasal inoculations. Repeated inoculations enhanced serum antibody titers, and annual reinoculation resulted in boosting of antibody titers. Maternal antibody interfered with virus replication and antibody production. Recombinant virus products induced antibody formation in dogs to human influenza-A virus, herpes simplex virus, and human hepatitis-B virus antigens. It was concluded that vaccinia virus would be safe and suitable as an eukaryotic expression vector in dogs.
A morphological study of selected white matter lesions was carried out in three dogs with canine distemper encephalomyelitis. Two dogs had experimental infections while the third was a spontaneous case. Two stages were identified in the process of demyelination. The earliest evidence of myelin injury was a ballooning change in myelin sheaths involving single or multiple axons. This was followed by a progressive stripping of compact sheaths by the cytoplasmic fingers of phagocytic cells which infiltrated and removed myelin lamellae. Some axonal necrosis also accompanied these changes. Where demyelination occurred, canine distemper viral nucleocapsids were found in astrocytes, macrophages, ependymal cells and infiltrating lymphocytes. In contrast, oligodendrocytes were conspicuous by their apparent lack of infection. Thus it seems that myelin loss cannot be ascribed to oligodendrocyte infection. Perturbed astrocyte function following canine distemper viral infection may cause oedema of myelin sheaths, leading to ballooning and primary demyelination. Cells which phagocytosed myelin were mainly identified as microglial cells with lesser involvement by astrocytes. Rarely, oligodendrocytes also acted as macrophages. Myelin debris was engulfed in bulk or as small droplets into coated pits. Remyelination was present in established plaques although not in great abundance, perhaps due to the diminished oligodendrocyte numbers and a relative increase in immature forms of these cells. These observations are compared to similar changes observed in other demyelinating diseases of animals and man.
Beagle dogs were experimentally infected with the Cornell A75-17 strain of canine distemper virus. At three time points post-infection (PI), immunoreactive myelin basic protein (MBP) was measured in cerebrospinal fluid (CSF). Levels were correlated with neuropathological findings, interferon in CSF and virus isolation from the brain. CSF from animals inoculated with Cornell A75-17 strain often showed detectable immunoreactive MBP late in the disease course. As anticipated from earlier morphological studies, CSF drawn around day 20 PI lacked MBP while subsequent samples were positive. Dogs with severe demyelination had elevated values of immunoreactive MBP while dogs with only mild inflammation had little or none. Release of MBP or MBP peptides into CSF of dogs with canine distemper may be a valuable laboratory test in studies of the natural history of this disease and in assessing the response to treatment. Whether an immune response to MBP plays an immunopathogenic role in the chronic, demyelinating phase of canine distemper encephalitis remains to be determined.
In this report, we describe a technique for identifying negative strand (genome) and positive strand (messenger) RNA of canine distemper virus (CDV) in dog tissues by using single stranded RNA probes. Plasmids (pSP64-P and pSP65-P) which contain insert DNA corresponding to the P gene of CDV were transcribed by SP6 polymerase in the presence of radioisotope to produce radiolabeled single stranded RNA probes. RNA transcribed from pSP65-P is complementary to the negative strand (genome) and RNA produced from pSP64-P is complementary to the positive strand (message) of CDV. The binding specificity of the single stranded RNA probes was determined on Northern-blots. The use of these RNA probes in hybridization assays resulted in greater sensitivity and specificity than that obtained from double stranded DNA probes (either whole plasmids or purified insert DNA) which were labeled by the nick translation reaction. We also describe the making of single stranded DNA probes by reverse transcription labeling of complementary RNA. The complementary RNA was produced by the transcription of cloned DNA (pSP64-P and pSP65P). Single stranded RNA probes and single stranded DNA probes were similar in sensitivity. The single stranded RNA and DNA probes were applied to ethanolacetic acid fixed tissue sections from dogs infected with CDV-A75/17. We used 32P-labeled probes in tissue hybridizations and 35S-labeled probes in in situ hybridizations to identify negative and positive stranded CDV RNA. In this report we demonstrate that single stranded RNA and DNA probes can be used successfully in tissue hybridization and in situ hybridization assays to study viral expression in this virus-host system.
Canine distemper virus attachment (hemagglutinin [H] equivalent) and fusion (F) antigens were purified by affinity chromatography with monoclonal antibodies. The purified antigens were used to immunize groups of three dogs. Radioimmune precipitation assays with sera from these animals showed that the F antigen preparation was pure and induced only an F polypeptide-specific antibody response but that the H antigen preparation had a slight contamination by the F antigen. Immunized animals were challenged with virulent canine distemper virus. Two animals in each group developed pronounced humoral and cellular immune responses after challenge. Among these infected animals, only the dogs immunized with H antigen developed symptoms, albeit mild. In contrast, three nonimmunized control animals developed severe disease, with a fatal outcome in two cases. The complete resistance against challenge in two dogs was interpreted to reflect in one case anti-F immunity and in the other case most likely a high level of anti-H immunity. It is suggested that the F antigen may be of particular interest for the development of morbillivirus and possibly other paramyxovirus subunit or synthetic vaccines, because it can induce immunity capable of blocking virus infection and in situations of virus replication prevent the emergence of symptoms.
Persistent infection with virulent canine distemper virus (CDV-SH) was established in 2 human lymphoblastoid B cell lines (Wi-L2 and Raji), and one human (HSB), one simian (1670) and one canine (CT-45-S) lymphoblastoid T cell line. Cell free virus from persistently infected T cell lines was avirulent for dogs but virulence was maintained during 31 cell passages in persistently infected B cell lines.
Syncytia formation has been observed in response to experimental Canine Distemper Virus (CDV) infection in specific-pathogen-free Beagle dogs. Multinucleated giant cells are most frequently found in white matter lesions of the brain and in the anterior uvea of the eye. Lymph nodes and the lung are occasionally positive, while the leptomeninges will also rarely harbour syncytia. The mechanism of syncytia formation and their possible importance in the pathogenesis of the disease are briefly discussed. Widespread tissue syncytia formation is a feature of CDV infection which should assist in identifying cases of non-suppurative encephalitis in the dog caused by this agent.
The clinical signs, hematologic changes, serum and fecal virus titers, specific antibody production and the occurrence of histologic lesions were studied in 22 nine-week-old seronegative beagle dogs inoculated by the oral and intravenous route with canine parvovirus. Approximately 30% of the dogs had clinical signs of pyrexia, depression, vomiting, and diarrhea irrespective of the route of inoculation. Events in the dogs inoculated intravenously preceded those in dogs inoculated orally by approximately two days. Only one dog died. Lymphopenia was the most consistent hematologic change. Viremia always preceded the initiation of fecal virus shedding. Viral titers in the serum and feces were significantly greater in symptomatic dogs compared to asymptomatic dogs. Termination of the plasma viremia coincided with the onset of the humoral immune response, but viremia persisted one day longer in symptomatic dogs. The severity of lymphoid tissue and intestinal infection, assessed by tissue immunofluorescence and histology, was also greater in symptomatic dogs. The severity of intestinal disease was highly correlated with the magnitude and duration of viremia.
After oral inoculation, the sequential distribution of canine parvovirus was studied in 14 nine-week-old seronegative beagle dogs. Two or three dogs were necropsied on days 1 through 6 after inoculation. Tissues were collected for virus isolation, immunofluorescence testing, and light microscopy. Virus was isolated from, and fluorescent cells were seen in the tonsil, retropharyngeal and mesenteric lymph nodes one and two days after inoculation. Virus infection of systemic and intestinal lymphoid tissues occurred as early as three days after inoculation and was associated with viremia. Intestinal epithelial infection was first seen four days after oral inoculation. All dogs were viremic before intestinal epithelial infection was found. Fecal virus excretion first occurred four days after oral virus inoculation. Intestinal virus infection and lesions became progressively more severe between four and six days after inoculation. The severity of intestinal lesions was variable and related to the severity of systemic lymphoid tissue lesions and the magnitude and duration of viremia. Four littermates of virus-infected dogs were passively immunized against canine parvovirus with convalescent canine serum 24 hours after oral virus inoculation. Neither clinical signs, lymphopenia, nor fecal virus excretion occurred in passively immunized dogs. Intestinal epithelial infection was not demonstrable by immunofluorescence testing when passively immunized dogs were necropsied four, five, and six days after virus inoculation.
Pairs of specific pathogen free dogs were immunized with two injections of heat inactivated canine distemper virus (CDV) or one injection of a live CDV or live measles virus (MV) vaccine. Three unimmunized dogs were used as controls. All 9 dogs were challenged with virulent CDV (Snyder Hill strain). The three unimmunized dogs developed severe signs of disease with a lethal infection in one. The two dogs immunized with live CDV vaccine developed a strong humoral as well as cellular immune response after immunization and were protected against virus replication. Animals immunized with either inactivated CDV or modified live MV failed to develop a measurable cellular immune response after immunization and had a comparatively weak humoral immune response to distemper antigens. They showed mild signs of infection after challenge and responded with strong anamnestic cellular and humoral immunity. The measles vaccine immunized dogs had a moderate serum titer of measles hemolysin-inhibiting antibodies which, after exposure to distemper virus, was boosted to high levels. It is proposed that this response plays a role in the mitigation of the virulent distemper infection in these animals.
Disease induced by 3 virulent strains of Canine Distemper Virus (CDV) was compared in specific pathogen-free Beagle dogs. All strains produced an encephalomyelitis but variation was observed in the severity, clinical course and resulting neuropathology. Infection with Snyder Hill strain of CDV was consistently acute; dogs either succumbed 14 to 19 days post-inoculation (PI) or recovered. Lesions in the neuraxis were those of a polioencephalomyelitis. In contrast, CDV strain A75-17 produced subacute to chronic disease in which demyelination was the predominant finding. Some dogs succumbed, generally around 28 to 42 days PI. Total recovery was again recorded for some members of the group. Others developed persistent central nervous system (CNS) infection but remained clinically stable until electively killed with barbiturate, up to 62 days PI. CDV strain R252 also induced delayed, predominantly white matter disease with a mixed pattern of mortalities, persistent infections and recoveries, similar to A75-17. Neutralizing antibody responses correlated with the disease course. Dogs which died had low serum titres or lacked serum antibody. Recovering dogs had the earliest and highest titres. A few dogs with persistent CNS infection had antibody in the cerebrospinal fluid also. Current concepts of the pathogenesis of canine distemper encephalomyelitis (CDE) are discussed and a basis for the strain-dependent clinical and pathological expression of CDE is proposed. Viral strain appears to be an important factor in this common disease of the canine CNS.
The pattern of virus-induced and allergic demyelinating encephalomyelitis in the dog were compared. The predominant pattern of myelin loss in canine distemper (CD) infection was focal, periventricular and was initially noninflammatory. In contrast, sensitization to myelin produced a uniform pattern of central nervous system (CNS) myelinolysis which was disseminated, inflammatory and perivascular. Ultrastructurally, virus-infected neuroglia participated in the demyelination in CD, whereas infiltrating haematogenous mononuclear cells predominated in the lesions of experimental allergic encephalomyelitis (EAE). Areas of predilection within the CNS differed, being influenced by viral spread in CD and by vascular factors in EAE. In CD, the paramyxovirus appears to play a central role in the process of demyelination. In contrast to EAE, however, these studies do not support the view that autoreactivity to myelin contributes to the pathogenesis of CD demyelinating encephalomyelitis.
Dogs that are persistently infected or that become moribund after exposure to canine distemper virus (CDV) have antibody that neutralized CDV when tested in dog lung macrophage cultures but failed to neutralize CDV when tested in epithelial, fibroblastic, or lymphatic cells. The antibody attached to protein A and was found in the immunoglobulin G fraction. The antibody bound complement and lysed CDV-infected target cells. The neutralizing activity in macrophages could be abolished (i) by pepsin digestion and removal of Fc portions from the antibody, (ii) by blocking the Fc receptors of macrophages with heat-treated normal dog serum, and (iii) by binding of protein A to Fc portions of the antibody. It was concluded that attachment of the CDV-antibody complex to Fc receptors of macrophages was essential for virus neutralization. If this attachment was hindered, the CDV-antibody complex became infectious for macrophages. In contrast, serum from recovering dogs neutralized CDV when tested in epithelial, fibroblastic, or lymphatic cells as well as in macrophages.
Myocarditis was produced in seronegative five-day-old pups by oral and by intraperitoneal inoculation of canine parvovirus. The disease was subclinical. Histologic lesions were compatible with, but less extensive than, those seen in naturally occurring canine parvoviral myocarditis. In pups necropsied 23 days after inoculation, scattered cardiac myocytes contained intranuclear inclusion bodies, and virus-infected myocytes were demonstrated by immunofluorescence. Degeneration and loss of cardiac myocytes usually was not associated with a cellular infiltrate. At 51 days after inoculation, the myocardium contained an extensive lymphocytic infiltrate which was sometimes associated with fragmented myocytes, and was often contiguous with areas of interstitial fibrosis. At 108 days after inoculation, inflammatory lesions had regressed, and there were multifocal areas of myocardial fibrosis.
Two types of killed canine distemper virus (CDV) vaccine and a modified-live CDV vaccine were clinically evaluated in four species of exotic carnivores. In 16 trials in which 13 red pandas (Ailurus fulgens) were given the killed vaccine, only 1 animal had a virus-neutralization titer that exceeded 1:100. A red panda given modified-live CDV vaccine deemed safe for gray foxes and ferrets died of bacterial pneumonia 16 days later. There was no pathologic evidence of canine distemper in that panda. The same modified-live vaccine proved to be immunogenic and safe in 12 bush dogs (Speothos venaticus), 5 maned wolves (Chrysocyon brachyurus), and 3 fennec foxes (Fennecus zerda) in which virus-neutralization titers often exceeded 1:512 and persisted for several months after vaccination.
Epidemiological studies suggest that multiple sclerosis (MS) might be triggered by an infectious agent. Uveitis has been observed in a small percentage of MS patients. Dogs with canine distemper encephalomyelitis, another demyelinating disease of the central nervous system, have an anterior uveitis which is usually mild and asymptomatic, and dogs with persistent CNS infection and chronic distemper encephalomyelitis harbour virus persistently in the uvea. These observations in dogs suggest that pathological and virological studies of the uveitis associated with MS would be worth while.