Behavior of hematopoietic stem cells in a large animal.

To study the behavior of hematopoietic stem cells in vivo, we transplanted glucose-6-phosphate dehydrogenase (G6PD) heterozygous (female Safari) cats with small amounts of autologous marrow. The G6PD phenotypes of erythroid burst-forming units and granulocyte/macrophage colony-forming units were repeatedly assayed for 3.5-6 years after transplantation to track contributions of stem cell clones to the progenitor cell compartment. Two phases of stem cell kinetics were observed, which were similar to the pattern reported in comparable murine studies. Initially there were significant fluctuations in contributions of stem cell clones. Later clonal contributions to hematopoiesis stabilized. The initial phase of clonal disequilibrium, however, extended for 1-4.5 years (and not 2-6 months as seen in murine experiments). After this subsided, all progenitor cells from some animals expressed a single parental G6PD phenotype, suggesting that blood cell production could be stably maintained by the progeny of one (or a few) cells. As the hematopoietic demand of a cat (i.e., number of blood cells produced per lifetime) is over 600 times that of a mouse, this provides evidence that an individual hematopoietic stem cell has a vast self-renewal and/or proliferative capacity. The long phase of clonal instability may reflect the time required for stem cells to replicate sufficiently to reconstitute a large stem cell reserve.

Perspectives on the epizootiology of feline enteric coronavirus and the pathogenesis of feline infectious peritonitis.

This review presents some current thoughts regarding the epizootiology of the feline coronaviruses; feline infectious peritonitis virus (FIPV) and feline coronavirus (FECV) with primary emphasis on the pathogenesis of these viruses in nature. Although the mechanism(s) whereby FIPV causes disease are still incompletely understood, there have been significant contributions to the literature over the past decade which provide a framework upon which plausible explanations can be postulated. Two concepts are presented which attempt to clarify the pathogenesis of FIPV and at the same time may serve as an impetus for further research. The first involves the hypothesis, originally promulgated by Pedersen in 1981, that FIPV is derived from FECV during virus replication in the gastrointestinal tract. The second involves a unique mechanism of the mucosal immune system referred to as oral tolerance, which under normal conditions promotes the production of secretory immunity and suppresses the production of systemic immunity. In the case of FIPV infection, we propose that oral tolerance is important in the control of the virus at the gastrointestinal tract level. Once oral tolerance is disrupted, FIPV is capable of systemic spread resulting in immune-mediated vasculitis and death. Thus, it may be that clinical forms of FIP are due to a combination of two events, the first being the generation of FIPV from FECV, and the second being the capacity of FIPV to circumvent oral tolerance.

Evidence for the maintenance of hematopoiesis in a large animal by the sequential activation of stem-cell clones.

To test if hematopoiesis can be maintained by the sequential activation of stem-cell clones, we performed autologous marrow transplantations with limited numbers of cells in cats heterozygous for the X chromosome-linked enzyme glucose-6-phosphate dehydrogenase (G6PD) and observed the G6PD phenotypes of erythroid and granulocyte/macrophage progenitors over time. The animals were the female offspring of Geoffroy male and domestic female cats. In repeated studies of marrow from control animals (n = 5) or experimental animals prior to transplantation (n = 3), the percent of progenitors with domestic-type G6PD did not vary. After transplantation, the peripheral blood counts, marrow morphologies, frequencies of progenitors, and progenitor cell cycle kinetics returned to normal. However, abrupt and significant fluctuations were seen in the G6PD type of progenitors from each cat during the 1-1.5 years of observation. These data cannot be explained if there were either a large or constant population of active stem cells and thus imply, in a large-animal system, that hematopoiesis was maintained through clonal succession. A stochastic model was developed to estimate the numbers of active clones and their mean lifetimes.

Comparative properties of feline coronaviruses in vitro.

Two feline coronaviruses were characterized to determine their biological properties in vitro and their antigenic relatedness to a previously recognized feline infectious peritonitis virus and canine coronavirus. The viruses, designated WSU 79-1146 and WSU 79-1683, were shown to have comparable growth curves with the prototype feline infectious peritonitis virus. Treatment of the feline infectious peritonitis virus strains with 0.25% trypsin indicated that they were relatively resistant to proteolytic inactivation when compared with the feline enteric coronavirus strain. This observation may serve as a useful in vitro marker to distinguish closely related members of the feline coronavirus group. Plaque assay results indicated that the feline infectious peritonitis virus strains produced large homogeneous plaques in comparison to the feline enteric coronavirus strain and canine coronavirus, which showed a heterogenous plaque size distribution. No naturally temperature sensitive mutants were detected in either of the feline coronavirus populations. Both of the viruses were antigenically related to feline infectious peritonitis virus and to a lesser extent to canine coronavirus by virus neutralization.

Lymphocytes and antibody in retrovirus-induced feline pure red cell aplasia.

The possible role of antibody and T-lymphocytes was investigated in the pure red cell aplasia (PRCA) associated with feline leukemia virus, subgroup C (FeLV-C), infection. In previous studies, erythroid colony-forming cells were undetectable in marrow culture of cats with PRCA. Yet erythroid burst-forming cells (BFU-E) remained, suggesting that BFU-E were able to differentiate in vitro but not in vivo. It was inferred that immunologic suppression may contribute to the pathogenesis of feline PRCA, and the interactions of antibody and T-lymphocytes with erythroid and granulocyte-macrophage progenitors were studied. Incubation of normal or PRCA marrow cells with PRCA serum or IgG concentrated from this serum and then complement (C') failed to decrease hematopoietic colony growth when compared to the results obtained with cultures of marrow cells incubated with C' alone. In crossover coculture studies, T-cells from Safari cats with PRCA had no inhibitory effect on colony growth from normal or autologous PRCA marrow cells. For the determination of whether feline PRCAs were associated with a clonal T-cell process, lymphocytes were obtained periodically from glucose-6-phosphate dehydrogenase (Glc-6-PD) heterozygous cats following FeLV-C infection and were expanded with a crude preparation of interleukin-2. The ratios of Glc-6-PD enzyme types in these samples did not change as cats developed anemia, suggesting that the inhibition of erythropoiesis was not associated with the clonal expansion of T-cells. These studies, therefore, do not support the premise that feline PRCA results from the interaction of antibody or T-cells with erythroid progenitors.

Diagnosis of feline viral infection.

A diagnosis of a specific viral disease in the cat involves a combination of an accurate history, careful observation of disease signs, demonstration of characteristic clinical pathologic changes, and isolation or identification of the virus. Isolation or identification of a virus from the patient does not establish that the disease observed was caused by the virus so isolated or identified; correlation and proper interpretation of all findings are necessary to establish a diagnosis. Virus identification may involve office laboratory tests, such as cytology or ELISA, or more specialized procedures. Whether specimens are to be sent out for specialized tests or office laboratory procedures are to be used, the veterinary practitioner must not only know what specimens are required but must also understand the test and be able to properly interpret the results in light of the patient's observed condition.

Isolation and identification of caliciviruses from dogs with enteric infections.

Caliciviruses were isolated from 7 dogs and 1 captured coyote with enteritis. There was a high fatality rate in dogs 4 to 16 weeks of age. The occurrence in these dogs of concurrent infection with known enteric pathogens such as Salmonella sp, canine parvovirus, canine coronavirus, and canine rotavirus did not allow making any conclusions regarding the pathogenicity of this newly recognized calicivirus. The caliciviruses were characterized by electron microscopy and were further identified as being closely related to feline calicivirus by immunoelectron microscopy with specific antibody.

Feline glucose-6-phosphate dehydrogenase cellular mosaicism. Application to the study of retrovirus-induced pure red cell aplasia.

Neoplasms result from the uncontrolled proliferation of abnormal or transformed cells. The early stages of this process are difficult to study because of the lack of sensitive and specific markers of clonal evolution in an experimental system. We have developed a cat model using cellular mosaicism for glucose-6-phosphate dehydrogenase (G-6-PD). Our findings confirm that the structural locus for feline G-6-PD is on the X-chromosome and demonstrate that it is randomly inactivated in somatic cells. Heterozygous cats have balanced ratios of G-6-PD enzyme types in peripheral blood cells and hematopoietic progenitors that remain stable over time. In our initial studies, we used the model to analyze the events surrounding marrow failure experimentally induced by selected strains of feline leukemia virus (FeLV). Two G-6-PD heterozygous cats, one F1 male hybrid and one domestic cat were infected with FeLV (C or KT) and developed pure red cell aplasia (PRCA). Colonies arising from the more mature erythroid colony-forming cell were not detected in marrow culture of anemic animals although erythroid bursts persisted, suggesting that the differentiation of early erythroid progenitors (BFU-E) was inhibited in vivo. The ratio of G-6-PD types in hematopoietic progenitors and peripheral blood cells from the heterozygous cats did not change when the animals developed PRCA. Thus, the anemia did not result from the clonal expansion of a transformed myeloid stem cell. With this experimental approach, one may prospectively assess clonal evolution and cellular interactions in other FeLV-induced diseases.

Pathogenicity studies of feline coronavirus isolates 79-1146 and 79-1683.

Two feline coronavirus isolates were characterized by their disease-causing potential in cats. The 79-1683 feline coronavirus isolate caused an inapparent-to-mild enteritis when given oronasally to specific-pathogen-free kittens and was not a cause of feline infectious peritonitis (FIP). Target tissues for the virus were the mature apical epithelium of the small intestine, mesenteric lymph nodes, tonsils, thymus, and (to a lesser extent) the lungs. Inoculated kittens shed high numbers of virus in their feces for 14 to 17 days, but remained infectious to susceptible kittens for longer periods of time, as evidenced by contact-exposure studies. Because the 79-1683 isolate induced only enteritis, it was designated feline enteric coronavirus (FECV) 79-1683. The 79-1146 feline coronavirus isolate induced effusive abdominal FIP in specific-pathogen-free kittens after oronasal and intraperitoneal inoculation. Clinical signs of disease appeared within 12 to 14 days in almost all inoculated kittens. Because this isolate caused FIP, it was designated FIP virus (FIPV) 79-1146. Cross-protective immunity was not induced by the various coronavirus infections. Kittens preimmunized with the UCD strain of FECV (FECV-UCD) or with FECV-79-1683 were not immune to infection with FIPV-79-1146. Likewise, kittens previously inoculated with FECV-79-1683 were not immune to infection with FIPV-UCD1. In fact, preexisting heterologous FECV-79-1683 immunity often accelerated and enhanced the severity of disease caused by inoculation with FIPV-UCD1.(ABSTRACT TRUNCATED AT 250 WORDS)

Congenital myasthenia gravis in 13 smooth fox terriers.

In 13 Smooth Fox Terriers with a congenital form of myasthenia gravis, clinical signs included intermittent, progressive muscle weakness that became more pronounced with exercise; muscle wasting; megaesophagus; and aspiration pneumonia. Neurologic abnormalities were apparent only during periods of weakness and included inability to retract the fore- and hindlimbs from painful stimuli. A decrement of the compound muscle action potential was evident during repetitive supramaximal nerve stimulation. Intravenous injection of a short-acting cholinesterase inhibitor evoked immediate improvement of clinical and electromyographic signs. Intracellular microelectrode studies of a biopsied external intercostal muscle revealed reduced amplitude of miniature end-plate potentials, as occurs in acquired myasthenia gravis. However, in contrast to acquired myasthenia gravis, antibodies directed against acetylcholine receptors were not demonstrable in serum and were not bound to acetylcholine receptors in muscle. Despite lack of complexing with immunoglobulin, the amount of acetylcholine receptor protein in biopsied external intercostal muscles from 9 affected pups was less than 25% of the amount in 5 unaffected littermates. The latter finding accounted for the reduction in amplitude of miniature end-plate potential and the failure of neuromuscular transmission. Treatment with a long-acting cholinesterase inhibitor in 6 cases resulted in temporary improvement in muscle strength.

Diarrheal condition in dogs associated with viruses antigenically related to feline herpesvirus.

Viruses with properties consistent with herpesvirus were isolated from dogs with diarrhea. The viruses were shown to be antigenically related to feline herpesvirus-1 (FHV-1) by virus neutralization tests. It was also observed that a canine herpesvirus (CHV) prototype, D004, and two field isolates from fatal CHV infections in 2-week-old and 6-week-old puppies were neutralized at a low level by antiserum to FHV-1. Reciprocal neutralization tests with CHV antiserum against FHV-1 were negative. These results indicated that viruses related to FHV-1 can infect the dog and that there appears to be uni-directional virus neutralization of CHV by FHV-1 antibody.

Characterization of a feline infectious peritonitis virus isolate.

A virus isolated in cell culture from the spleen of a cat with feline infectious peritonitis was identified by physicochemical, morphological and antigenic criteria as a coronavirus. The feline infectious peritonitis virus was compared in vitro with canine coronavirus, a reported enteric pathogen of dogs. The feline isolate was characterized, by chloroform sensitivity and resistance to 5-iododeoxyuridine, respectively, as containing essential lipid and an RNA genome. Other traits of the isolate included resistance to acidic conditions, heat lability, and resistance to trypsin. Electron microscopy showed viral particles with a structure consistent with that of the prototype of the coronavirus group, infectious bronchitis virus. Indirect immunofluorescence with canine coronavirus monospecific antiserum showed the viral isolate to be antigenically related to canine coronavirus. Specific-pathogen-free cats inoculated by various routes with cell-culture-propagated virus had both clinical symptoms and lesions consistent with feline infectious peritonitis.

A muscle disorder of Labrador retrievers characterized by deficiency of type II muscle fibers.

A muscle disorder was detected in 5 Labrador Retrievers. The age of onset was less than 6 months. The clinical signs, which were exaggerated with exercise, cold, or excitement, included abnormal head and neck posture, and a stiff, forced, hopping gait. A marked deficiency of skeletal muscle mass resulted in poor conformation. Both black and yellow Labrador Retrievers were affected, and pedigree studies indicated the disorder may have been inherited as an autosomal recessive trait. Some relief of the clinical signs was afforded by treatment with diazepam. Marked creatinuria and low serum creatine phosphokinase activity were the only consistent, abnormal laboratory findings. Histologic and cytochemical examinations of muscle biopsy specimens revealed a relative predominance of type I and a deficiency of type II skeletal muscle fibers. Electromyographic studies of the affected dogs indicated a myotonic defect.