William J. Hadlow

Virologic and neurohistologic findings in dairy goats affected with natural scrapie.

Virologic and neurohistologic findings in three dairy goats that became affected with scrapie while living with naturally infected Suffolk sheep were essentially like those in affected sheep. Virus, detected by mouse inoculation, was widespread in non-neural sites, particularly in lymphatic tissues and intestine. In most sites, titers of virus ranged from 3.0 to 3.5 log10 mouse intracerebral LD50/30 mg of tissue. Virus was in nervous tissue in much higher titer. Ranging from 5.1 to 5.8 log10, the highest mean titers were in the diencephalon, midbrain, medulla oblongata and cerebellar cortex—sites of the most severe histologic changes. Although these changes were like those in naturally affected Suffolk sheep, they differed somewhat from those in goats affected with the experimental disease. Spongiform alteration of neuropil was minimal, and the more rostral parts of the brain, such as corpus striatum, globus pallidus and septal area, had few changes. Concentrations and distribution of virus in non-neural tissues were consistent with the conclusion that scrapie virus no doubt can be maintained by contagion in a herd of goats living apart from infected sheep.

Some Properties of the Scrapie Agent and Its Behavior in Mice

Discussion and summary The agent isolated by Chandler in mice inoculated intracerebrally with brain tissue from goats affected with experimental scrapie can be passed serially in mice by this route. In the Rocky Mountain Laboratory stock of Swiss mice it causes a relentlessly progressive, and invariably fatal, disease of the central nervous system, usually after an incubation period of 4 to 5 months. The disease is characterized clinically by incoordination of gait, muscular atrophy, and drowsiness and neuropathologically by prominent astrocytosis, degeneration of neurons, and spongy alteration of the neuroparenchyma. That the agent causing this disease in mice is the transmissible agent of scrapie may be questioned. At present, identification of the scrapie agent can be made only by observing its pathogenic capacity in susceptible sheep and goats. In these hosts it induces a distinctive clinicopathologic picture whose essential features also characterize the infection in mice. Thus, the long incubation period, the slowly progressive course of the disease, and the degenerative changes in the central nervous system all are consistent with the view that the agent under study is the transmissible agent of scrapie. Moreover, first passage mouse brain has induced scrapie in goats inoculated intracerebrally(4). At this laboratory, goats inoculated intracerebrally or subcutaneously with a 10-1 suspension of fourth passage mouse brain have been observed for too brief a period (5 months) to permit any conclusion about the ability of this inoculum to cause typical caprine scrapie. The high concentrations of the agent in the brain, spinal cord, spleen, and thymus of affected mice make it appear unlikely that a toxic substance is being passed. That the agent passes through a gradacol membrane of 100 mμ average pore diameter, but not one of 30–100 mμ, suggests it is a medium-sized virus.

Experimental Scrapie in the Mouse: Electrophoretic and Sedimentation Properties of the Partially Purified Agent

: Some biochemical and biophysical properties of the scrapie agent in a partially purified fraction P5 from murine spleen are described in this communication. The agent was stable in the nonionic detergents Triton X‐100 and Nonidet P40 and stable in the nondenaturing, anionic detergents sodium cholate and sodium N‐lauroyl sarcosinate. In contrast, sodium dodecyl sulfate (SDS) inactivated the agent at high concentrations (1% or >) when the detergent‐to‐protein ratio approached 1.5 g SDS/g protein. The agent was resistant to inactivation by nucleases and proteases, even in the presence of 0.1% SDS. A broad peak of infectivity was exhibited in modified colloidal silica (Percoll) density gradients. Maximal titers were found at a Percoll density of 1.10 g/cm3 in the presence and absence of 0.05% SDS. Gel electrophoresis of the agent in the presence of 0.1% SDS resulted in inactivation of >95% of the agent loaded onto the gel. Free‐flow electrophoresis showed that >99% of the agent in fraction P5 migrated toward the anode, but not as a discrete species. Sedimentation analysis of the agent in fraction P5 in the presence of 1% lysolecithin showed that the agent has a sedimentation coefficient of <300S but >30S. Heating P5 preparations caused the agent to associate with cellular elements and form aggregates with sedimentation coefficients >10,000S. Removal by differential centrifugation of the large forms of the agent produced upon heating permitted characterization of a discrete subpopulation of scrapie agent particles. Rate‐zonal sucrose gradient studies showed that >>95% of the infectivity in this subpopulation sedimented as uniform particles with a sedimentation coefficient of 240S.

Studies on salmon poisoning disease of canines: I. The rickettsial relationships and pathogenicity of Neorickettsia helmintheca☆

Abstract The agent of so-called “salmon poisoning disease” of dogs on the Pacific Coast of the USA, first reported by Cordy and Gorham (1950) , is an intracytoplasmic, rickettsia-like, sometimes pleomorphic microorganism found particularly in the reticulo-endothelial cells of lymphoid tissues of infected Canidae. It is transmitted in nature to members of the Canidae by ingestion of fish containing encysted cercariae of the intestinal fluke, Nanophyetus salmincola. The natural, developmental cycle of this trematode includes snails, Goniobasis pliciera silicula, and fish of the family Salmonidae. The infection is transmissible in series by blood and by fresh or frozen node tissues of infected to susceptible dogs as well as by suspension of adult flukes. There is an incubation period of 8 to 10 days followed by several days of an acute febrile episode. Mortality is high. When there is occasional recovery, the dogs are solidly immune. Reasons are presented for assigning this pathogen to the family Rickettsiaceae by modifying the vector character of the family from “arthropod-borne” to “invertebrate-borne” agents, and for having given this the name of Neorickeltsia helmintheca Philip, Hadlow, and Hughes (1953) .

Carcinoma of the Anal Sac Glands in Ranch Mink

During a 14-year period, carcinoma of the anal sac apocrine glands was found in 52 pastel and 8 sapphire mink (Mustela vison) kept for studies on slow viral diseases. The pastel mink varied in age from 72 to 135 months (mean age 108 months), the sapphire mink from 63 to 100 months (mean age 81 months). All but one pastel mink were females. The primary tumor varied in size from masses that caused bulges in the perineum to those that were found only after microscopic examination of the anal sac glands. Although the primary tumor grew mainly by expansion with little local infiltration, 41 of the 60 tumors had metastasized to the regional lymph nodes and sometimes also to more distant sites. The striking propensity of the carcinoma to metastasize while still small, even microscopic, often resulted in massive secondary growths, notably in the iliac lymph nodes. Hypercalcemia did not accompany the carcinoma. Its varied microscopic appearance included solid, glandular, squamous cell, and spindle or round cell components. Combinations of them formed mixed or complex histologic patterns, no doubt largely attributable to neoplastic proliferation of myoepithelial cells and squamous metaplasia of the apocrine gland epithelium. Although its cause remains obscure, the carcinoma appeared to arise from small foci of hyperplastic apocrine glands, sometimes in relation to both anal sacs. The tumor is a common and distinctive expression of neoplasia in older ranch mink.

Ocular Lesions in Mink Affected with Aleutian Disease

Uveitis, characterized by infiltrates of lymphocytes and plasma cells, was the principal ocular lesion in 122 sapphire and pastel mink affected with experimental Aleutian disease. It was present to various degrees in all but five mink examined five to 164 weeks after inoculation (intraperitoneal or intranasal) with any of four North American strains of Aleutian disease virus. The uveitis, mostly iridocyclitis, was accompanied often by protein-rich fluid in the anterior chamber and less often by fibrin and cells in the vitreous body. Cellular infiltration of the limbus, seldom pronounced, also occurred in about 20% of the mink. In 11 mink with moderate or severe uveitis, the retina was detached by pools of protein-rich fluid. Infiltrates of lymphocytes, plasma cells, and a few histiocytes often were found in the orbital soft tissues, occasionally in association with retrobulbar arteritis. In general, the ocular lesions were more severe in sapphire than in pastel mink. The uveitis accompanies glomerulonephritis, the principal lesion of Aleutian disease, much more regularly than do several other lesions of the disease. Like the glomerulonephritis, it probably results from the deposition of circulating immune complexes.

Cerebrocortical Degeneration in Goats Inoculated with Mink-passaged Scrapie Virus

Widespread spongiform degeneration of the cerebral cortex occurred in four African pygmy goats that became affected with scrapie after intracerebral inoculation with scrapie virus (Suffolk sheep brain origin) that had been passed three times in ranch mink. The occurrence of such cerebrocortical degeneration was a distinct departure from the topographic pattern of neuropathologic changes that characterizes scrapie in sheep and goats. But the cortical lesion was identical to the one found in goats that became affected with a disease otherwise indistinguishable from scrapie after intracerebral inoculation with transmissible mink encephalopathy (TME) virus that had been passed twice in mink. If TME originated from infection with wild scrapie virus, as is generally thought, then the viruses used in these two instances would be equivalent in their passage history in this aberrant host. Given this similarity, the common occurrence of the cortical lesion is thought to be consistent with the view that TME virus almost certainly is scrapie virus whose biologic properties became altered by chance passage in ranch mink.

Tissue Culture Studies of the Virus of Progressive Pneumonia, A Slow Infectious Disease of Sheep

Summary Properties of progressive pneumonia virus (PPV) were studied in sheep lung cell monolayers. Growth studies revealed that, with a multiplicity of infection of 2 TCID50 virus/cell, virus replicated to a titer of 106.6 TCID50 virus/ml in 4 days. Typical polykaryocytes were first seen at 1 day post inoculation (PI) and became larger and more numerous as the virus titer increased. Inoculation of high concentrations of virus produced cell fusion within 3 hr PI. Indirect immunofluorescence revealed homogeneous cytoplasmic fluorescence in infected cells as early as 1 day PI. The number of cells showing fluorescence and the intensity of the fluorescence increased steadily with increase of virus titer. The genetic material for PPV is probably single-stranded RNA. The various properties indicate that PPV is closely related to maedi, zwoegerziekte, and visna viruses.

Salmon Poisoning Disease of Canines. II. Further Observations on Etiologic Agent

Summary To elucidate the method of persistence of the rickettsialike agent of salmon poisoning disease of dogs, Neorickettsia helminthoeca, the authors failed to infect dogs in 2 tests by injection of eggs laid by flukes from infected dogs. In 5 tests by injection of dogs with fluke stages from dissected livers of Oregon snails, 3 resulted negatively, while in 2 others, the dogs developed characteristic severe infections, confirmed pathologically, after unusually prolonged incubation periods. Flukes and node tissues from antibiotic-treated, recovered dogs, and flukes from a refractory raccoon were shown to remain infectious. As little as 250 mg (one commercial capsule at one feeding) of aureomycin or terramycin were shown to save 15-pound beagles when given as late as the fourth day of fever compared to fatal infections in controls. In one titration experiment, node suspension was found to be infectious in a dilution of 10-6.7. Although the agent has been assigned to the Rickettsiaceae, it appears genetically distinct from Ehrlichia canis and Colesiota spp.

Common Occurrence of Urethral Cysts in Older Female Ranch Mink

Cysts were found often in the proximal urethra of female sapphire and pastel mink, Mustela vison, examined at necropsy during studies on slow viral diseases. Their prevalence, the same in both color phases, was age-dependent. They occurred in less than 2% of females under 2 years old but in more than 60% of those over 7 years old. The cysts varied from ovoid vesicles 3 to 4 mm long to multilocular masses 10 to 15 mm across that greatly distended the proximal urethra and sometimes occluded it. Small cysts were not accompanied by clinical signs, but large ones often caused persistent urinary incontinence and occasionally, urine retention. The cysts contained fluid that varied from water-clear to dull yellow. They arose by expansion of small urethral glands normally present in female mink and destroyed much of the urethral wall by pressure atrophy. Although the cysts became larger and more prevalent as the mink aged, the stimulus that caused them to form was not apparent. They appear to have no counterpart in other animals.