[CANCER RESEARCH 29, 33—39,January 1969J

SUMMARY

The irises of seven chickens with lesions of spontaneous oculas leukosis were examined by light and electron microscopy.Histologically, the abnormal contents of the irises includednecrotic striated musculature, mononudear cells, and granulocytes. Observed by electron microscopy, viral particles wereembedded in the basement membrane of striated muscle cells,in membrane-bound vesicles in muscle cells, in proximity topigmented epithelial cells, and in the lumen and basementmembrane of endothelial cells of capillaries of the iris. Necrosis of striated muscle cells occurred where large masses of viralparticles were observed extracellularly and in vesicles in thecell. Particles budded from the cell membrane and the membrane surrounding vesicles.

It appeared that depigmentation of the irises of chickenswith ocular leukosis was correlated with necrotizing changes ofthe striated muscle cells in the irises.

INTRODUCTION

Discoloration of the irises of chickens with ocular leukosishas been observed by several workers; the disease has beentransmitted experimentally, with the production of neural andvisceral lesions as well as ocular changes (7, 9, 10). In one cxperiment, the ocular lesion developed five days after the injection of 0.01 to 0.02 ml of a specffic isolate into the anteriorchamber of the eye (9). As a result, the pupil was dilated andelliptical or irregular in shape. The iris was notched and grayinstead of orange.

The irises of chickens with lesions of ocular leukosis havebeen examined histologically (1) but not with the electronmicroscope. Thus, the present electron microscopic study is reported: Viral particles were observed in the irises of chickenshaving typical lesions of ocular leukosis.

MATERIALS AND METHODS

Five male and two female, 20-week-old chickens having bilateral lesions of ocular leukosis, were culled from a commer

1Supported in part by a grant (H-6580) from the National Heart Institute, Florida Agricultural Experiment Stations Journal Series No.3010.

Received June 24, 1968; accepted September 19, 1968.

ical breeder flock. Four of these birds had partial paralysis ofthe legs or wings. Eyes were secured from two normal chickens. At autopsy, small pieces of the sciatic nerves and irises ofeach chicken were fixed in 1@Y7oformalin, embedded in paraffin, cut at 5 11,and stained with hematoxylin-cosin stain. Anadjacent portion of iris was fixed in 2.5% buffered glutaraldehyde, postfixed in 1% 0504, and embedded in Araldite (6).No other tissues were examined with the electron microscope.Ultrathin sections on grids were stained with lead citrate (8)prior to being examined with a Philips EM200 electron microscope.

RESULTS

Gross Pathology. The irises of chickens that had ocular leukosis were gray. The pupils of these eyes were dilated and ciliptical or irregular in shape. The irises of birds with normaleyes were orange; the pupils were even and circular. The sciaticnerves of the four chickens with partial leg paralysis wereedematous and yellow, as contrasted to the slender, white sciatic nerves of birds without ataxia. One of the birds with grayirises had white, nodular, tumorous masses in the liver. Another of the chickens having ocular lesions had a large ovariantumor.

Histopathology. Small numbers of granulocytes and mononuclear cells consisting of lymphocytes and plasma cells werepresent in the irises ofleukotic chickens. Striated muscle fibersin the irises were fragmented and necrotic (Fig. 1). The sciaticnerves of partially paralyzed chickens were sparsely infiltratedby small lymphocytes. Tumors in the liver and ovary werecomposed of diffuse masses of cells with the characteristics oflymphocytes, plasma cells, and reticulum cells, many of thelatter containing mitotic figures.

Electron Microscopy. The irises from normal chickens contamed striated muscle cells, collagenous fibers, myelinatednerve fibers, and capillaries. Striated muscle cells containinglipid, glycogen, a nucleus, mitochondria, and sarcoplasmicreticulum had infoldings of the sarcolemma and were lined bya prominent basement membrane. The border of the iristoward the lens was lined by a layer of pigmented epithelialcells three to five cells thick. On their surfaces, these cells hadelongated, twisted microvilli which intertwined with those ofadjacent cells. The border of the iris toward the cornea waslined by a single layer of nonpigmented epitheial cells.

Although the iris of the leukotic eye contained collagenousfibers and myelinated nerve fibers similar to those observed in

33JANUARY 1969

Electron Microscopy of the Irises of Chickens with Spontaneous

Ocular Leukosis'

Charles F. Simpson

Department of Veterinary Science, University ofFlorida, Gainesville, Florida 32601

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Charles F. Simpson

the normal eyes, degenerative changes were seen in striatedmuscle cells. Viral particles were usually embedded in basement membranes lining muscle cells (Fig. 2). For the mostpart, they accumulated in packets of three to twelve or more,especially in the invaginations of the cell membrane (Fig. 3).There was thickening and increased osmiophilia of the sarcolemma (Fig. 4) where the viral particle and cell membranewere in close apposition. It seems likely, as suggested by others(2, 4), that such thickening of the sarcolemma might be attributed to pinocytosis (Fig. 4 insert). Budding from the sarcolemma seemed to be involved in the separation of the virusfrom the cell cytoplasm (Fig. 5). Striated muscle cells alsocontained viral particles within membrane-bound vesicles. Itappeared that such particles emerged as buds from the vesicular membranes (Fig. 6) and the peripheral cell membrane.

There appeared to be a correlation between severity ofmyopathy and extent of viral invasion of the muscle cell.Where there were few intravesicular particles, there was nodisruption of myofibrils or alteration of their crossbanding(Fig. 7). Separation of myofibrils and disruption of banding(Fig.8)occurredwhereextensiveaccumulationsofextracellular particles and moderate numbers of intravesicular particleswere observed. Necrosis and disappearance of myofibrils (Fig.9) occurred in cells containing large masses of viral particles,both extracellularly and within vesicles. In addition, necroticmuscle cells contained myelinated figures, probably degenerated lipid droplets (Fig. 9). Mitochondria were altered in cellsthat were heavily laden with virus; such organelles were swollen, misshapen, and contained fragmented cristae (Fig. 10).

Viral particles were not observed within membrane-boundspaces in pigmented epithelial cells, but sizeable clusters ofvirus were trapped in the invaginations of the plasmalemmaformed by branching microvilli on the surfaces of these pigmented cells (Fig. 1 1). As a result, viral particles could bemistakenly interpreted as being present within membranebound vesicles in the cells.

Viral particles were also seen in the capillaries of the iris.Particles were observed not only in lumens of these bloodvessels, but they were also embedded in the basement membrane of endothelial cells and in pericytes of the capillaries(Fig. 12).

Viral particles had a uniform ultrastructural appearance, regardless of location. They varied from 110 to 130 m@zin diameter, had an inner ring and an outer membrane, and had acentral nucleoid which varied in size from 40 to 46 rni. Theinner ring was separated from the nucleoid, and the outermembrane had surface projections (Fig. 13)

Viral particles were not seen in the nucleus of muscle cells. Afew plasma cells, granulocytes, and lymphocytes were presentin the vicinity of necrotic muscle cells.

DISCUSSION

Irises having lesions typical of ocular leukosis were examinedin the present work. Previous investigators have reported thatocular leukosis, “grayeye,― resulted from infiltration of theiris with lymphocytes, plasma cells, and granulocytes, resultingin a grayish, deformed, and paralyzed iris (1, 3). These cellswere scarce in the ocular lesions studied by light and electron

microscopy in the investigation reported herein. Instead, thesignificant observation was the presence of a multitude of viralparticles in membrane-bound vesicles and around striatedmuscle cells, in the close vicinity of pigmented epithelial cells,and in the lumen, basement membrane, and pericytes of capillanes. Muscle cells heavily infected with virus, both intravesicular and extracellular, were necrotic. Thus, it was concluded that necrotizing changes of the striated muscle werecorrelated with color change of the iris, as well as notching orrupture. Comparisons with other works could not be madebecause there do not appear to be any previous reports ofelectron microscopic studies of the irises of leukotic chickens.

The viral particles observed in irises of leukotic eyes appeared to be similar to those of the leukosis-sarcoma group ofviruses which emerge as buds from the cell membrane (5, 12,13) and exhibit pinocytosis (4). The agent in the iris was ultrastructurally similar to that described in the periosteum ofchickens with experimental osteopetrosis (11).

ACKNOWLEDGMENTS

The technical assistance of J. W. Carlisle and L. Mallard is acknowledged.

REFERENCES

1. Biester, H. E., and Schwarte, L. H. Diseases of Poultry, Ed. 5, pp.527—530. Ames: The Iowa State University Press, 1965.

2. Bowers, B. Coated Vesicles in the Pericardial Cells of the Aphid(Myzus persicae Sulz). Protoplasma, 59: 351—367, 1964/65.

3. Campbell, J. G. A Proposed Classification of the Leukosis Complexand FowlParalysis. Brit. Vet.J., 117: 316—325,1961.

4. Febvre, H., Rothschild, L. Arnoult, J., and Haguenau, F. In vitroMalignant Conversion of Rat Embryonic Cell Lines with the BryanStrain of Rous Sarcoma Virus. Internat. Conf. on Avian TumorViruses. Natl. Cancer Inst. Monograph, I 7: 459—477, 1964.

5. Heine, U., Bonar, R. A., Becker, C., and Beard, J. W. LysosomeSystem of Avian Leukemic Myeloblasts. Internat. Conf. on AvianTumor Viruses. Natl. Cancer Inst. Monograph, I 7: 677—700, 1964.

6. Luft, J. H. Improvement in Epoxy Resin Embedding Methods. J.Biophys. Biochem. Cytol., 9: 409—414, 1961.

7. Pappenheimer, A. M., Dunn, L. C., and Cone, V. A Study of FowlParalysis(Neuro-lymphomatosisGallinarum). Storrs Agri. Exp. Stat.Bull., 143: 187—290, 1926.

8. Reynolds, E. S. Use of Lead Citrate at High pH as an ElectronOpaque Stain in Electron Microscopy. J. Cell BioL, 17: 208—212,1963.

9. Sevoian, M., and Chamberlain, D. M. Avian Lymphomatosis II.Experimental Production of the Ocular Form. Vet. Med., 57:608—609,1962.

10. Sevoian, M., and Chamberlain, D. M. Avian Lymphomatosis III.Incidence and Manifestations in Experimentally Infected ChickensofVarious Ages. Avian Diseases, 7: 97—102, 1963.

11. Simpson, C. F., and Sanger, V. L. Electron Microscopy of thePeriosteum in Experimental Osteopetrosis. Cancer Res., 26:590—595, 1966.

12. Vogt, P. K. Avian Tumor Viruses. Advances in Virus Res., 11:293—385, 1965.

13. Zeigel, R. F., Theilen, G. H., and Twiehaus, M. J. Electron Microscopic Observations on RE Virus (Strain T) that Induces Reticuloendotheliosis in Turkeys, Chickens, and Japanese Quail. J. Natl.Cancer Inst., 37: 709—729,1966.

34 CANCER RESEARCH VOL.29

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Avian Ocular Leukosis

Fig. 1. Striated musculature (M) is fragmented in the iris of a bird having ocular leukosis. The cells (arrows) in that area are lymphocytes, plasmacells, and granulocytes. H & E, x 125.

Fig. 2. Several packets ofviral particles (arrows) are embedded in the basement membrane of a striated muscle cell. x 15,000.Fig. 3. Viral particles (V) are present in invaginations of the sarcolemma of muscle cells. X 25,000.

Fig. 4. There is thickening and increased osmiophiia of the muscle cell sarcolemma (arrows) where it is in close apposition with viral particles(V). x 30,000. Insert. The thickened sarcolemma(arrow) has almost surrounded a viral particle. x 45,000.

Fig. 5. Sequence of increasing separation of virus from the muscle cell sarcolemma by the process of budding (arrow) as shown in steps athrough d. x 100,000.

Fig. 6. Virus particles seem to emerge as buds from the sarcolemma (arrow) of a striated muscle cell and from the membrane lining intracellularvesicles (V). x 55,000.

Fig. 7. Disruption of myofibrils does not occur if there are only a few intravesicular particles present (arrows). The delicate, granular material isglycogen (G). There are many extracellular particles (V) embedded in the basement membrane ofthe muscle cell. x 20,000.

Fig. 8. Disruption of myofibrils and bandings (B) occurs when there are numerous extracellular particles ( V) and a moderate number ofintravesicular particles (arrows) in a muscle cell. X 20,000.

Fig. 9. Necrosis of the muscle cell and disappearance of fibrils (F) occur when there are large masses of virus, both extracellular ( V) andintravesicular (arrows). X 18,000. Insert. A myelinated figure in the cytoplasm (M), and virus (V) in the basement membrane. X 12,000.

Fig. 10. Lipid (L) in cytoplasm of a muscle cell with extracellular virus (II has a normal appearance; mitochondria (M) are swollen and thecristae fragmented. x 18,000.

Fig. 11. In pigmented epithelial cells, viral particles (1') are present in invagination of the plasmalemma, formed by branching microvilli (arrow)on the surface of the cells. x 30,000. Insert. The structure of branching microvilli (arrow) on the surface of pigmented epithelial cells can beobserved. X 12,000.

Fig. 12. Viral particles (V) are free in the lumen and are embedded in the basement membrane (arrows) of endothelial cells of a capillary. x12,000.

Fig. 13. Viral particles embedded in the basement membrane of a muscle cell have inner (I) and outer (0) membranes and a nucleoid (N). X100,000.

JANUARY 1969 35

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1969;29:33-39. Cancer Res   Charles F. Simpson  Ocular LeukosisElectron Microscopy of the Irises of Chickens with Spontaneous

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