Vet. Pathol. 12: 178-185 (1975) Amyloidosis Induced in Hamsters by a Filarid Parasite (Dipetalonema viteae) W. A. CROWELL and C. L. VOTAVA Departments of Veterinary Pathology and Veterinary Parasitology, College of Veterinary Medicine, University of Georgia, Athens, Cia. Abstract. Amyloidosis was induced in hamsters infected with the filarial nematode parasite, Dipetalonema viteae. The incidence of amyloidosis was 64% in a group inoculated with 150 larvae and 54% in the group receiving 150 larvae in each of two inoculations. Amyloidosis was not seen in control animals. Microfilariae probably served as the antigenic stimulus in the pathogenesis of amyloidosis, since those animals in which amyloidosis was formed had microfilaremias that were significantly greater (k0.05) both in number and duration than those in infected animals that did not develop amyloidosis. Other reports of amyloidosis associated with filariasis are reviewed. Amyloidosis can be induced by many agents [l, 31 and recently has been associated with filariasis [4, 91. This association may be of importance in naturally occurring filariasis in man and animals. The increased incidence of amyloidosis seen in dogs might be correlated with the increased incidence of dirofilariasis [l 11. The present experiment was designed to substantiate the hypothesis that microfilariae of filarial parasites induce amyloidosis. Dipetalonema viteae, a filarial nematode parasite of rodents [2, 8, 12-14], was chosen because results comparing adult and microfilaria numbers to pathologic changes were available [9]. D. viteae lives in the subcutaneous tissues of the host, and after a prepatent period of approximately 8 weeks, microfilariae can be recovered from the blood. These levels of microfilariae increase to a peak at 10-12 weeks and then gradually decrease, apparently due to the immune response of the host [13].
CROWELL/VOTAVA 179 Materials and Methods Sixty male hamsters (Mesocricetus uurutus), 4-6 weeks of age, were caged individually and maintained on a standard rodent diet (Mouse Chow, Ralston Purina Co., St. Louis, Mo.). Hamsters of this age were utilized so that they would be less than 10 months old at the termination of the experiment, since hamsters develop amyloidosis spontaneously after 1 year of age [6]. After a 2-week period of acclimation, the hamsters were randomly divided into three groups and numbered by toe-clipping. Group A served as uninoculated controls that were killed at the end of the experiment. Each hamster in group B was inoculated subcutaneously with 150 washed, infective third-stage larvae of D. viteue prepared as previously reported [8, 141. Each hamster in group C was inoculated initially with 150 infective third-stage larvae and 2 months later with an additional 150 infective third-stage larvae. Two months after the initial inoculation, blood was collected from each hamster for microfilarial counts. Counts on 20 mms blood samples were done each week thereafter until two or more consecutive negative counts were seen. Mean microfilarial levels were computed from the positive counts. The mean microfilarial levels and the duration (in weeks) of the positive microfilarial counts for amyloid-positive and amyloid-negative animals within group B and C were compared by analysis of variance. R0.05 was chosen as the level of significance. Previous studies have indicated that D. viteue adult worm burden does not play a role in microfilaria levels or pathological changes in glomeruli [9]. Therefore, adult worm burdens were not determined in this study. Postmortem examinations were done on all infected and control hamsters when they died or were killed. Tissue samples were fixed in buffered 10% formalin. These samples were processed for histologic examination. Kidney, liver, and spleen tissues were stained with Congo red and standard toluidine blue. These tissues were examined with a polarizing light microscope and considered positive for amyloid if green birefringence was demonstrated in Congo red tissues and red birefringence with toluidine blue tissues. The amount of amyloid present in tissues was subjectively quantitated from negative (0) to positive (+, ++, *). Electron microscopy was used to confirm the presence of amyloid fibrils in the liver and kidneys of some animals. Samples for electron microscopy were obtained from the formalin-fixed tissue. The samples were rinsed not less than three times in cacodylate buffer before fixation in cacodylate-buffered 1% osmium tetroxide for 1 h. The samples were dehydrated through graded series of alcohols and embedded (epon and araldite, Ernest Fullam, Inc., Schenectady, N.Y.). At least three blocks of each sample were sectioned with a ultramicrotome, collected on 300-mesh copper grids, stained in uranyl acetate and lead citrate, and examined in an e1,ectron microscope. Sections (1 pm thick) were placed on glass coverslips and stained with alkalinized 2% methylene blue. These sections were utilized for orientation. Results Two outbreaks of severe diarrhea occurred during the experiment. Overall mortality was approximately equal in all groups, but during the first out-
180 CROWELL/VOTAVA Table I. Microfilaremia and occurrence of aniyloidosis in three groups of hamsters Group Number Mean (range) Mean (range) Amyloidosis, % of microfilarial duration of animals count microfilaremia, per 20 mm3 weeks A Control (un i n fec ted) 15 0 B Single infected (D. viteae) 14 72.3 (1-628) 10.8 (2-18) 64 C Double infected ( D. viteae) 13 52.5 (1-276) 8.3 (2-24) 54 Table If. Comparison of microfilaremias in D. viteae-infected hamsters with amyloid present (-\ or absent (-)I Group Number Mean (SE) Mean (SE) of animals microfilarial duration of count per 20 mm3 microfilaremia, weeks B + Amyloid 9 B - Amyloid 5 C + Amyloid 7 C - Amyloid 6 103.6' (40.1) I2.7++ (I.6) 16.0 (6.2) 7.6 (1.6) 71.71 (21.5) 10.3++ (2.9) 30.0 (14.4) 6.0 (1.0) +,++=These values are significantly different (Pi0.05) from the corresponding values. break mortality occurred only in infected animals in group B and C (3 and 6 animals, respectively). Mortality during the second outbreak occurred in all groups, but was highest in group A (controls). The cause of the two outbreaks of diarrhea was not ascertained. No pathogenic bacteria or fungi were isolated from these hamsters. Clinical signs and lesions in many of these hamsters suggested proliferative ileitis. Parasitologic and pathologic data from these hamsters were excluded from this report, and only data from those hamsters that survived 5 months or longer were compiled. Parasitologic and pathologic data are shown in tables I and 11. Pathological alterations in selected tissues are shown in figures 1-4. No amyloid was found in control animals, but 9 of 14 (6400) animals in group B and 7
Arnyloidosis and Filariasis 181 of the 13 (54%) animals in group C had amyloidosis. Amyloid was found in the liver, kidney, spleen, or all of these (fig. I, 2). The subjective quantity of amyloid did not differ in group B or C animals. Amyloid fibrils were also demonstrated by electron microscopy in the liver and kidneys of some animals (fig. 3, 4). Chronic glomerulonephritis and focal hepatitis with occasional granulomas occurred in infected animals and have been described previously [9]. Glomeruli of infected animals were characterized by thickened basement membranes, increased cellularity, subendothelial granular deposits, and, in many glomeruli, amyloid fibril deposition (fig. 2-4). These changes are compatible with a diagnosis of membranoproliferative glomerulonephritis and amyloidosis. Spleens from infected animals were occasionally, but not consistently, enlarged. Microscopically, most of the spleens from infected animals had lymphoid and reticuloendothelial cell hyperplasia. Infected animals with amyloidosis had significantly greater numbers of microfilariae (PcO.05) and significantly longer duration of microfilaremias (P<0.05) than infected animals that did not have amyloidosis. Discussion The present study supports three previous studies in our laboratories that have noted the association of filariasis and amyloidosis. These include Litomosoides carinii in jirds (Meriones unguiculatus) [4], D. viteae in hamsters [9], and Brugia phangi in jirds [lo]. The high incidence of amyloidosis in both D. viteae infected groups (64 and 54%, respectively) compared with the absence of amyloidosis in thecontrol group supports our hypothesis that D. viteae can induce amyloidosis. The difference in incidence between infected groups is probably because of differences in microfilaremias, group B microfilaremias being greater in density and duration than those of group C. The paradox is that group Chad lower microfilaremias, even though these animals were superinfected with twice the number of infective larva; however, it previously has been demonstrated that D. viteae adult worm burden is not correlated with microfilariae levels [2,9]. The presence of microfilariae appears to be the major contributing factor to amyloidosis in this study. Hamsters with amyloidosis had significantly higher and significantly longer microfilaremias than those infected animals that did not develop amyloidosis. We do not believe the two outbreaks of diarrhea influenced the development of amyloidosis in this experi-
I82 CROWELL/VOTAVA Fig. 1. A section, I pm thick, of liver of hamster with amyloidosis. There are large amounts of amyloid (A) adjacent t o the hepatocytes (H) and vessels (V). Occasional plasma cells (arrows) are seen near the vessels. Methylene blue. Fig. 2. A section, I pm thick, of kidney of hamster with amyloidosis. The glomerulus has increased cellularity and thickened basement membranes (arrows). Methylene blue.
Amyloidosis and Filariasis I83 Fig. 3. Electron micrograph of glomerular capillary of hamster with amyloidosis. The lumen (L) is greatly reduced in diameter because of subendothelial and infiltrative deposition of amyloid fibrils (A). Epithelial foot processes (f) are fused in focal areas. One mesangial cell (M)is seen. Fig. 4. Electron micrograph of glomerular capillary wall. The basement membrane is fragmented (arrows) and contains subendothelial granular deposits (d) and amyloid fibrils (A). Epithelial foot processes (f) are not fused, and the capillary lumen (L), endothelium (E), and urinary space (U)appear normal.
I84 CROWELL/VOTAVA ment because all groups had animals with diarrhea, and known affected animals were excluded from this report. Although the pathogenesis of amyloidosis is still unknown [5,7], chronic antigenic stimulation is often incriminated in secondary amyloidosis. Microfilariae may act as a persistent antigenic stimulus since there is evidence that an immune response occurs in hamsters infected with D. viteae [12, 131. The type of immune response needs further elucidation. Acknonledgement Supported in part by Animal Disease Research Grant 21-31-GC286-000 No. 160. References 1 ABRUZZON, J.L.: Effect of a gold salt on Leishmania-induced amyloidosis in hamsters. Arthritis Rheum. 11: 812 (1968). 2 BEAVER, P. C. ; ORIHEL, T. C., and JOHNSON, H. C. : Dipetalonema vifeue in the experimentally infected jird, Meriones rrnguiculutus. 11. Microfilaremia in relation to worm burden. J. Parasit. 60: 310-315 (1974). 3 COHEN, A.S.: Amyloidosis. New Engl. J. Med. 227: 522-530, 576583,628-638 (1967). 4 CROWELL, W.A.; MALONE, J. B., jr.; CHAPMAN, W. L., jr., and THOMPSON, P. E.: Arnyloidosis in mongolian gerbils infected with Litomosoides carinii. J. Am. vet. med. Ass. 163: 596-598 (1973). 5 FRANKLIN, E.C.: The complexity of amyloid. New Engl. J. Med. 290: 512-513 (1974). 6 GLEISER, C. A.; VAN HOOSIER, G. L.; SHELDON, W. G., and READ, W. K. : Amyloidosis and renal paramyloid in a closed hamster colony. Lab. Anim. Sci. 21: 197-202 (1971). 7 GLENNER, G.G.; ELIN, D., and TERRY, W.D.: The immunoglobulin origin of amyloid. Am. J. Med. 52: 141-147 (1972). 8 JOHNSON, M. H. ; ORIHEL, T. C., and BEAVER, P. C. : Dipetalonema vireae in the experimentally infected jird, Meriones rrnguiculutus. 1. Insemination, development from egg to microfilaria, reinsemination, and longevity of mated and unmated worms. J. Parasit. 60: 302-309 (1974). 9 KLEI, T. R.; CROWELL, W.A., and THOMPSON, P.E.: Ultrastructure glomerular changes associated with filariasis. Am. J. trop. Med. Hyg. 23: 608-618 (1974). 10 KLEI, T. R.; CROWELL, W. A., and THOMPSON, P.E.: Immunological observations on Erugia pahungi infected jirds. 3rd Int. Congr. Parasitology, Munich 1974. 11 LEWIS, R.: in BRADLEY and PACHECO Canine heartworm disease, the current knowledge, p. 42 (Institute of Food and Agricultural Sciences, University of Florida, Gainesville 1972). 12 PACHECHO, G. : Infection and superinfection of jirds (Meriones unguiculafus) und hamsters (Mesocricerus auratrrs) with Dipetalonema viteae. J. Parasit. 56: 255-256 (1970).
Amyloidosis and Filariasis 185 I3 WEISS, N. : Parasitologische und immunbiologische Untersuchungen iiber die durch Diperalonema viteae erzeugte Nagetiefilariose. Acta Trop. 27: 219-259 (1 970). 14 WORMS, M. J.; TERRY, R. J., and TERRY, A.: Dipetalonema vitei, filarial parasite of the jird, Meriones libycus. I. Maintenance in the laboratory. J. Parasit. 47: 963-970 (1961). Dr. W. A. CROWELL, Department of Veterinary Pathology, School of Veterinary Medicine, Louisiana State University, Baron Rouge, LA 70803 (USA)