Implantation of Schistosomatium douthitti into the eyes of rats

EXPERIMENTAL

PARASITOLOGY

Implantation

7,

152-164 (1958)

of Schistosomatium the Eyes of Rats’ Chauncey

Departwlent

douthitti

G. Goodchild

of Biology, Emory University, Emory University, (Submitted

into

for publication,

27 April

Georgia

1957)

Blood flukes survive in some media other than blood. Early work on in vitro cultivation of human schistosomes was reviewed by Newsome and Robinson (1954), and Robinson (1956) who perfected an all-glass gravity flow apparatus in which Schistosoma munsoni adults lived and remained active in fresh horse serum for periods of up to two months. Schistosomes also survive and mature in the peritoneal cavity of experimental hosts. Yolles, Moore and Meleney (1949) reported persistence of young S. mansoni in the celom of rabbits and hamsters after intraperitoneal injection of cercariae. Although worms persisted for 9 weeks in the former and 10 days in the latter, progressive development to the epsilon or zeta stage (Faust and Meleney, 1924) occurred only in the rabbit. Moore and Meleney (1955) injected cercariae of S. mansoni intraperitoneally into hamsters and white mice. In the former, young adult worms were recoverable in the celom for only 14 days; in the latter, parasites were found 168 days after inoculation; development kept pace with that in the blood vascular system for the first 14 days, but lagged behind afterwards. Sexual maturity was attained in most intraperitoneal males and in a few females; the females revealed sperm in the seminal receptacles and ova in the obtype. Male and female worms attained only half the length intraperitoneally that they achieved in the mesenteric veins. In the present study (cf. also, Goodchild, 1956) immature and mature Schistosomatium douthitti, a normal parasite of small rodents, were transplanted into the anterior chamber of the eye of Wistar strain albino rats. Behavior, development, and reproduction of these implanted 1 This investigation was supported in part by a research grant (U. S. P. H. S. E-795) from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health, Public Health Service. 152

FLUKE

IMPLANTATION

IN R.4T EYES

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flukes were studied in wivo through the transparent cornea. Growth rates, organogenesis and organoclasis were also studied comparatively in worms which had been placed in the eye with those which had developed in the usual vascular sites. Studies of this type contribute to our understanding of the nutritional needs of blood flukes. MATERIALS

AND METHODS

The strain of Schistosomatium douthitti used in the present investigation was obtained originally through the courtesy of Dr. Irving G. Kagan, University of Pennsylvania, and Dr. Robert B. Short, Florida State University. The parasite has since been maintained in the snail Lymnaea patusttis and in Swiss mice and golden hamsters. Difficulties in maintaining adequate stocks of breeding and infected snails were experienced early in the study. A chief difficulty seemed to be the low mineral content of North Georgia water (2 grains hardness per gallon). Natural waters in this area are not heavily populated with snails (Clench and Turner, 1956), and Lymnaea palustris and other suitable molluscan hosts for X. douthitti are absent. A partial solution to these troubles was had by pulverizing Aqua-Pura Aquarium Health Cubes2 and adding 5 grams of the powdered cube to 5 gallons of tap water which was then aerated vigorously for 48 hours to dissolve the minerals and eliminate chlorine. The resulting water had 12 grains of hardness, and supported a satisfactory growth and reproductive activity in the snails. Snails were fed a variety of foods including lettuce, dried maple leaves, artificial alginate food (Moore et al., 1953; Lee and Lewert, 1956), cooked string beans and various prepared fish foods. Miracidia were recovered from livers and intestines of infected hosts by standard techniques (Kagan, Short, and Nez, 1954) and were placed in contact with snails en masse or in small vessels. Emergent cercariae from these snails were handled with hair loops (Kagan et al., 1954) and placed on the shaved abdomen of white mice spread-eagled with adhesive tape; some mice were infected via the tail route (Olivier and Stirewalt, 1952). Mice were exposed to 80 cercariae each. Adult worms from these cercariae were used in comparative growth studies and for intraocular implantation. Donor mice were killed by an overdose of ether and parasites removed under oligoseptic methods. To establish normal growth rates parasites were relaxed in distilled water, fixed in Gilson’s fluid and stained with 2 Barnett

Products

Company,

El Monte,

California

154

GOODCHILD

Semichon’s carmine. Parasites destined for implantation were selected from adult worms pooled in 0.85 % saline. Extremes of size were avoided; parasites which subjectively seemed about average for the group were selected. Wistar rats were recipient hosts. These animals were etherized and the upper cornea punctured with a sharp scalpel (Goodchild, 1954). The flukes were placed into the anterior chamber by means of a small hypodermic syringe (x cc) equipped with a No. 22 gauge needle. The needle was ground to make a 30 degree angled point in the middle of the shaft rather than on one side. The inside was carefully polished, with a wire coated with fine abrasive, to remove burrs which made implantation difficult. Introduction of large flukes was accomplished easily; some difficulties were experienced in inoculation of small flukes. These latter tended to pop back out through the incision when the small quantity of suspending saline was discharged from the syringe. With slow depression of the piston even very small flukes could usually be placed deep into the anterior chamber. Eyes in post-implanted rats were not sutured; there was prompt adhesion of the conjunctiva and cornea and flukes were never lost because of lack of cornea1 sutures. Rats were given subcutaneously 0.1 cc of S.R.D.3 (penicillin and dihydrostreptomycin-streptomycin) and 0.3 cc of Cortisone acetate4 (25 mg per cc) to control infection and reduce ocular inflammation. Antibiotics and cortisone were administered subsequently, if needed, to help keep the eye clear. Periodically, during the interval flukes remained in the eye, the rat was lightly etherized and the implanted eye examined under the stereobinocular microscope. Details of movement, copulation, egg laying, changes in morphology including total size, general condition of the eye including cloudiness, appearance of cellular masses in the anterior chamber (upon which flukes were repeatedly seen to feed), engorgement of iridal blood vessels, and in a few instances death and histolysis of the fluke, were noted. At the termination of the implantation period the rat was etherized and the eye removed. The flukes were recovered, washed in saline, relaxed in distilled water, fixed in Gilson’s fluid, and stained with Semichon’s carmine. Flukes were measured from outline drawings made with the camera lucida or with projection apparatus. a Parke, Davis and Co., Detroit, Michigan. 4 The Upjohn Company, Kalamazoo, Michigan.

FLUKE IMPLANTATION

IN RAT EYES

155

RESULTS

Schistosomatium douthitti continue alive and active in the anterior chamber of the eye. Implanted parasites have lived for as long as 161 days in this ectopic site. These flukes were alive and active at the time the host was killed. It is certain that they will live longer, intraocularly, if permitted. Flukes are able to creep over the inner surfaces of the anterior chamber and frequently succeed in crawling through the pupil into the posterior chamber. In event of the latter verification of their continued existence was rendered more difficult since they had to be viewed through the iris. During the course of these experiments 48 Wistar rats had a total of 20 male, 36 female, and 26 sexually undetermined blood flukes implanted into their eyes. The latter were too young to be sexed under the stereoscopic microscope. One rat died 10 days after implantation; eleven others died from ether during the periodic observations. In these 12 rats 9 had living flukes when examined. In 16 of the 36 surviving rats only dead flukes were recovered at the time of ocular extirpation and examination. In 29 rats from which living flukes were recovered, 15 (74 %) males, 23 (57 %) females, and 1 sexually undetermined flukes were recovered. The 1 undetermined one remained 22 days in the eye. Obviously some of the 26 sexually undetermined flukes originally implanted became sexually differentiated and are included in the totals of male and female worms recovered. Of the 82 worms of all sorts known to be implanted 39 (47.5 %) were recovered at autopsy. In 10 rats eyes became continuously cloudy; since these eyes resisted clearing treatment they were removed, fixed whole and sectioned. The most striking immediate effect of implantation upon parasites was the change in the nature of cecal contents. Flukes removed from blood vessels of donors have reddish or brownish blood or hematin in the gut. During the first day intraocularly this material is often cast out and is gradually replaced with a transparent or creamy material which surges back and forth by gut peristalses or during locomotion. A similar milkiness in gut contents was noted by Yolles, Moore, and Meleney (1949) in 6-week-old Schistosoma mansoni recovered from the peritoneal cavity of rabbits. They considered the white opaque material, “may have been mesothelial cells and cellular debris,” That this may indeed be the source of the white opaque material in ocular flukes was confirmed when flukes were seen repeatedly to feed on clot,s of cells which appeared in the eye. The aqueous humor became clouded at

156

GOODCHILD

some time in nearly all operated rats. Frequently this opacity was seen early, but this was not a consistent pattern, since in other rats the eye was crystal clear initially and then days to weeks later might cloud. The latter effect suggests that this may be a sterile inflammation induced by parasitic irritation. Furthermore, there was no causal relationship between death of parasites and hosts and appearance of intraocular opacity. Flukes and rats thrived during these periodic flare-ups; in fact flukes ingested this material which certainly was a component of the creamy cecal contents. Reproductive behavior was noted in implanted flukes. Male worms holding females at the time of implantation usually released them during the first day or two. Occasionally, however, male worms were seen to regrasp the same or other females. Immature virgin females 8 days old when implanted with males showed sperm in their seminal receptacles after being in the eye 8 to 10 additional days. Other females retained egg forming and egg laying potentialities. Twelve females out of 23 recovered showed eggs in the uterus. Twelve eyes had many ripe eggs; eggs from 3 of these hatched to produce free swimming miracidia when placed in water. Two batches of these miracidia were placed in contact with small L. palustris, but, to date, none of these snails has shed cercariae. The miracidia swam normally and their failure to infect snails is unexplained. The most striking effect of implantation upon S. douthitti is the alteration in the growth pattern, but in order to ascertain these changes the normal growth rates of our strain of parasite had to be determined. Figure 1 shows the growth rate of male S. douthitti in the normal hepatic and vascular sites, and represents lengths of 93 males (ranging in age MM 6 5 4.

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FLUKE

IMPLANTATION

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IN RAT EYES

from 4-149 days) taken from bisexual infections in Swiss mice. Figure 2 gives comparable data for 131 female flukes from the same host and sites. The vertical lines, through the point averages, in the 2 graphs represent standard deviations. The solid curves in Figs. 1 and 2 have been arbitrarily drawn through maximum growth points; these arbitrary curves have been used as base curves in Fig. 3. Figure 3 gives results of growth after implantation into the eye. The single dots represent single specimens which were recovered from the eye. The origin on the base curve of the line drawn to the dot represents age (and expected size) at the time of implantation. By inspection it will be seen that, except for one specimen, all flukes failed to grow at the usual rate, or failed to retain the size that they already had reached. Twenty days of age seems to be a critical period : flukes younger than 20 days when implanted increase in size, often at near-normal rates; at twenty days they appear, roughly, to be able to hold in the eyes the size attained in the normal sites; beyond approximately 30 days they mostly diminish in size. Figure 3 seems to suggest a band of size between the limits of 1 to 2 mm which implanted flukes can reach. Flukes below these limits when implanted will grow to attain them, flukes larger when implanted shrink in size to these limits. Statistical analysis of these results indicates a logistic type of growth curve for normal in situ development. In Swiss mice length increases were similar in both sexes, beginning to approach maxima at 60-70 days. Worms grown in the eye were classified by sex, age at the time they were implanted, length of time they remained implanted, and age when removed from the eye. Analysis of statistical significance of length MM 6. 5 4. 3

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differences from normal and experimental habitats was based on logarithms of length only. Average lengths of worms in each group were compared with average lengths of normal worms of the same age as the experimental worms at the time of implantation and also at the time of recovery. If average lengths of experimental worms at recovery were significantly greater than average lengths of normal worms at the age the experimental worms were implanted it may be concluded that growth took place. Comparison with lengths of normal worms of the same age as the experimental worms at time of recovery from the eye indicate whether or not growth in the eye proceeded at the normal rate. Results of these comparisons using Fisher’s t-test for testing significance were prepared in table form, but the table is not included here. The results indicate, as was surmised from visual inspection of the graphs, that worms implanted at an early age, 7-10 days, show approximately normal growth for a period of 2 to 3 weeks, although most fail to reach

DAYS

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Male

and female

DAYS S. douthitli in anterior

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of eye

FLUKE

IMPLANTATION

IN

RAT

EYES

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the full length of worms grown intravascularly. Worms implanted at older ages (X1-21, and 29-30 days) appear to show little if any growth. After 2 to 3 weeks in the eye they appear smaller than normal worms of the same age as the experimental ones at time of implantation. Morphologically, implanted flukes differ in appearance after a few days in the eye from normal flukes of the same total age. The disappearance of blood and hematin from the ceca has already been noted. Muscles seem still to be differentiated and functional since flukes from eyes move vigorously when placed in warm saline. Fixed and stained parasites appear slightly more pellucid, as though the parenchyma, perhaps by losing stored food, becomes more transparent; this is evident in some flukes after 25 days in the eye. Other flukes less than 25 days in the eye exhibit near-normal opacity and granularity. The gut ceca in most flukes contain cells, identified as monocytes and polymorphonucleocytes, in the esophagus and anterior gut; in a few flukes these cells are also present posteriorly. In a few parasites the ceca become distended with a colloidal flocculent material often tan or golden color in carmine stained specimens. The female reproductive system exhibits changes after implantation into the eye. Up to about 25 days the system continued functioning, with oogenesis and yolk cell elaboration continuing. From 3 to 50 or more eggs may be present in the uterus of these implants regardless of the age at the time of implantation. Beyond about 25 days in the eye, again regardless of the age at implantation, the ovary and yolk glands become cachectic with loss of morphological integrity and function. The yolk glands become diffuse and agranular; no yolk cells are produced. The ovary becomes moribund, shrinks in size and becomes less compact. The uterus is devoid of eggs. These specimens do not die; they merely cease reproducing. Young virgin females implanted at 8 days of age into the eye with males reveal sperm in the seminal receptacle after an additional 8 to 10 days. Females are also clasped in the gynecophoral canal. The changes in the implanted male reproductive system parallel somewhat those of the female. If implanted after 7 to 10 days in the donor, differentiation and functional activity ensue; sperm cells are plentiful in the follicles and in the seminal vesicle from about 19 to 39 days. Flukes left longer than about 50 days in the eye gradually lose reproductive organ integrity and function. In these parasites the testicular follicle becomes pale, less discrete, and meiotic activity ceases.

160

GOODCHILD

After 80 to 100 days in the eye, flukes, although somatically normal, show continuing atrophic changes in the reproductive system; testes become further reduced in size and may show only isolated, pale patches of cells without meiotic activity. In the oldest male implant (total age 203 days, of which 161 days were in the eye) the whole body was pale and morphologically degenerate; the testes, seminal vesicle, and cirrus were completely absent. Only 1 exception to these generalities occurred; a specimen 40 days old, of which 12 days had been in the eye, was pale, flabby and atrophic with diffuse and nonfunctional testes; sperm were lacking in testes and in the seminal vesicle. DISCUSSION

Blood flukes survive, but do not thrive, in non-vascular sites. In the present study flukes have continued alive and active in the aqueous humor for 161 days; longer survival was prevented because the hosts were killed. Lee and Chu (1935) kept Schistosoma japonicum alive in vitro for 82 days in various animal sera or human ascites fluid which was changed every week or two. Newsome and Robinson (1954), and Robinson (1956), as mentioned previously, kept S. mansoni alive in vitro for periods of up to 2 months; morphology and behavior of these flukes was not given.6 Ross and Bueding (1950) confirmed that S. mansoni will survive in vitro for 18 days in serum, for 12 days in serum ultrafiltrate, but failed to achieve survival for more than 18 hours in a defined synthetic medium. Survival in serum ultrafiltrate led them to state, “. . . protein and other non-dialysable components of serum are of little significance to the survival of the worms.” Yolles, Moore, and Meleney (1949), and Moore and Meleney (1955) found S. mansoni adults in the peritoneal cavity of rabbits, hamsters and white mice. These flukes kept pace with extraperitoneal ones for only 14 days; later they were stunted as compared to vascular flukes. Intraperitoneal flukes 42 days old were not yet mature; by 84 days in one host they were “less advanced” than peritoneal worms from other hosts autopsied at 63 and 70 days. Sexual development was also delayed intraperitoneally. Males at 42 days had sperm in the seminal vesicle; females, only one-fourth as long as extraperitoneal ones, were still 6 Senft and Weller (1956. Proc. Sot. Ezpll. Biol. and Med., 93:16-19) reported growth (up to 4.58 mm) and regeneration of S. mansoni in bovine amniotic fluid, beef embryo extract, horse serum, and antibiotics. AddiCon of mouse red cells to their in vitro medium resulted in feeding and increased movements.

FLUKE

IMPLANTATION

IN

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sexually immature. Some females at 56 days were sexually mature, had mated, but showed no formed eggs. By 84 days males were sexually mature, as were most of the females; some of the latter had an egg in the ootype. A similar pattern was noted for flukes living 98 through 168 days in the celom. Failure of normal growth and sexual maturation in extravascular sites is attributed to lack of ingestion of blood (Moore and Meleney, 1955). These authors noted, as did El-Gindy (1950), and as was seen in the present study, that ceca of extravascular flukes contained a creamy, opaque material which consisted of leucocytes and frequently a colloidal flocculent material. Ingested red blood cells in normal flukes were rapidly destroyed, probably in the esophageal region. Uniform fragmentation products of erythrocytes ranging in size from 1 to 0.6 microns were regularly seen in the gut lumen, attached to the surface of the epithelial cells, and also in the interior of the latter. Leucocytes resist digestion; intact white cells were routinely seen in the gut of normal and implanted flukes. Even though the white rat is not considered a good laboratory or natural host since eggs do not burst through the gut mucosa into the lumen to be eliminated in the feces, nevertheless, normal growth rates and large size are attained in this host in the vascular system (Price, 1931). It was considered probable that the eye of the rat was as favorable a site for growth as the eye of white mice; the latter were attempted as an implantation host, but obvious size difficulties prevented their successful use. The intraocular fluid, in which S. douthitti achieved considerable longevity, has been intensively investigated. An excellent summary of these findings has been given by Duke-Elder and Goldsmith (1951). The intact eye contains aqueous humor largely devoid of large-sized molecules; they are present in only 5 to 15 mg %, but are specifically identical to serum proteins. Small-sized diffusible molecules are present, but in amounts which differ from seral concentrations. Sugar reaches only 75 to 80% of blood levels; urea and creatine are also slightly deficient in amounts. Electrolytic concentrations also vary; most negatively charged ions are more abundant in the aqueous than positively charged ones. However, sodium is more abundant in aqueous than in serum. Some other substances, e.g., lactic acid, hyaluronic acid, and ascorbic acid may achieve much higher concentrations in aqueous than in blood. The paracentetic eye, on the other hand, in which the flukes

162

GOODCHILD

actually were placed, becomes filled with secondary aqueous which differs from primary aqueous in several important aspects. Protein concentration increases to approximate that of plasma, and sugar, urea and other non-electrolytes do likewise; electrolytes, lactic acid, hyaluronic acid, and ascorbic acid decrease in amounts, Later, these changes disappear and the aqueous once more assumes its original composition. There is certainly the suspicion that early favorable growth and maturation responses of the implanted fluke are supported by these protein and sugar increases in early paracentesis, and that after 20 days or so, with a return to normal, the flukes fail to get sufficient nourishment to support them. The contention of Ross and Bueding (1950) that sugars and proteins are of little significance to survival of the worm may be true; however, these nutrients may be very important to the continuation of normal functioning. We are currently testing the effect of increasing aqueous sugar by making rats artificially diabetic with alloxan. In preliminary experiments growth of implanted parasites seems to be improved in rats with blood sugar levels of 200 to 300 mg %, which is above the normal level. Parallel increases in aqueous sugar have also been noted in these diabetic rats. In inflammatory eyes protein concentrations also increase (Duke-Elder and Goldsmith, 1951); this may be a partial explanation of observed differences in length and organ integrity in implants since some eyes became inflamed. Even with periodic cortisone treatment reoccurrences of inflammation persisted. Unfortunately the cellular and bacterial composition of such cloudy aqueous has not yet been studied. Inferentially, however, an aqueous leucocytosis occurred in cloudy eyes since flukes removed from these eyes revealed many white cells in their ceca. Flukes removed from clear eyes had few or no white cells in the gut. The nature of the tan or golden flocculent material evident in ceca of some implants is unknown; morphologically it resembles the material in which the hematin granules float in vascular flukes. In a few instances sympathetic clouding of the non-operated eye occurred simultaneously with that of the operated one; furthermore, these spontaneous events might take place weeks after the paracentetic operation. SUMMARY

Blood flukes, Schistosomatium douthitti (Cort), continue to live when transplanted from veins of donor hosts into the anterior chamber of

FLUKE

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eyes of Wistar-strain white rats. Flukes have remained alive and active for as long as 161 days in the eye. Indefinite survival seems probable. Flukes attain sexuality when implanted young; they retain active sexuality if implanted when mature, but after about 25 days in the case of female worms and about 50 days in males reproductive activity ceases and worms atrophy sexually. Growth in implanted young males and females (J-20 days in donor) proceeded at near normal rates for an additional 20 days. Young mature males and females (20-30 days in donor) held their implanted lengths for an additional 20 days. Older males and females (30 days plus in donor) decreased in size after implantation. ACKNOWLEDGMENT The author wishes to express grateful appreciation to Dr. Robert Chief, Statistical Section, Communicable Disease Center, Atlanta, assistance in the statistical analyses embodied in the paper.

E. Serfling, Georgia for

REFERENCES CLENCH, W. J., AND TURNER, R. D. 1956. Freshwater mollusks of Alabama, Georgia, and Florida from the Escambia to the Suwannee River. Bull. Florida State Mus. (Biol. Sci.) 1, 97-239. DUKE-ELDER, S., AND GOLDSMITH, A. J. B. 1951. Recent Advances in Ophthalmology. C. V. Mosby Co., St. Louis. (Published in London by J. and A. Churchill, Ltd.). EL-GINDY, M. S. 1950. Biology of Schistosorna~ium douthitli (Cort, 1914) Price, 1931, (Trematoda) in its hosts. (Unpublished Ph.D. thesis, University of Michigan) (Microfilm B 1770, Ann Arbor, Michigan). FAUST, E. C., AND MELENEY, H. E. 1924. Studies on schistosomiasis japonica. Am. J. Hyg. Monogr. Ser., No. 3, l-339. GOODCHILD, C. G. 1954. Survival of gorgoderine trematodes in experimentally altered environments. J. Parasitol. 40, 591-602. GOODCHILD, C. G. 1956. Transfaunation of Schistosomatium douthitti. J. Parasitol. 43 (suppl.), 27-28. KAGAN, I. G., SHORT, R. B., AND NEZ, M. N. 1954. Maintenance of Schistosomatium douthitti (Cort, 1914) in the laboratory (Trematoda: Schistosomatidae). J. Purusitol. 40, 424339. LEE, C. U., AND Cue, H. J. 1935. Simple technique for studying schistosome worms in vitro. Proc. Sot. Exptl. Biol. and Med. 32, 1397-1400. LEE, C., AND LEWERT, R. M. 1956. The maintenance of Schistosoma mansoni in the laboratory. J. Infectious Diseases 99, 15-20. MOORE, D. V., THILLET, C. J., BARNEY, D. M., AND MELENEY, H. E. 1953. Experimental infection of Bulinus truncatus with Schistosoma haematobium. J. Parasitol. 39, 215-221. MOORE, D. V., AND MELENEY, H. E. 1955. Development of Schistosomu mansoni in the peritoneal cavity of mice. J. Parasitol. 41,235-245.

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NEWSOME, J., AND ROBINSON, D. L. H. 1954. Investigation of methods of maintaining Schistosoma mansoni in vitro. Ann. Trop. Med. and Hyg. 48, 194-200. OLIVIER, L., AND STIREWALT, M. A. 1952. An efficient method for exposure of mice to cercariae of Schistosoma mansoni. J. Parasitol. 38, 19-23. PRICE, H. F. 1931. Life history of Schistosomatium douthitti (Cort). Am. J. Hyg.

13, 685-727. ROBINSON, D. L. H. 1956. A routine method for the maintenance of Schistosoma mansoni in vitro. J. Helminthol. 29, 193-202. Ross, 0. A. AND BUEDINC, E. 1950. Survival of Schistosoma mansoni in vitro. Proc. Sot. Ezptl. Biol. and Med. 73, 179-182. YOLLES, T. K., MOORE, D. V., AND MELENEY, H. E. 1949. Post-cercarial development of Schistosoma mansoni in the rabbit and hamster after intraperitoneal and percutaneous infection. J. Parasitol. 36, 276-294.