Experimental infection of tapeworms and oribatid mites with Nosema helminthorum

EXPERIMENTAL

PARASITOLOGY

Experimental Mites

7,

306-318 (1958)

Infection of Tapeworms and with Nosema heEminthorum’

Oribatid

A. S. Dissanaike Department of Parasitology, London School (Submitted

of Hygieneand Tropical Medicine2

for publication,

16 July 1957)

Nosema helminthorum is a microsporidian hyperparasitic in anoplocephalid cestodes of the genus Moniem’a. A detailed description of this protozoan and the question of its nomenclature have been dealt with elsewhere (Dissanaike 1957a & 195713).The present paper deals with the results obtained during attempts at elucidating its life-cycle. Moniez (1887), who discovered this parasite, was of the opinion that transmission was through the eggs of the worm, but as this appeared to be an unlikely mode of transmission it was decided to see whether a simpler method obtained in nature. The simplest that suggested itself was that lambs, already infected with Moniezia, accidentally swallowed the spores present in the grazing-ground, and that thereby the worms became infected. In an attempt to see what part the oribatid mite played in transmission, a few experiments were performed with these mites and these results are also included here. Material and Methods

As it was not practicable to carry out any experiments with lambs, mice and rats already infected with Hymenolepis nana were mainly used. It was first decided to seewhether H. nana in these rodents could be infected with the microsporidian by merely feeding the spores to the animals. Subsequent experiments were done to find out the exact mode of entry of the parasite into the tapeworm tissues. Tapeworms of other animals and Taenia saginata of man were also exposed to infection with this microsporidian. Mice and rats already infected with H. nana were available from stock. Spores were introduced by feeding the animals forcibly with infected segments of Mcmietia, or by contaminating their food with these segments. f Part of a thesis approved by the University of London for the award of the Ph.D. degree. 2 Present address: Department of Parasitology, Faculty of Medicine, University of Ceylon, Colombo, CEYLON. 306

MICROSPORIDIAN

INFECTlON

OF TAPEWORM

AND

MITE

307

The two human cases were made available through the kind permission of Professor A. W. Woodruff, and with the help and co-operation of his staff in the Chamberlain Ward of the Hospital for Tropical Diseases. Two patients admitted for treatment of tapeworm infection were fed concentrated suspensions of spores in gelatin capsules (No. 0). A dog was experimentally infected with Taenia hydatigena by feeding it with cysticerci from sheep (Cysticercus tenuicollis). After it began to show eggs of the cestode in its stools, its meat food was mixed with heavily infected segments and spore-suspensions. Jackdaws known to be infected with the cestodes Hymenolepis sp. and Anomotaenia sp. were given homogenized suspensions of infected segments containing spores, by injection through a fine catheter into the gullets. From 2-10 ml of these suspensions were injected into each jackdaw. Mites were fed with spores by introducing the infected segments or suspensions of spores into culture tubes containing the mites. Some of the spore-suspensions and infected segments used in all the above instances were kept for long periods in the refrigerator without their viability or infectivity being affected, as shown by their ability to infect H. nana of mice. In examining the experimentally infected worms for evidence of infection several methods were employed. As a rule the worms were crushed, segment by segment, to see whether spores or developmental stages could be detected. It was found quite accidentally that Newton’s Crystal Violet stain for chromosomes was retained by the spores even though the other structures had lost the stain on treatment with alcohol. Whole worms stained in this way showed up the spores in a striking manner (Fig. B), and the presence of even a single spore could be detected by this means. Finally, infected worms were detected by serial sections of representative segments, a method which was most reliable when dealing with infections of short duration where only developmental stages were present. In infected mites too this was the only way of ascertaining whether spores found in them were in the tissues or merely spores swallowed and remaining in the lumen of the gut. Mites used in these experiments were extracted from turf samples and dried leaves using a modification of Rayski’s technique (1945) .3The mites were kept during experiments in culture tubes as suggested by Rayski. 3 Samples of turf and collections of leaves were obtained from St. Albans Kent, the latter being kindly brought by Mr. D. Minter of the Department Entomology. Mr. C. H. Fernando kindly sent me some mites from Oxford.

and of

308

DISSANAIKE

Experimental Injection of Hymenolepis nana Experiment 1. Five mice infected with H. nanu were selected from the

stock. Three were kept as controls and the other two were given spores of N. helminthorum on two consecutive days. Five days after the first feed one experimental mouse was sacrificed and 20 worms recovered from it. Fifteen of these were carefully examined by crushing, and five showed ovoidal spore-like structures which bore a strong resemblance to immature spores of N. helminthorum but were smaller. Dry-fixed smears of these stained in Giemsa stain showed many sporonts with a pre-metachromatic granule so characteristic of N. helminthorum. Sections of the five remaining worms showed spores and earlier developmental stages scattered in various places in the parenchyma of three worms. Table I shows the results for the second mouse. Mature spores were obtained, from the worms in this mouse, which measured 5.C-6.2 P by 2.9-3.5 P (Av. 5.7 by 3.2 /L). None of 98 worms recovered from the three control mice were infected. A portion of the small intestine of the second mouse (experimental) with worms in situ was sectioned, but no trace of any infection of the mucosa was seen in spite of the fact that several scolices (containing spores) were seen embedded in the mucosa. Experiment 6. In this experiment two mice were fed spores while one was kept as a control: the results are summarized in Table II. They confirm the fact that H. nuna can be infected with N. helminthorum. The sectioned worms showed all the stages of development as seen in Moniezia including the fusiform second phase schizonts (Dissanaike, 1957b.). Experiment 5. In this experiment an attempt was made to seewhether refrigeration and centrifugation of spores have any adverse effects on their infectivity and viability. It is seenfrom Table III that sporesstored TABLE I Results of Experiment N~t~rfe;fG~~ spores

MOUSI?

1 2 Control Control Control

1 2 3

5 12 -

Number of worms examined

20 123 33 30 35

1 Results (Positive worms)

Smears

Newton’s

Sections

5/15 g/M o/20 -

33/59 o/10 o/30 o/35

3/5 o/3 -

MICROSPORIDIAN

INFECTION

OF

TAPEWORM

AND

309

MITE

TABLE II Results of Ezperiment Z? Results (Positive worms) MIXIS Smears

1 2 Control

23 15 14

10

19 -

Newton’s

Sections

m

9/11

3/3 O/4

5/B

lO/lO 3/4 -

o/10

TABLE III Results of Experiment 3 Results (Positive worms) MOU% SlllCVS

1 (Fed spores and infected segments kept in refrigerator in saline and water for 2-3 weeks) Control for 1 2 (Fed spore-suspensions after centrifugalisation at 2,500/ min. for 6 minutes) Control for 2

13

-

6

-

Newton’s

13

l/5

6

O/3

O/3

5

4/5

-

8

O/8

-

l/8

in this way in water or in saline for long periods remain infective, and could therefore be used with advantage whenever the need arose. Experiment 4. In this and some subsequent experiments with mice, no control animals were used. Here, an attempt was made to seewhether worms infected for a long time show appropriate changes in the tissues due to the prolonged infection. As the results in Table IV indicate, the TABLE IV Results of Erperiment MOWIT

1 2

Number of days after feeding spores

26 27

4 Results (Positive worms)

24/24 25125

310

DISSANAIKE

percentage of positive worms is greater; it was also found that all the infected worms could be easily detected under the lower powers of the microscope, as spores could be seen in all of them. Sections of some of these worms showed the tissues filled with spores, but though the cortex and medulla were heavily infected the genital organs remained free (Fig. c>* The stools of these mice showed spores in large numbers after the second week. These spores obviously were liberated from detached and disintegrated segments. About half an inch of the small intestine of one of these mice was sectioned serially but no trace of infection of the mucosa was detected. Experiment 5. It was decided to seewhether spores obtained from one experimentally infected H. nana could infect worms in other mice and also whether the spore size was altered when such an infection occurred. As Table V shows, this type of infection is possible, the spore size being 5.7-6.0 p by 3.0-3.4 cc(Av. 5.8 by 3.2 P). Experiment 6. The results of the above experiments suggested that once the sporoplasm emergesfrom the spore in the intestine of the vertebrate host, it must work its way inwards through the cuticle of the worm. There is little doubt, as I have pointed out earlier (Dissanaike, 1955) that the filament cannot penetrate the thick cuticle of tapeworms, but that the sporoplasm must work its way in by secreting a histolytic substance. That the microsporidia can penetrate the thick cuticle of helminths in this manner has been suggested by Thorne (1940) for Duboscquia penetrans parasitic in the free-living nematode Pratylenchus pratensis.

The purpose of this experiment was therefore to see whether this cuticle-penetrating stage could be observed. Three mice were used. They were first forcibly fed segments of Monieziu and subsequently a sporeTABLE V Results of Experiment 6 MOW?

Nxz OfN;$UiylllS

1 2

12 14

5 4

3 Control

32 -

6 5

Results (Positive

worms)

Smears

Newton’s

Sections

o/4 l/4

-

l/l -

-

O/l

All infected o/4

MICROSPORIDIAN

INFECTION

OF

TAPEWORM

TABLE VI Results of Experiment MOW.?

1 2 3

Duration,

20 25 68

hours

No. of worms sectioned

1 2 2

AND

MITE

311

6 Observations

No stages seen No stages seen Smears of 2 worms showed Sections sporont stages. showed few scattered solitary sporonts

laden emulsion was added to the drinking water. The results are indicated in Table VI. These results indicate that within 68 hours, the development in H. nuna can proceed to the sporont stage. Furthermore in this instance, no marked schixogonic multiplication had taken place and the sporonts had developed very soon after the initial infection. This again confirms what I have pointed out earlier (Dissanaike, 1957b) that overcrowding with developmental stages of the parasite is not the important factor in bringing about spore-formation. In a few sections, a more or less rounded deeply staining body was seen just under the cuticle between the sub-cuticular cells. This appeared to be an early stage of the parasite, but as nuclear details were not recognizable this was regarded as inconclusive. Two other mice were fed in the above way and sacrificed much earlier, in 234 and 5 hours respectively. There were over 100 worms in the first mouse. Some of them were fixed in Carnoy’s Fluid for sectioning. Several pieces of small intestine with the worms in situ were also sectioned after fixation in Carnoy’s Fluid. Before this the contents of the various parts of the intestinal tract were examined in detail under the microscope in order to see the condition of the spores in various parts of the gut. The stomach showed a large number of spores free from the segments. About 50% of these were empty, but there were no traces of the filaments: The upper parts of the small intestine showed a decidedly higher proportion of empty spores, while in the last part, where the worms were present, there were hardly any spores that had not extruded filaments. In some of these worms the spores with their extruded filaments were seen in an entangled mass. The second mouse, killed after 5 hours, showed the same conditions,

312

DISSANAIKE

and the three worms recovered from it were sectioned. In none of the worms sectioned singly or in situ in the intestine were any developmental stages of the parasite seen. Experiment 7. One rat infected with H. nana was fed infected segments of Moniezia and sacrificed after 11 days. All 6 worms recovered from it showed spores and other developmental stages, chiefly sporonts. The spores had the same appearances and they measured 5.7-6.3 p by 2.9-3.5 /.t (Av. 5.9 by 3.2 /.L). A single control rat killed on the same day showed no infection in any of the 3 worms recovered from it. Experimental Infection of Taenia saginata of Man

Arrangements were made to seewhether Taenia saginata of man could be infected with Nosema helminthorum. Two patients were available for this purpose. The first patient was fed a concentrated suspension of spores as well as some heavily infected segments kept in the refrigerator for about 3 weeks. These were administered in gelatin capsules (No. 0), two capsules being given in the morning and two in the evening of the same day. Segments passed by the patient on the day the capsules were given were collected and examined thoroughly as controls. Some segments passed by the patient 4 days after, as well as the complete worm passedon the 5th day (after the vermifuge was given) were also collected. The worm proved to be a Taenia saginata. Numerous segments were examined after crushing in saline but no spores or developmental stages were found. The remaining segments were sectioned after fixation in Carnoy’s fluid, representative segments from twenty different portions of the worm being taken for this purpose. No evidence of infection was found. The second patient was fed similarly but on the morning following the first administration of the spores, a concentrated suspension of spores from a sieved centrifuged saline extract was given in flavored milk. The patient was given a vermifuge on the 3rd day and the worm was passed on that day. Unfortunately no control segments were available for examination. This worm too proved to be a Taenia saginata and was examined as above. No spores or vegetative stages were seenin the smears, but sections of mature and gravid segments in many regions of the worm revealed the presenceof early division stages (Fig. D). These were mostly spherical and between 1.4-2.0 ~1in diameter, with deeply staining cytoplasm and a compact nucleus. They closely resembled the stages seen

MICROSPORIDIAN

INFECTION

OF’ TAPEWORM

AND

MITE

313

in the early infections of Moniezia and in experimentally infected H. nana of rats and mice. All these stages were seen in the cortical regions and were invariably situated at some distance from the cuticle (about 106 II). This suggested that the original sporoplasm travelled a fair distance inward before starting to divide. In several places chains of schizonts were seen (Fig. E). The short duration of the infection in this instance accounts for the absence of sporonts or spores. The number of days required for spore formation, therefore, is over 3, but it was not practicable to keep the patient for longer periods without treatment. It would have been better to administer the spores to the patient at the out-patient stage so that by the time he came for treatment, the infection would have been old enough for spores to have formed. This is a necessary step to prove conclusively that the stages seen are definitely those of a microsporidian. There was little doubt, however, that what was seen resembled the early stages of Nosema helminthorum. Attempts at Infecting Other Tapeworms

Since the above results indicated that N. helminthorum has no host restriction it was decided to expose other tapeworms to infection with this parasite. Taenia hydatigena of the dog, and Anomotaenia sp. and Hymenolepis sp. from the jackdaw were exposed in the manner detailed earlier, but no infections were obtained although the worms were very carefully examined by all the methods described above. Experimental

Injection of Two Oribatid Mites

During attempts to determine the role of oribatid mites in the transmission of N. helminthorum, the literature was first searched to see whether any natural infection of these mites with microsporidia had been reported. Although several other protozoa have been found in various species of these mites (Nicolet, 1855; Michael, 1881; Wellmer, 1911; Thor, 1930; and Warren, 1944) there is no record of any microsporidia from them. Furthermore, many of these mites examined by crushing and serial sectioning showed no evidence of any natural infection with a microsporidian. An attempt was then made to see whether these mites could be infected with N. helminthorum experimentally. The results of these experiments are summarized in Table VII. Two out of 65 mites fed on spores showed infection. One was Ceratoppia Irip& and the other Xenillus tegeocranus. The spores obtained from these mites were

314

DISSANAIKE TABLE Ezperiments

VII

with Oribatid

Mites

Results Number Igiven spore! s

Oribatid

X.

5 3 2

24 Hours

i 2 20 8 1 2 1 7 1 1 2 2 1 2

tegeocranus

C. bipilis Belba sp. Platynothrus peltifer Euzetes globula

Sections

--

.-

Platynothrus peltifer “Notaspids” Xenillus tegeocranus “Notaspids” Hoploderma magna X. tegeocranus Ceratoppia bipilis Ceratoppia bipilis ceratoppia bipilis Scheloribates sp. X. tegeocranus X. tegeocranus

Duration

48 Hours 5 6 7 8 8 9 11 11

Days Days Days Days Days Days Days Days

12 Days

Controls X. tegeocranus E. globula P. peltifer

2; -ve 1; -ve 1; -ve 5; -ve 3; -ve 1; -ve 2; -ve 1; infected -

5; -ve 2; -ve 2; -ve

3; 2; 2; 4; 2; 15; 5; 1; 1; 1; 5; 1; 2; 2; 1; 2;

-ve -ve -ve -ve -ve -ve -ve infected -ve -ve -ve -ve -ve -ve -ve -ve

15; -ve 4; -ve 3; -ve

A number of small mites tion WY&~not practicable.

with

pointr ed pteromorphs

were lsbelled

“N, otaspids”

a~ detailed

identifica-

identical in shape and appearance with those of N. helminthorum in Monieziu, but were of a smaller size. For instance, the spores from C. bip&s measured in the fresh state were 2.7-3.1 /L by 1.25-1.5 ~1(Av. 2.9 p by 1.4 II) ; the spores from X. tegeocrunus measured from sections were 2.2-2.5 k by 1.0-1.25 ~1(Av. 2.4 by 1.17 P>. It was not possible to say from which part of C. bipilis the spores came because they were seen only after the mite was crushed. They were probably from the gut, for sections of X. tegeocrunus showed the spores in the epithelial cells of the midgut and ceca. The infection was so heavy that many cells were hypertrophied and filled with spores (Fig. A). Some of these cells had ruptured, liberating the spores into the gut lumen. No developmental stages of the parasite were seen in these sec-

MI(‘KOSPORIDIrlS

ISFIWTIOS

OF

TAPE\VOIIM

ASD

MITE

:215

tions, nor was there any evidence of the infection having spread t’o ot,her structures in the surrounding body cavity. Sporonts seen in dry-fixed smears from crushed Ceratoppia bipilis showed t#he characteristic pre-metachromatic granule. n1scrss10s

The fact that’ Hymenolepis nana of mice rind rats and 2’acnia sayinata of man could be experimentally infected with AVosc~mahelminthorum show that this microsporidian has a high infectivity for (Lestodes belonging to different families. It is clear that the failure to detect infection in :I number of other cest’odes is due to one or more of t#he following hypotheses. (a) The infection probably was est,ahlished in some, but escaped dct,cction in spite of careful exnminat,ion. (1~) Conditions in the digestive t’rncts of the vertebrate hosts in which t’he tapeworms failed t,o become infect’ed may have been unsuitable for the successful emergence of the sporoplasm from the spores. Even if this did occur, it may have been in t’hnt, part, of the digestive trwt. where there were no worms. (c) The pnrt~icular worms that failed to become infected may not have been suscept8ihle to infection with this microsporidian, or the microsporidian was not infective to t,hese tapeworms. The first of these explanations is unlikely for the simple reason that, many t’housands of spores were used in each experiment and the opportunities for infection were great. Henw, at, least) one of these areas should have been detected at least in the serial se&ions from different part,s. In Z’amia hydatigena of the dog, the durnt~ion of infect’ion was sufficient for mature spores t)o he present iii large numbers. The same was true for t.he cestodes of the jnckdaws. The infection in the first) human case, even t,hough the duration was greater than in the sewnd (MC, may have escaped detectioll; more probably, the early stages may have developed but degenerated soon aft’er, sincr cwlditions in 1’. saqinata were not suitable for furt)her development,. This same expl:mat,ion could also apply to t’he cestodes of the dog and the j:wkdnws. .1nother possible reason why !Z’.sayinata in the first pat,ient showed no infection may have been t’hat proper conditions for the successful emergence of the sporoplasm and it’s ent’ry int,o t’he t,issues, did not, exist. in the intestinal tract of t,his patient,. The above experiment’s also suggest, that’ Moni&a in nature becomes

FIG. A. Transverse section of Xenilllrs tegeocranus showing spores of Nosewa helminthorunl in cells of midgut. Arrow indicates hypertrophied cell that has ruptured liberating spores into lumen. (X169) FIG. B. Anterior end of Njlnzenolepis ~LCUKIexperimentally infected with N. helminthowm, showing spores stained hy Newton’s (Crystal violet stain. (X52.5) FIG. C. Section of a heavily infected Hymenolepis nana showing cortex and medulla packed with spores. (X75) FIG. D. Sect.ion of ‘I’aenia saginala showing developmental stages of N. helnrinthoruw 3 days aft,er infection. (x450) FIG. 15. Same as Fig. D, showing ringed area under higher power, indicating chain of whizonts. (x1350) 316

MICROSPORIDIAN INFECTION OF TAPEWORM AND MITE

317

infected in a similar way, by the already infected lamb swallowing spores from the grazing ground. The infection of two oribatid mites, Ceratoppia bipilis and Xenillus tegeocranus brings out the interesting fact that the infectivity of this microsporidian is not confined to cestodes. Furthermore, the results obtained with Nosemuhelminthorum tend to show that there is no generic host restriction as shown by Kudo for certain other microsporidia (1924a, 1924b, 1925). In fact cestodes belonging to widely different families can be infected as well as oribatid mites. One is therefore prompted to conclude with Becker (1933) that ‘La particular parasite will develop normally in as many hosts as provide for it adequate environmental conditions and mode of entry”. SUMMARY

1. Hymenolepis nana of mice and rats has been successfully infected with Nosema helminthorum. In this cestode the microsporidian shows all the stages of development as seen in naturally infected Moniezia. The spores are, however, of a smaller size and the cycle is probably of shorter duration. 2. One specimen of Taenia saginata in the human host was experimentally infected with this microsporidian, but only early developmental stages were seen. 3. Two oribatid mites, Ceratoppia tip&s and Xenillus tegeocranus were also infected with this organism. The spores developed in the midgut and caeca and were very much smaller than those developing in H. nana. 4. Although the cuticle-penetrating stages of this parasite could no+ be demonstrated, there is little doubt that in the experimentally infected worms as well as in naturally infected Moniezia the sporoplasm enters the host tissues through the cuticle. ACKNOWLEDGEMENTS I am deeply grateful to Professor P. C. C. Garnham for his invaluable help and guidance in this work; to Professor J. J. C. Buckley and Dr. P. L. Le Roux for their interest and suggestions; to Dr. G. 0. Evans of the British Museum (Natural History) for the identification of the oribatid mites; to Dr. C. Rayski of Edinburgh University for his suggestions on the extraction and culture methods for these mites; to Mr. W. Cooper, Mr. F. R. N. Pester and Mr. R. Killick for their technical assistance at all times; and finally to Mr. W. T. Bush and his staff for the photomicrographs. REFERENCES BECKER, E. R. 1933. Host-specificity Trop. Med. l&505-523.

and specificity

of Animal Parasites. Am. J.

318

DISSANAIKE

A. S. 1955. Emergence of the Sporoplasm in Nosema helminthorum. Nuture 176, 1002-1003. DISSANAIKE, A. S. 1957a. Protozoa Hyper-parasitic in Helminths with some observations on Nosem.a helminthorum Moniez, 1887. J. Helminthol. 31, 47-64. DISSANAIKE, A. S., (1957b). The Morphology and Life Cycle of Nosema heZminthorum Moniez, 1887. Parasitology. 47, 335-346. KUDO, R. R. 1924a. Studies on Microsporidia parasitic in mosquitoes III. On Thelohania Zegeri Hesse (Th. illineisensis Kudo). Arch. Protistenk. 49,147-162. KUDO, R. R. 1924b. A Biologic and Taxonomic Study of the Microsporidia. Illinois Biol. Monogr. 9, No. 2-3, l-268. KUDO, R. R. 1925. Studies on Microsporidia parasitic in mosquitoes V. Further observations upon Stempellia (Thelohania) magna Kudo, parasitic in Culex pipiens and C. territans. Biol. Bull. 48, 112-127. MICHAEL, A. D. 1881. British Oribatidae vol. I. Ray Society Publication, London. MONIEZ, R. 1887. Observations pour la revision des microsporidies. Compt. rend. 104, 1312-1314. NICOLET, M. H. 1855. Histoire naturelle des Acariens qui se trouvent aux environs de Paris. Arch. Mus. Hist. nat. Paris, 7, 381482. RAYSKI, C. 1945. Oribatid mites as Intermediate hosts of Anoplocephaline Cestodes. Ph.D. Thesis, University of Edinburgh. THOR, S. 1930. tfber einzellige Parasiten in verschiedenen Acarina I. 2. Parasitenk. a, 551670. THORNE, G. 1940. Duboscquia penetrans, n.sp. (Sporozoa, Microsporidia, Nosematidae) a parasite of the Nematode Pratylenchus pratensis (de Man) Filipjev. Proc. Helminthol. Sot. Wash., D. C., 7, 51-53. WARREN, E. 1944. Observations on the Anatomy and Histology of a Myrmecophilous mite (Urodinychus sp.) and an account of certain of its sporozoan parasites. Ann. Natal. Museum. 10, 359406. WELLMER, L. 1911. Sporozoen ostpreussischer Arthropoden. Schriften phys. ksnigsberg. iiken. Ges. 62, 103-164. DISSANAIKE,