PINOTTII

possedent cette curieuse membrane; h savoir dans quelle mesure elle est comparable h la “membrane pkriplasmique” des Ephelota.

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1. Bretschneider, L. H. 1959. Die submikroskopische Struktur der Pellicula von Epidinium rcaudatum (Ophryoscolecidae) . Proc. Acad. Sci. Amsterdam, S.C. 62, 542-55.

2. Carasso, N., FaurC-Fremiet, E. & Favard, P. 1962. Ultrastruc- ture de I’appareil excreteur chez quelques cilies p6ritriches. J . Microsc.

3. Corliss, J. 0. 1958. The systematic position of Pseudomicro- thorax dubius, ciliate with a unique combination of anatomical fea- tures. J . Protozool. 5 , 184-93.

4. - 1958. The phylogenetic significance of the genus Pseudo- microthorax in the evolution of holotrichous ciliates. Acta Biol. Acad. Sci. Hung. 8, 367-88.

3. - 1961. The Ciliated Protozoa: Characterization, Classifi- cation, and Guide t o the Literature. Pergamon Press, London & New York.

6 . Dragesco, J. & Hollande, A. 1965. Sur la prksence de tricho- cystes fibreux chez les Peridhiens; leur homologie avec les tricho- cvstes fusiformes des ciliCs. C . R . Acad. Sci. 260, 2073-76.

1, 455-68.

- 7 . Engelmann, T . W. 1862. Zur Naturgeschichte der Infusions-

8a. Faurt-Fremiet. E. 1966. La frange ciliaire des Nassulidae (Ci- thiere. Z . Wiss. 2001. 11, 347-93.

liata Cyrtophorina) et ses possibi1itG bvolutives. C.R. Acad. Sci. (sous presse) .

8b. - 1967. Le genre Cyclogramma Perty, 1852. J . Protozool. 14. (in press)

9. FaurC-Fremiet, E. et AndrC, J. 1966. Ultrastructural study of the ciliate Pseudomicrothorax dubius Maupas and its systematic and evolutionary implications. ,4mer. SOC. 2001. (Abstr.) Amer. 2001. 6 , 593.

10. - Armatures pharyngiennes des CiliCs Gymnostomatida. (en preparation).

11. FaurC-Fremiet, E., Favard P. & Carasso, N. 1962. Etude au microscope ilectronique des ultrastructures d’EpistyZis anastatica. J . Microsc. 1, 287-312.

12. FaurC-Fremiet, E. & Rouiller, C. 1959. Le cortex de la vacuole contractile et son ultrastructure chez les cilib. J . Protozool. 6 , 29-37.

13. Fresenius, G. 1858. Beitrage zur Kenntniss Mikroskopischen Organismen. Abh. Senkenb. Nat . Gesellsch. 2, 200-42.

14. Grain, J. 1966. Etude cytologique de quelques cilies holotriches endo-commensaux des ruminants et des equidks. Protistologica 2 ( l ) ,

15. Hovasse, R. 1965. Trichocystes, corps trichocystoides, cnido- cystes et colloblastes. Protoplasmatologia IIb’F, pp. 1-57.

16. Kahl, A. 1926. Neue und wenig bekannte Formen der holo- trichen und heterotrichen Ciliaten. Arch. Protistenk. 55, 215-438.

59-141; (Z), 5-51.

MALARIA 473

17. - 1930-35. Wimpertiere order Ciliata. In Die Tierwelt Deutschlands, Fixher, G., edit. Jena.

18. Kriiger, F., 1936. Die Trichocysten der Ciliaten im Dunkel- feldhild. Zoologica 34, 1-83.

19. Levander, K. M. 1900. Zur Kenntniss des Lebens in den stehen- den Kleingewassern auf Skareninseln. Acta SOC. Fauna Flora Fenn. 18.

20. Maupas, E. 1883. Contribution i l’etude morphologique et anatomique des infusoires ciliPs. Arch. Zool. Exp. Gen., 2em’ S . 1,

21. Mermod, G. 1914. Recherches sur la faune infusorienne des tourbikres et des eaux voisines de Sainte-Croix (Jura vaudois). Rev. Suisse Zool. 22, 114 pp.

22. Noirot-Timothee, C. 1958. L’ultrastructure de la limite ecto- plasme-endoplasme et des fibres formant le caryophore chez les ciliCs du genre Isotrichu Stein. C . R . Acad. Sci. 247, 692-5.

23. Penard, E. 1922. Etudes sur les infusoires d’eau douce, 1 vol. Georg & C’”, Genkve. 331 pp.

24. Pitelka, D. R. 1961. Fine structure of the silverline and fibril- lar systems of three tetrahymenid ciliates. J . Protozool. 8, 75-89.

25. - 1963. Electron-Microscopic Structure of Protozoa, Per- gamon Press. 269 pp.

26. Prelle, A. 1961. Contribution ?L 1’6ttude de Leptopharynx costa- tus (Mermod) (infusoire d i 6 ) . Bull. Biol. France Belg. 95, 732-52.

27. - 1965. Quelques aspects de l’ultrastructure du d i 6 Lep- topharynx costatus Mermod. Pvotistologica 1, 23-7.

28. Puytorac, P. de 1961a. Complement i l’btude de l’ultrastruc- des cilib du genre Metaradiophrya Heid. Arch. Anat. Microsc. 50, 35-58.

29. - 1961b. Observations sur l’ultrastructure d’dnoplophrya commune, cili6 parasite du ver Eophila savignyi. C.R. SOC. Biol. 155, 783-6.

30. Robertson, J . D. 1959. The ultrastructure of cell membranes and their derivatives. Biochem. SOC. Symp. , no. 16, 3-43.

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33. Rouiller, C., Faurk-Fremiet, E. & Gauchery, M. 1956. The pharyngeal protein fibres of the ciliates. Proc Stockholm Conf. Elec- tron Microscopy 1956, pp. 216-18.

34. - 1956. Les tentacules d’Ephelota ; etude au microscope Plectronique. J . Protozool. 3, 194-200.

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36. Thompson, J. O., Jr. & Corliss, J. 0. 1958. A redescription of the holotrichous ciliate Pseudomicrothorax dubius with particular at- tention to its morphogenesis. J . Protozool. 5, 175-84.

37. Wrzesniowski, A. 1870. Uber Infusorien aus der Umgebung Warchau. Z . Wiss. Zool. 20, 467-511.

427-664.

J. Protozool. 14(3) , 473-416 (1967).

Water Balance in Pinottii Malaria of Pigeonsm

REGINALD D. MANWELL and WARD B. STONE

Department of Zoology, Syracuse University, Syracuse, New York

SYNOPSIS. Pinottii malaria of the pigeon is a fulminating and usual- ly fatal infection lasting only a few days and characterized by greatly increased water consumption in most, tho not all cases. Injection of hemoglobin solutions derived from laked pigeon erythrocytes causes a similar hemoglobinuria, and is accompanied by marked diuresis and a correspondin: rise in water consumption. However, it appears

likely that there are other causal factors as well as this one for the rise seen in malaria. A sharp increase in blood uric acid may be one. Urea and blood sugar levels do not change significantly, except that the latter often falls sharply a few hours before death. Tissue sec- tions reveal kidney injury, but not of a sort to account for polyuria.

INOTTII malaria of pigeons has so far received rela- naries and very young chicks are all good laboratory hosts; Ptively little attention, altho known for about 13 years in all of them the parasite causes serious and often fatal and potentially a very useful research tool. Pigeons, ca- infections. The disease itself presents numerous problems,

not the least being why it is so often fatal in those host * Aided by NIH Grant AI-05182.

474 PINOTTII

species. Of more than 600 pigeons we have infected, only 4 have recovered spontaneously. In these i t was possible to discover persisting infection, d t h o parasitemia was too low for microscopic detection and relapses were not observed.

We have been especially interested in the clinical aspects of pinottii malaria in the pigeon because it seemed likely that they might shed light on why it is so uniformly lethal. One such aspect is water balance, with which this paper is chiefly concerned.

MATERIALS AND METHODS

The strain of Plasmodium pinottii we have used is the original one isolated by Muniz and Soares, who described the species from the Brazilian “toucan” (a large-billed bird of the woodpecker family). In its natural host it is said not to be especially pathogenic. We ob- tained it in December 1964 from Dr. P. C. C. Garnham of the Lon- don School of Hygiene and Tropical Medicine; he in turn secured the strain from its discoverers.

Since it no longer produces gametocytes it can only be transferred by blood inoculation. Parasitized blood may be withdrawn by intra- cardial puncture (which pigeons withstand very well) or from super- ficial veins such as those under the wings. Methods for doing this have already been described(7). Fresh birds were inoculated intra- muscularly (breast muscles), since survival is longer than when intra- venous injection is used. Death may be expected on the 8th or 10th day.

Our pigeons were obtained from local sources and were of various breeds and ages, and of both sexes. Except for the frequent presence of parasites such as Ascaridia columbae, Capillaria columbae, and (tho less common) Haemoproteus columbae, the birds were appar- ently healthy. Since H. columbae is so closely related to the malaria parasites, pigeons infected with it were not used. Some pigeons were also undoubtedly carriers of pigeon pox, since acute pox infections developed in some individuals after prolonged stays in the laboratory. Such birds were also discarded whenever they could be identified.

Experimental birds were usually kept in an air-conditioned aviary in which the temperature was maintained at about 65 F and illumi- nation arranged to give an artificial day of 12 hours of light and 1 2 hours of darkness. However, many birds were transferred after in- fection to the laboratory for closer observation, and here there was neither air-conditioning nor light control.

Parasitemia was followed by making Giemsa-stained blood films at about 2 p.m. daily, and making counts under an oil immersion 95 X apochromatic objective and a 10 X ocular, using a Howard disk. Water balance was studied by measuring the amount of water con- sumed daily by each pigeon, and also that excreted by many of the birds. Graduated inverted tubes filled with water were fitted to the cages, in each of which a single pigeon was confined; sheets of heavy aluminum foil were applied to the cage bottoms to collect the urine.t Water consumption can be measured very precisely this way, since pigeons-unlike most other birds-actually drink rather than filling the bill and buccal cavity with water and allowing it to trickle down the throat.

When it became clear that water consumption and water excretion were both often greatly increased as the disease became severe other determinations were made, especially blood urea and uric acid levels, since it seemed possible that changes in these might be related to water balance. The test described by Skeggs(9), as adapted for the Technicon Auto Analyser, was used to measure urea. Folin’s method (3,4), using a Klett-Summerson Colorimeter, was employed for uric

t Altho we are referring to the total cloaca1 product as “urine,” we think this is a justifiable use of the term in view of the relatively small amount of fecal material it contains. Bartholomew and Cade (2) so regarded i t in their discussion of the “Water Economy of Land Birds.” Autopsies of pigeons dying of malaria also never re- vealed any accumulation of fluid in the intestines.

MALARIA

acid determinations. The urine was also examined both for erythro- cytes and occult blood, but with negative results for the former.

Since hemoglobinuria is marked in the later stages of the disease, it seemed possible that this was also related to water balance. To test this possibility, moderate quantities of blood (6 to 12 ml) were withdrawn from each of 15 birds, centrifuged and the supernatant plasma discarded, and the sedimented red cells laked by the addition of distilled water. This was then made isotonic with blood and the volume restored by the addition of suitable amounts of concentrated jab; it was then reinjected, each bird receiving its own hemo- globin and cellular debris. Care was taken to avoid introducing clots, however small. It was found to be very important not to reinject a greater volume than previously removed.

Sections of kidney from acutely ill pigeons were examined for evi- dence of renal damage. Bilirubin blood levels were determined by Malov and Evelyn’s method(5).

RESULTS

The general characteristics of pinottii malaria in pigeons have already been described(7). I t is only necessary to say here that the prepatent period after intramuscular injection of parasitized blood is usually about 4 days, and the patenlt period another 4-6 days, with death occurring in most cases between 2 p.m. and midnight (which is also the time a t which the peak of parasite reproduction takes place),

Many birds (but not all) have a greatly increased water consumption as the disease advances (Table 1). Some birds drank almost half a pint (215 ml) in 24 hours at the height of their infection, altho others had little or no abnormal thirst, but the average consump- tion showed a consistent rise as the infection progressed.

The amount of water consumed by healthy birds was also found to vary considerably, but their intake aver- aged only 10.5 * 0.4 (SEm) ml per 100 g body weight as against 14.5 % 1.1 ml for infected birds even on the initial day of parasitemia, when very few parasites were to be seen in the peripheral blood. As the disease devel- oped, except for the 2nd day of parasitemia, there was a daily increment of about 5 ml per 100 g body weight until death, with a corresponding increase in urinary excretion. Further evidence that little of this water is retained in the tissues is seen in the relatively small weight changes (Table 1 ) .

Examination of the urine for abnormal constituents which might help to explain the thirst and diuresis re- vealed only large amounts of hemoglobin, and even this was not present until rather late in the disease.

I t was therefore of considerable interest to find that the pigeons which had been injected with laked blood developed marked thirst and polyuria, with concomitant hemoglobinuria lasting 10 or 12 hours, thus mimicking quite exactly the condition observed in pinottii malaria. I t therefore seems likely that the greatly increased water consumption of malarious pigeons may be caused by the need to excrete the parasite-freed hemoglobin liberated in great quantities when reproduction occurs. However, other factors must also be involved since water con- sumption increases before hemoglobinuria is evident and while parasitemia may still be low. At this stage of the disease, fever, often high, may be a contributing cause.

PINOTTII MALARIA 475

TABLE 1. Water consumption of healthy and malarious pigeons.

A. Healthy Birds I- Water consumption (ml per day) -,

Per 100 g body weight No. of birds Readings Mean Range Mean Range Mean SEni Range

Weights ( g )

18 102 341.4 195-410 37.1 9-87 10.5 f 0.40 2.6-23

B . Malarious Birds (During Course of Infection) ,.-Water consumption (ml per day) ----,

Per 100 g body weight KO. of parasit- birds einia Mean Range Mean Range Mean SEin Range

Day of Weights ( g )

18 1 347.3 18 2 18 3 18 4 350.4 13 5 5 A 1 7

(Total readings-91) C.

18 3-6

200-430 51.5 16-110 14.5 f 1.1 49.8 17-120 13.6 f 1.1 60.2 14-166 17.0 f 2.3

19 5-43 5 77.2 25-153 22.6 2.1 93.9 25-215 27.1 f 3.6 94.0 17-155 32.5 f 9.4

120 27.9

Malarious Birds (Day Before Death) 81.1 17-166 23.7 2 3.1

D. Malarious Birds (Day of Death) 18 4-7 351.1 200-430 92.9 17-215 27.7 -c 2.9

7.0-25.6 6.1-34.8

7.0-58.8 5.9-54.1 4.7-54.5

5.2-4 7.4

4.7-58.8

4.7-54.5

Another factor which may influence urinary excretion, and hence water consumption, is uric acid. The amounts present in the urine have not ‘been measured, but blood levels (Table 2 ) rise steeply as the disease advances, probably as the result of parasite-caused destruction of red cells, the proteins of which must be disposed of.

The uric acid level in the blood of healthy birds was found to average 9.3 0.6 mg (SEm) per 100 ml, in- creasing with rising parasitemia until a level of 64.7 -+ 5.9 per 100 ml was reached terminally. I t is clear that the problem of ridding the body of this product of nitrogen metabolism is one with which the malarious bird copes inadequately. The accumulation of uric acid in the blood may well be a contributing cause of death.

Urea blood levels changed little, as might be expected, since this substance is not an important end product of nitrogen metabolism in birds. For 10 healthy birds the range was 4-10 mg per 100 ml (mean 7.2 -+ 0.63 SEm), as against 4-12 mg per 100 ml (mean 8 -t- 0.4 mg) for 24 experimentals a t the time of death.

Bilirulbin levels were 0.32 k 0.16 mg per 100 ml in non-malarious pigeons (range 0.10-0.65) and 1.65 + 1.24 mg per 100 ml in pigeons dying of the disease (range 0.20-3.55). This is a small and possibly not a real difference, but it is of interest, since if malaria pig- ment is metabolized into bilirubin and iron (as it is in mammals), one might expect a sharp rise.

Urine pH was slightly acid (range 6.0-6.8) in both healthy and malarious birds. Glucose was never present, a finding which might be expected since blood levels did not change significantly except for a precipitous fall shortly before death in many, but not all, pinottii-infected pigeons (this subject will be considered more in detail in a later paper). The urine also remained protein-free until it

became positive for occult blood in the later stages of the disease.

Examination of sections of kidney from acutely ill pigeons revealed cloudy swelling of the cells lining the tubules, and large focal areas of leucocytes and other cells loaded with pigment. The glomeruli appeared essen- tially normal.

DISCUSSION

I t is remarkable how little is known of the physiologic change induced in the host by the malaria parasite. Sadun et a1.(8) commented on this in a recent study of the “pathophysiology” of Plasmodium berghei in mice; perhaps’less is known for birds than for mammals. We axe aware of no studies dealing with water balance in malaria in either type of host.

Despite the apparent role of parasite-freed hemoglobin in provoking marked diuresis in pinottii-infected pigeons, i t seems unlikely that this is the chief cause of Ithe ab- normal thirst and water consumption. Some malarious pigeons had no such thirst and may even have drunk less than the normal amount. Among those which did take more than the normal amount there was much vari- ation, and increased consumption was often noticeable while parasitemia was still low and *before the appearance of either occult blood or hemoglobin in the urine.

I t is of interest, too, that blackwater fever patients, in whom there is also intense hemoglobinuria, usually excrete abnormally large amounts of urine( 6) , tho lthe increase is relatively much smaller than in malarious pigeons and- as in pigeons-does not occur in all cases; furthermore, anuria often follows in human cases, but we have never observed it in malarious pigeons.

476 PINOTTII MALARIA

TABLE 2. Uric acid levels in pinottii malaria of pigeons.

A. Correlation with parasitemia (during life) Uric acid (mg per 100 ml) Number of ‘Parasitemia

specimens (per cent) Mean SEm Range

12 less than 1 10.9 f 1.2 5.6- 19.2

10 21-40t 37.8 f 9.6 7.7-111.5 10 41-70t 77.4 k14.8 24.7-193.2

11 1-20 12.4 & 1.4 6 . k 24.5

9 71-96 t 51.1 6.8 9.8- 76.44

B. At death 23 64.7 f 5.9 9.8-76.0

C. In health 22 9.3 t 0.6 4.8-13.2

D. Rapidity of rise Parasitemia per Parasiteniia per

10,000 R.B.C. Uric 10,000 R.B.C. Uric Bird No. (4th day) acid (5th day) acid

1265 510 14.6 2830 70 1317 1460 10.7 6800 64.2 1324 2200 9.2 3450 65 1325 1200 6.4 4400 77.6 1377 540 12.2 3400 111.5

account for the striking rise in blood uric acid levels observed late in the disease. Some liver damage probably always occurs in acute malaria of any type. The Kupffer cells are characteristically engorged with pigment, there are “severe vacuolation of the liver cells (and) degenerative and necrotic changes” as well as “haemorrhages from small blood vessels, capillaries, and sinusoids”( I ) , and enlarge- ment of the organ. While Al-Dabagh (from whom this is quoted) was writing primarily of blood-induced galli- naceum malaria in chicks, he indicated that what he said would probably apply also to other avian malarias.

Much of the uric acid in the blood of pinottii-infected pigeons doubtless arises from the purines present in large amounts in the nuclei of parasite-destroyed erythrocytes, but some may also come from a uric acid riboside said to be a constituent of red cell cytoplasm. I t is quite possible that the high plasma levels of uric acid may stimulate urinary flow.

Since the destruction of large numbers of erythrocytes also liberates much potassium, this may also play a significant role in renal secretion, but of this we have yet no knowledge.

* Totals of 47 malarious and 22 healthy birds were used; one de- termination was made on each except for 5 birds on which 2 were made (see “ D ” above).

t Deaths occurred in each of these groups. $ Only one bird had a normal uric acid (9.8) at death.

The nature of renal damage was not such as to account for the polyuria seen in pinottii-infected pigeons. Although pathologic changes of various kinds in the kidneys of malarious birds have ,been reported, they are often contradictory, and i t seems likely that they vary both with the host species and the species of parasite (as they do in man). Al-Dabagh( 1) stated that “renal lesions seem to occur much less frequently in avian malaria than they do in simian and human malaria.”

Conceivably irritation or injury to the water-regulating center of the pituitary could play a causal role, but of that there is a t present no evidence.

Since uric acid is synthesized both in the liver and kidney of birds(lO), damage to either or both could

REFERENCES

1. Al-Dabagh, M. A. 1966. Mechanisms of Death and Tissue In- jury in Malaria. Shafik Press, Baghdad.

2. Bartholomew, G . A. and Cade, T. J. 1963. The water economy of land birds. Auk 80, 504-39.

3. Folin, 0. 1933. Standardized methods for the determination of uric acid in unlaked blood and in urine. J . Biol. Chem. 101, 111.

4. - 1934. The preparation of sodium tungstate free from molybdate together with a simplified process for the preparation of a correct uric acid reagent. J . Biol. Chem. 106, 311.

5. Malov, H. T. and Evelyn, K. A. 1937. The determination of bilirubin with the photoelectric colorimeter. J . Biol. Chem. 119, 481- 90.

6. Mannaherg, J. 1905. “Malarial Diseases” in Nothnagel’s Prac- tice. Saunders, Philadelphia and London.

7. Manwell, R. D. & Stone, W. B. 1966. The role of anemia in pinottii malaria of pigeons. J . Parasit. 52, 1145-9.

8. Sadun, E. H., WiKims, J. S., Meroney, F. C., & Hutt, G . 1965. Pathophysiology of Plasmodium berghei in mice. Ex#. Parasit. 17,

9. Skeggs, L. T. 1957. An automatic method for colorimetric analy- sis. Am. J . Clin. Path. 28, 311-22.

10. Sturkie, P. D. 1965. Avian Physiology. 2nd Ed. Comstock Pub. Assn., Ithaca, N. Y.

277-86.