NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
230
MUCOPOLYSACCHARIDES IN EIMERIA TENELLA
[ Vol. XXIV, Part IV,
Young second generation schizonts appeared as dark blue masses stuffed in
the submucosa with their nuclei stained intense blue. Just prior to segmentation
into merozoites they reflected a purplish hue and in the cytoplasm there appeared
a large number of violet granules [Plate XII, fig. 4]. Merozoites contained several
(2-7) brilliant violet granules mostly concentrated just anterior to the nucleus.
(Plate XII, figs. 2 & 3). Rarely one could notice one or two granules posterior to
the nucleus also.
Microgametocyte appeared as a rough purplish mass. Comma-shaped bodies
of microgametes took brilliant violet colour.
Karyosome of young gametocytes stained intense blue, and metachromatic
granules were seen scattered at the periphery of the organism (Plate XII, fig. 4.).
Mature macrogametes presented purplish granular material dispersed in their cyto-
plasm which after fertilisation condensed into definite metachromatic granules in
the zygote protoplasm.
Cytoplasm and nucleus of a majority of oocysts stained light blue, but a small
proportion of them took deeper stain at both these sites.
Parasitised caeca were greatly infiltrated with mast cells, which were laden
with coarse, intensely violet granules, especially so between the early fourth day to
the seventh day of infection.
Lison's method, though strictly adhered to, did not reveal any superiority
over that of Brachet. The latter technique gave as good results, besides being
simpler than the former.
Mucin present in goblet cells and lumen of caecum provided an excellent control
for efficient metachromatic staining.
Metachromasy was not affected by ribonuclease treatment (Plate XII, fig. 3)
showing thereby that it was truly due to mucoid sulphate and not due to
ribonucleic acid. This observation is quite contrary to that of Lewert [1952] who
showed that metachromasy in Plasmodium cynomolgi was due to ribonucleic acid
which disappeared with ribonuclease treatment.
Polysaccharide sulphates were not dissolved by water as the metachromasy
in tissues fixed in Zenker's remained unaffected supporting thereby the conclusion
deduced by Monne and Harde [1952] in their studies on sea urchin eggs that meta-
chromasy was not due to ribonucleic acid. Metachromasy was also not affected
by the use of Bouin's fixative (Plate XII, fig. 1).
B. Distribution of hyaluronic acid type polysaccharide (HAP)
By Hale's method HAP is made visible by a modified prussian blue test. The
material is in the first instance treated with colloidal acetic iron in order to bind
hyaluronic acid with iron ; the latter being made demonstrable by ferrocyanide
test as green or bluish green deposit.
A dot-like structure stained bluish-green appeared within the nucleus of a
freshly penetrated sporozoite. With the process of development into trophozoite
greenish material spread out into cytoplasm of the parasite forming bluish matrix
in which were embedded emerald-green granules. In developing schizont, the
Indian J. vet. Sci., Vol. XXIV, Part IV ]
PLATE
XII
Photomicrograph showing metachromasy Eimeria tenella
(Photomicrographs showing metachromasy of Eimeria tenella as revealed by toluidine blue
Coloured Filters have been used wherever necessary to produce proper photographic effect. Hours
within parentheses denote the stage of infection.)
Fig. 1. Second generation merozoites (120 hrs. × 2250). Tissues were fixed in Bouin's fluid
(aqueous). Note the presence of metachromasy after this treatment.
Fig. 2. Second generation merozoites (120 hrs. × 2250). Tissues were fixed in Bouin's fluid
(aqueous). Note intensely metachromatic granules in the Golgi region (antenuclear).
Fig. 3. Second generation merozoites (120 hrs. × 2250). Tissues were fixed in Bouin's fluid
(aqueous). Sections were treated with ribonuclease prior to staining. Note the persistence of
metachromasy after enzyme digestion.
Fig. 4. Earliest gametocytes (108 hrs. × 1920). Note metachromatic granules at their
periphery. Also note the metachromasy in second generation ripe schizonts (bigger and smaller type
on the left and right bottom of the micrograph respectively).
Fig. 5. Sporozoite (35 hrs. × 2250). Note metachromatic granules in the cytoplasm.
236
MUCOPOLYSACCHARIDES IN EIMERIA TENELLA
[ Vol. XXIV, Part IV,
of the fittest but not extinction of both the competitors. Other (nonpathogenic)
species of Eimeria of poultry, have struck a decent compromise and attained balance
with the host, thus ensuring perpetuation of their species. But it seems that E.
tenella shall take considerable time to arrive at such a compromise.
SUMMARY
Mucoid sulphates occur in the cytoplasm, and hyaluronic acid type polysaccha-
ride (HAP) is more active in the nucleus (especially the karyosome), and also forms
the protective covering of the oocyst.
It is believed that by virtue of their hydrotropic property, mucopolysaccharides
are of immense value to the organism (E. tenella) and perform the following important
functions :
1. They inhibit intra-cytoplasmic clotting and create physiological 'liquefac-
tion' of the nucleus during multiplication and gamete formation.
2. They promote contact between enzymes and substrate by maintaining fluid
condition of the protoplasm.
3. They help to create a fluid zone round the parasite after its penetration into
the host cell. The fluid condition thus produced, promotes osmosis between the
host and the parasite.
4. They protect second generation merozoites and subsequent forms against
the highly coagulating property, the host tissue has acquired at this stage of infec-
tion.
5. HAP in the oocystic wall ensures safe passage of oocysts through acidic
medium of proventriculus of birds.
6. In this protozoon, there is highest concentration of mucopolysaccharides
at the end of the second generation schizogony—a stage, when the parasite acquires
its immunogenic character also. Whether there exists any relationship between
the antigenicity and the mucopolysaccharides in this coccidium still remains to be
determined experimentally.
An intimate association of mucoploysaccharides with the Golgi element of the
second generation merozoites prompts the suggestion that the former are, perhaps,
metabolised by the latter, and that they occur in the form of a mucolipid complex
in this protozoon as in metazoa.
ACKNOWLEDGEMENT
Our sincere thanks are due to the Indian Council of Agricultural Research
for the award of Research Fellowship (Parasitology, 1952-54) to one of us (B.S.G.) ;
and to the Director, I.V.R.I. for providing the necessary laboratory facilities.
REFERANCES
Abel, J. J. and Kubota, S. (1919). J. Pharmacol., 39 : 386
Brachet, J. (1953). Quart. J. micr. Sci., 99 : 1
Bradford, M. J ; Herrick, C. A., and Wolfe, H. R., (1947). J. Parasit., 33 : 393
December, 1954 ]
B.
S.
GILL
AND
H.
N.
RAY
237
Ehrich, W. E. ; Siefter, J., Auburn, H. E. and Begany, A. J. (1949). Proc. Soc. Biol., 70 : 183
Goodrich, H. P. (1944). Parasitology, 36 : 72
Grey, H. and Lunt, L. K. (1914). Amer. J. Physiol., 34: 322
Hale, C. W. (1946). Nature ; 57 : 802
Holmgren, and Wilander, O. (1937). Z. Mekroskop.—Anat. Forsch., 42 : 242
Kunitz, M. (1950). J. gen. Physiol., 24 : 15
Levine, P. H. (1949). J. Parasit., 26 : 337
Lewert, R. M. (1952). J. infect. Dis., 91 : 125
Lison, L. (1935). Archiv. Biol. Liegl., 46 : 599
Lorand, L. (1950). Nature ; 166 : 694
Macheboeuf, M. (1937). Actualetes sci. Industr., 488, Exposes Biol.
M'onne, L. (1946). Ark. Zool., 38, 16
—
(1949). Ibid., 42. 4 and 9
—
and Harde', S. (1950). Ibid., 2, 1. 28
—
(1952.) Ibid., 3, 21
Oliver, J., Bloom, F. and Magnieri, C. J. (1947). J. Exp. Med., 86 : 107
Pratt. I. (1938). Doctorate Thesis, Univ. Wisc. (Quoted by Bradford, et al., 1948)
Riley, J. F. (1953). Science., 118 : 332
Runnstrom, J. (1945). Advanc. Enzymol. 9, 241
—
(1949). Exptl. Cell. Res. Suppl. 1 : 469
—,
Tiselius, A. and Lindvall, S. (1945). Ark. Zool., 36, 22
Shaffiroff, B. G. P., Doublet, H. Shiffert, R. and Tui, C. (1943). Amer. J. Physiol., 138 : 753
Sherwood, N. P., Stoland, O. O., and Nelson. H. G. (1941). J. Bact., 42 : 286
Smetana, H. (1933). Archiv. Path., 15 : 175
Sylven, B. (1941). Acta Chir. Scand. 86 (Suppl.), 66 : 151
Van den Valden. (1909). Arch. exp. Path. Pharmak., 61 : 17
104
AR/54
10
PHOSPHATASES AND THEIR SIGNIFICANCE IN EIMERIA
TENELLA RAILLIET AND LUCET, 1891
By B. S. GILL and H. N. RAY*, Indian Veterinary Research Institute, Mukteswar,
Kumaon, U. P.
(Received for publication on 29 July 1954)
(With Plate XIII)
R AY and Gill [1954] reported their preliminary observations on the distribution
of alkaline phosphatase in various endogenous stages of Eimeria tenella.
The enzymic studies were extended to acid phosphatase and 5-nucleotidase. These
observations and significance of the three phosphatases are reported in this article.
Acid phosphatase has been reported from Entamoeba histolytica [Carrera and
Changus, 1948 ; Carrera, 1950], but so far it has not been reported from coccidia.
5-Nucleotidase has been described from various mammalian tissues [Reis,
1937a, 1937b ; Gulland and Jackson, 1938 ; Newman, et al., 1950 ; Bragdon and
McManus, 1952], but it has not been described from protozoa so far.
PROCEDURE
White Leghorn chicks, 1-2 weeks old, kept coccidia-free, were infected per os
with large doses of a 'pure-line', sporulated brew of E. tenella. They were sacrificed
at 4-hourly intervals from the 1st to 8th day of infection. Their caeca were harvested
with least loss of time. Small pieces (2-3 mm.) of caeca, and smears of mucosal
scrapings were promptly fixed in chilled acetone.
Glick's revised technique [1949] was used for demonstrating acid phosphatase.
Technique of McManus, et al., [1952—cited by Bragdon & McManus, 1952] was
employed for locating 5-nucleotidase both in sections and smear preparations.
OBSERVATIONS
Both acid phosphatase and 5-nucleotidase were present in karyosome at all
stages of the coccidium. In microgametes they were located in the comma-shaped
body. The former enzyme was also present in nuclear membrane and in some cases
also in the Golgi region (ante-nuclear area 2-3 µ long-reported elsewhere) of the
second generation merozoites.
Unlike alkaline phosphatase, no regional difference in the reaction of healthy
and parasitised caecal tissue for acid phosphatase and 5-nucleotidase could be
observed.
DISCUSSION
Phosphatases are a group of enzymes which catalyse (1) hydrolysis and synthesis
of monoesters, diesters, pyrophosphates, phosphoamides, and anhydrides of organic
*At present Professor of Protozoology, School of Tropical Medicine, Calcutta.
239
104 AR/54
11
Indian J. vet. Sci., Vol. XXIV, Part IV ]
PLATE
XIII
Photomicrograph of second sexual generation of Eimeria tenella.
(Photo-micrographs depicting the enzyme reactions of the second asexual generation of Eimeria
tenella)
Fig. 1, 3 and 6 are from smear preparations of caecal mucosa, and the rest are the micrographs
taken from sections. Hours within parentheses denote the stage of infection.
Fig. 1. Merozoites (Acid phosphatase, 120 hrs. × 1500). Note positive reaction in their
karyosomes, and in the Golgi (antenuclear) region.
Fig. 2. Schizont (Acid phosphatase, 100 hrs. × 900). Note the reaction in karyosomes of the
parasite and in nucleolus of the host cell.
Fig. 3. Merozoites (5.nucleotidase, 120 hrs. × 1200). Note the presence of reaction only in
the karyosome.
Fig. 4. Schizont (5-nucleotidase, 100 hrs. × 900). Note the reaction in the karyosomes of the
parasite, and nucleolus of the host cell.
Fig. 5. Schizont (Alkaline phosphatase, 90 hrs. × 900). Note the reaction in karyosomes of
the parasite, and also in the nucleolus of the hypertrophied host cell nucleus.
Fig. 6. Merozoite (Alkaline phosphatase, 120 hrs. × 1000). Note the reaction in karyosome
and the Golgi region. Both the small and bigger type of merozoites are seen.
244
PHOSPHATASES
IN
EIMBRIA TENELLA [ Vol. XXIV, Part IV
REFERENCES
Al Hussaini, A. H. (1948). Nature, 161, 274
Bourne, G. H. (1943). Quart. J. Exp. Physiol., 82, 1
Brachet, J. (1947). Growth, 7, 309
Bragdon, D. E. and McManus, J. F. S. (1952). Quart. J. micr. Sci., 93 ; 87
Carrera, G. M. (1950). Proc. Soc. Exp. Biol. Med, 73 ; 682
Carrera, G. M. and Changus, G. W. (1948). Ibid, 71 ; 610
Danielli, J. F. (1953). Cytochemistry—A critical approach. John Wiley & Sons, Inc. N. Y.
Davidson, J. N. (1940). The Biochemistry of Nucleic Acids. John Wiley & Sons, Inc. ; N. Y.
Dempsey, E. W. and Wislocki, G. B. (1946). J. Cell. Comp. Physiol. ; 27 ; 159
Dounce, A. L. (1951). In Simmer, J. B., and Myrback, K. (1951). The Enzymes, I, 2, 245
Emmel, V. (1945). Anat. Rec. 791, 39
—
(1946). Ibid, 95, 159
—
(1947). Ibid, 95, 423
Folley, S. J. and Kay, H. D. (1936). Egrabbnn. Enzymforsch., 5, 159
Glick, D. (1949). Technique of Cyto-and Histochemistry. I. P., N. Y.
Gulland, J. M. and Jackson, E. M. (1938). Biochem. J., 32, 597
Holmgren, H. and Wilander, O. (1937). Z. Mikroskop-Anat. Forsch., 42, 242
Jeker, (1936). Pflugers Arch., 237, 1
March, J. B., and Drabkin, D. L. (1947). J. Biol. Chem. 168, 61
Montagna, W ; Chase, H. B., Malone, J. D. and Mebragno, H. P. (1952) Quart. J. micr. Sci., 93, 241
Moog, F. (1946). Biol. Rev., 21, 41
Newman, W. L., Feign. I., Wolfe, A. and Kobat, E. A. (1950). Amer J. Path ; 26, 254
Ray, H. N. and Gill, B. S. (1954). Ann. Trop. med. & Parasit., 48, 8
Reis, J. (1937). a. Enzymologia, 2, 110, 183
—
b. Acta. Biol. exp. Warsaw., 11, 122
Riley, J. F. (1953). J. Path. Bact. 65, 461
—
and Drennan, J. M. (1949). Ibid, 61, 245
Robison, K. (1923). Biochem. J., 17, 286
Roche, J. (1942). Chim. Acta., 29, 1253
— (1947).
Ann. Nutrition Alimentation, 1, 3
— (1951).
In Simmer, J. B. and Myrback, K. The Enzymes, 1. I. A. P. Inc. N. Y.
—
and Curtois, J. (1943). Eposis Ann. Biochem, Med., 4, 219
Schmidt, G. and Tannhauser, S. J. (1943). J. Biol. Chem., 149, 369
Sylven, B. (1947). J. Bone and Joint Surgery, 29, 973
Wislocki, G. B. and Dempsey, E. W. (1946). Anat. Rec. 90, 249
SPECIATION AND VIABILITY OF POULTRY COCCIDIA IN
120 FAECAL SAMPLES PRESERVED IN 2.5 PER CENT
POTASSIUM DICHROMATE SOLUTION
By B. S. GILL, Indian Veterinary Research Institute, Mukteswar-Kumaon, U.P.
( Received for publication on 29 July 1954 )
E IGHT valid species of Eimeria parasitising poultry have been reported from
various parts of the world. Examinations conducted by Ray [1939-53] on
occasional faecal samples sent by Provincial Veterinary Officers have recorded
E. tenella, E. maxima, E. acervulina, E. praecox and Wenyonella gallinae from Indian
poultry. However, in the absence of any systematic and detailed survey, information
truly representative of coccidial population and their relative incidence in India is
not available. While working in the Protozoology Section of the Indian Veterinary
Research Institute, Mukteswar (1952-54) an opportunity was offered to the author
to undertake such a survey, and also to determine survival periods of various coccidia
in the commonly used preservative, i.e., 2.5 per cent aqueous solution of potassium
dichromate.
Morphology of unsporulated and sporulated oocysts, sporulation time and
prepatent periods, were the diagnostic criteria kept in view for the purpose of
speciation.
MATERIAL AND METHODS
Faeces were received from the States of Bombay, PEPSU, Punjab, Ajmer,
U. P. and M. P., which were collected both from apparently healthy flocks and from
flocks where actual outbreaks of coccidiosis had taken place.
Each sample was washed free of the preservative with water, and a part of it
was floated with brine to recover oocysts for microscopical examination. Rest of
the sample was fed to chicks, (4-14 days old) raised ' coccidia free '. These chicks
had been segregated from general brooders into single cages a week prior to infection.
Their faeces had remained continuously negative for oocysts during the pre-infective
period. This measure was adopted to eliminate all chances of an adventitious
infection vitiating the viability test, as infection picked up in a general brooder
would have revealed itself during that week. Faeces from infected birds for each
24-hour period were collected from the 4th to the 14th day of infection and suspended
in 2.5 per cent potassium dichromate for sporulation at room temperature.
OBSERVATIONS
Diagnostic survey of the material revealed the presence of Eimeria tenella,
E. acervulina, E. mitis, E. maxima, E. praecox, E. hagani and Wenyonella gallinae
in it ; order of frequency of their occurrence was : 70, 50, 48.4, 30.8, 29.2, 2.5 and 1.7
245
104 AR/54
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December, 1954 ]
B.
S.
GILL
247
tests. Average survival time of different coccidia in the preservative has been found
to be 1-2 years. Viability has been seen to sharply decline during the third year
of preservation such that by the end of the third year whole population of different
coccidia was rendered non-viable.
It was also noticed that with the age of preservation prepatent periods of E.
tenella, E. mitis and E. maxima were abnormally lengthened : 24-48 hours above the
usual periods. However, on further passage in susceptible poultry their prepatent
periods returned to the normal cycles.
ACKNOWLEDGEMENT
The author is indebted to Dr. H. N. Ray for suggesting the problem and helpful
criticism, and to the Indian Council of Agricultural Research, New Delhi, for the
award of Research Fellowship in Parasitology (1952-54).
REFERENCES
Dickinson, E. M. (1946). Poultry Sci., 25 : 319
Ray, H. N. (1939-53). Annual Reports of the Indian Veterinary Research Institute
COMPARATIVE FLOATING EFFICIENCY OF COPPER
NITRATE, COMMON SALT AND ZINC SULPHATE SOLU-
TIONS AS LEVITATING MEDIA IN A MODIFIED
LANE'S (1923-24) D.C.F. TECHNIQUE FOR
POULTRY COCCIDIA
By B. S. GILL, Indian Veterinary Research Institute, Mukteswar-Kumaon, U. P.
( Received for publication on 29 July 1954 )
F ULL realisation of pathogenicity of intestinal protozoa and helminths to man
and animals necessitated the devising of coprologic technique for concentrating
cysts or ova in fæces for establishing diagnosis. Literature upto 1924- has been
competently reviewed by Lane who also devised his direct centrifugation floatation
(D.C.F.) technique in 1923-24, various modifications of which have been used by
both medical and veterinary workers [Sheather, 1923 ; Young, 1923-24 ; Andrews
and Tsuchiya, 1931 ; Faust, et al., 1938 ; Swartzwelder, 1938 ; Otto, et al., 1941 ;
Farr and Luttermoser, 1941 ; Farr and Allen, 1942 ; Ripsom and Herrick, 1945 ;
Morehouse and Mayfield, 1945 ; Watson, 1947 ; Garcia and Pesigan, 1948 ; Koutz,
1948].
The literature extant on the subject revealed to the author many a short-
coming in the various procedures described so far. For poultry coccidia only
common salt [Fish, 1931 ; 1932], sugar and sodium nitrate [Koutz, 1948] solutions
have been used as floating media while many other chemical solutions which have
given good floats of helminth ova and protozoal cysts from human fæces, have not
been tried on poultry coccidia. Further, the comparative floating efficiency of
brine and syrup has not been investigated. Methods employed were not so simple
as advocated by the respective authors. The suspension to be examined under
hæmocytometers for counts [method employed by Beach, 1943 ; Farr, 1944 ;
Grumbles, et al., 1948] should be finely strained and much diluted. Besides entailing
extra labour and care this method has given varying counts with the same sample
[unpublished observations of the author and of D. K. Ray, 1953 in the laboratory
of the I. V. R. I.]. Levine's [1939] method required special vials and buckets to
receive them in the centrifuge, and unless the suspension was absolutely free from
fæcal debris, enough light might not pass through 25 mm. high column of the fluid
to reach the objective of microscope. Further, the method would be of no avail
when fæces contained much blood as in cæcal coccidiosis.
Before investigating various problems related to poultry coccidia, necessity
was felt for finding an efficient and simple method for concentrating oocysts. This
necessity initiated the present study. The following criteria were kept in mind
while assessing the suitability of a levitating medium to be employed.
1. It should give a maximum total count of oocysts in a few centrifugations ;
2. It should concentrate a high percentage of oocysts on first centrifugation ;
249
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FLOATING EFFICIENCY OF LEVITATING MEDIA [ Vol. XXIV, Part IV,
3. It should cause no appreciable shrinkage and loss in viability of oocysts
when the latter remained in it for a reasonable length of time ;
4. It should give consistent results when used over and over again on the
same fæcal sample, i.e. it should be reliable ;
5. It should be cheap and easily available ;
6. It should be highly soluble in water ; and
7. It should give cool and clear visual microscopical field to obviate eye
strain as far as possible.
MATERIAL
Four fæcal samples containing sporulated and four samples containing un-
sporulated oocysts were tried, but for any one of the series of experiments the same
sample was used throughout. For ' A ' series of trials noted in Table I, fæcal samples
preserved in 2.5 per cent aqueous solution of potassium dichromate and for the rest
four series fæces freshly voided by artificially infected chicks were employed. In
both the cases, there was mixed infection with Eimeria tenella, E. mitis and E.
præcox ; but the oocysts were sporulated in the former series of trials and un-
sporulated in the latter series.
Aqueous solutions of common salt (NaCl), zinc sulphate (ZnSo 4 ) and copper
nitrate (CuNO 3 ) were tried in these investigations. Specific gravity in the various
solutions was kept constant (1,200) to serve as a common denominator in these
comparative quantitative studies. Their solutions were prepared as follows :
(a) ZnSO 4 , 331.4 gm. plus 1 litre of water [Faust, et al., 1939]
(b) Common salt, 380 gm. plus 1 litre of water
(c) CuNO 3 , 100 gm. plus 344 ml. of water
Specific gravity of each solution was checked with a standard hydrometer and
if found different was adjusted accordingly.
In the beginning, it was intended to include magnesium sulphate and sugar
solutions in this investigation, but having seen their behaviour with the rabbit
coccidia [unpublished data] both the solutions were dropped in these trials due to
the following reasons :
Both the media gave ' statistically significant ' lower floats as compared to the
solutions of Cu NO 3 , Zn SO 4 and Na Cl. Sugar floated a lot of fæcal debris along
with oocysts, attracted flies in the laboratory, and because of its high refractive
index produced a glaringly shiny microscopical field thus making difficult prolonged
observations, besides adversely affecting the eyes. Oocysts in it, turned black
and soon were out of recognition—a great disadvantage when one is speciating the
various coccidia. Lastly, the speed of recovery of oocysts was very slow probably
because of its viscous nature. MgSO 4 solution crystallised soon under the coverslip
thus its preparations could not be kept for prolonged observations.
Ordinary centrifuge tubes 15 c.c. capacity, with rims made perfect by grinding
their tops against carborandum stone and cover slips of no particular specification,
but absolutely grease-free were used.