NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
118
Helmintholoyy in Relation to Veterinary Science
Cameron, T. W.M. (1932). Imp. Bur. of Agri. Parasit. Notes and Memoranda
7, pp. 1-8.
Fourie, P. J. J. (1931). 17th Rep. Director Vet. Ser. & Anim. Husb. U. S. Afri.
pp. 495-572.
Le Roux, P. L. (1930). J. S. Afri. Vet. Med. Assn. 1, 4, pp. 43-65.
Martin, C J. and Ross, C. I. (1934). J. Helm. 12, 3, pp. 137-142.
Wells, H. S. (1931). J. Parasit. 17, 4, pp. 167-182.
ESTIMATION OF TOTAL SULPHATE IN BLOOD SERUM
BY
N. C. DAS GUPTA
Animal Nutrition Section, Imperial Veterinary Research Institute, Izatnagar.
(Received for publication on 11th April 1938)
TOTAL sulphates are usually estimated by hydrolysing the substance and
precipitating the sulphates as barium or benzidine salts. The nephelometric
method of Denis and Reed [1926-1927] suffers from the disadvantage that the
turbidity of the suspension of barium sulphate is dependent on changes in the
pH. and composition of the blood filtrate.
Wakefield [1929] hydrolysed blood filtrate with hydrochloric acid and
estimated the sulphates as benzidine sulphate. He used two drops of concen-
trated hydrochloric acid to 5 c.c. of blood filtrate and kept the tube in boiling
water for fifteen minutes. It was allowed to cool and benzidine solution was
then added. The present author has found that with blood serum of cattle, the
concentration of acid used and the time allowed for hydrolysis are not sufficient
for complete hydrolysis. The hydrochloric acid present is also partly precipitat-
ed as benzidine chloride, which being insoluble in acetone cannot be removed by
washing. Fiske [1921] studied the difficulties which are encountered when
benzidine is used to separate sulphate quantitatively from other inorganic
compounds such as phosphates and chlorides. Hubbard [1930] also mentioned
about the interference of benzidine chloride when sulphate was precipitated
in the hydrolysed liquid.
The present work was started to find out a method of avoiding these
difficulties and to work out a suitable technique for the estimation of total
sulphates in blood serum.
1. EXPERIMENTAL WORK
Hydrolysis.—Tests were made to find out the effect of the concentration
of hydrochloric acid and the duration of boiling on hydrolysis of protein-free
serum filtrates.
(a) Effect of the concentration of acid.—Duplicates did not agree in many
cases when lower concentrations of acids, viz., 0.1 c.c. conc. hydrochloric acid
in 5 c.c. filtrate were used for hydrolysis. Better results were obtained with
higher concentrations. Two c.c. serum filtrate samples were hydrolysed for two
hours with different quantities of conc. hydrochloric acid 0.1 c.c. acid always
gave slightly lower results. 0.2 c.c. acid was also sometimes insufficient for
complete hydrolysis. Good results were obtained with 0.3 c.c. acid but as the
presence of more acid during hydrolysis did not interfere with the sulphate
precipitation, if the technique explained in the sequel were followed, the
writer prefered 0.4 c.c. acid to avoid any risk of incomplete hydrolysis.
( 119 )
120
Estimation of Total Sulphate in Blood Serum
TABAL I
Effect of the amount of acid on hydrolysis
Amount of conc. hydrochloric acid used for
2 c.c. filtrate
0.1 c.c
.
.
.
.
.
0.2 c.c
.
.
.
.
.
0.3 c.c . . . . .
0.4 c.c
.
.
.
.
.
S found by the macro method
Mg. per cent S as total sulphate
Sample 1
3.94
5.44
5.40
5.44
4.81
5.10
5.44
5.40
5.59
Sample 2
5.49
5.68
5.75
5.81
5.56
5.81
5.81
5.76
5.96
Table I shows that the extent of hydrolysis obtained with 0.3 c.c. and
0.4 c.c. acid compares favourably with that obtained by the macro method,
where 400 c.c. of serum filtrate, equivalent to 80 c.c. serum, were hydrolysed
with 20 c.c. concentrated hydrochloric acid and the sulphate was precipitated
and weighed as barium sulphate.
(b) Effect of time on hydrolysis.—The effect of the duration of hydrolysis
with the same concentration of acid, 0.4 c.c. for 2 c.c. filtrate, was tested
with some samples. The following results in Table II show that half hour
and one hour boiling gave lower results. There was no difference between
two hours and four hours' boiling. Therefore even with this increased
concentration of acid, blood filtrate samples should be hydrolysed for at least
two hours to ensure complete hydrolysis.
TABLE II
Relation between time and amount of hydrolysis
Duration of hydrolysis
Half hour
.
.
.
.
.
One hour
.
.
.
.
.
Two hours
.
.
.
.
.
Four hours
.
.
.
.
.
S found by the macro method
Mg. per cent S as total sulphate
Sample 1
4.63
4.90
5.44
5.40
5.59
Sample 2
4.90
5.56
5.95
5.81
5.49
5.10
5.75
5.75
5.96
Sample 3
2.81
..
2.84
2.87
2.38
..
2.84
2.84
2.91
N. C.
DAS
GUPTA
121
2.PRECIPITATION OF BENZIDINE SULPHATE
(a) Interference caused by the presence of hydrochloric acid.—It was found
that large quantities of chlorides separated when 5 c. c. of 0.5 per cent benzi-
dine in acetone was added to 2 c.c. of the hydrolysed sample. Even if one
drop of concentrated hydrochloric acid is used for hydrolysis, higher results
are obtained on account of the interference of chlorides. Sulphates estimated
in the same sample after removal and without removal of the drop of hydro-
chloric acid used for hydrolysis gave 3.05 per cent and 4 .3 per cent S respec-
tively. In some cases when the quantities of acid were small, it was noticed that
the chlorides might not separate with the addition of the benzidine solution. It
remained in solution in the liquid (about 70 per cent acetone by volume).
But, while washing, after the first decantation, the traces of the chloride
solution left adhering to the sulphate precipitate or sticking to the sides of
the tube became insoluble as soon as acetone was added for washing the
precipitate. Higher concentrations of hydrochloric acid kept both benzidine
sulphate and benzidine chloride in solution, the solubility increasing with the
hydrochloric acid concentration.
This difficulty was overcome by frequent tests in this laboratory which
showed that if all the free hydrochloric acid was removed by evaporation in
vacuum at 100°C, the quantities of chlorides left in the hydrolysed sample
did not interfere with the results when pure acetone was used for washing.
TABLE III
Relation between concentration of hydrochloric acid and precipitabilty
of benzidine salts
Concentration of hydrochloric acid
No hydrochloric acid
.......
0.25 c.c. N hydrochloric acid
.....
0.5 c.c. N hydrochloric acid
......
1.0 c.c. N hydrochloric acid
......
1.8 c.c. N hydrochloric acid
......
Mg. per cent S as
total sulphate
5.44
5.96
5.81
4.67
0.61
Two c.c. serum filtrate samples were hydrolysed and evaporated to dryness in
vacuum to remove the hydrochloric acid used for hydrolyses. 0.2 c.c. of 20
per cent tri-chloracetic acid and different quantities of N hydrochloric acid
were added to the residues and the volumes made up to 2 c.c. Five c.c. benzidine
in acetone were added to each sample. Table III shows that higher results
were obtained when either 0.25 c.c. or 0.5 c.c. N hydrochloric acid
was present due to the precipitation of benzidine chloride. One c.c. N
hydrochloric acid kept some benzidine sulphate in solution. No precipitate
122
Estimation of Total Sulphate in Blood Serum
separated with 1.8 c.c. acid, the solution in the tube was quite clear; the
figure obtained, viz., 0.61 mg. S, was due to the soluble benzidine salts adhering
to the sides of the tube made insoluble by the pure acetone used for washing.
(b) Effect of acidity of the liquid on the benzidine sulphate precipitate.—
The trichloracetic acid present in the serum filtrate was also removed
along with the hydrochloric acid used for hydrolysis when the hydrolysed
sample was dried under vacuum. The dried substance left in the tube
was practically neutral. This was dissolved in water and several attempts
were made to precipitate sulphate in this neutral solution but they were not
successful. The liquid should be acidic. The following table shows the opti-
mum acidity required for the complete precipitation of benzidine sulphate :—
TABLE IV
Amount of acid added to the
dried sample
2 c.c. water ; solution neutral .
2 c.c. 0.5 per cent trichloracetic
acid
2 c.c. 1 per cent trichloracetic acid
2 c.c. 2 per cent trichloracetic acid
2 c.c. 3 per cent trichloracetic acid
2 c.c. 4 per cent trichloracetic acid
By macro method . .
Mg. S per cent as total sulphate
Sample
A
No pre-
cipitate
..
..
3.33
..
3.26
3.50
Sample
B
No precipi-
tate
10.00
..
5.26
..
5.10
9.62
..
5.26
..
5.10
5.15
Sample
C
No precipi-
tate
..
2.78
2.79
..
2.29
..
..
2.78
..
..
2.91
Sample C
+
0.01 mg.
phosphorus.
No precipi-
tate
..
7.81
2.94
2.84
2.75
..
6.25
2.84
2.84
2.63
2.91
Five c.c. of 0.5 per cent benzidine in acetone was added for precipitation.
There was no precipitate when the solutions were neutral. But sulphates
separated as soon as the solution was made acid with one drop of 20 per cent
trichloracetic acid. The above figures show that best results were obtained
when 2 c.c. of 2 per cent acid was used. Four per cent acid gave decidedly
lower results.
(c) Interference caused by the presence of phosphates.—The results
obtained with less acid in Table IV are of interest. With sample B
much higher results (100 per cent) were obtained when the liquids were
just acid. Similarly in the case of sample C, one per cent acid gave
N. C.
DAS
GUPTA
123
good results with the normal sample, viz., 2.78 mg. S per 100 c.c. serum, but
when the inorganic phosphorus content was nearly doubled by the addition of
0.01 mg. phosphorus in each tube (1 c.c. serum filtrate equivalent to 0.2 c.c.
serum was taken for each estimation in the case of sample C. 0.01 mg. extra
phosphorus in 0.2 c.c. serum is equivalent to 5 mg. extra phosphorus per
100 c.c. serum), one per cent acid figures were 7.81 and 6.25 mg. S per cent,
that is more than double the quantity present in the sample. The higher
results in both the samples B and C were due to the separation of benzidine
salts of phosphoric acid on account of insufficient acid. High concentration
of acid tended to hold benzidine sulphate in solution and low ones to permit
the precipitation of phosphates. With samples of normal phosphorus content
(5 mg. inorganic phosphorus per cent) one per cent acid might be
sufficient in certain cases to keep the phosphates present in solution, but
with samples of abnormally high phosphorus content it was safer to use three
per cent acid.
The following observations in Table V confirm the results obtained in
Table IV :—
TABLE V
Mg. phosphorus in
the tube
0.01 . . .
0.02 . . .
0.08 . . .
Acidity of the 2 c.c. phosphate solution in terms of per
cent trichloracetic acid
Neutral
No precipitate
No precipitate
No precipitate
One per cent
Slight precipi-
tate
Precipitate .
Precipitate .
Two per cent
No precipitate
No precipitate
Slight precipi-
tate
Three per cent
No precipitate
No precipitate
No precipitate
Pure potassium phosphate solutions were used for the above tests. Five c.c. of
0.5 per cent benzidine in acetone were added to each tube. The table shows
that, like sulphates, phosphates also did not precipitate in neutral solutions.
Two per cent acid was not sufficient when 0.08 mg. phosphorus was present.
(d) Time required for the quantitative precipitation of sulphate.—
Cuthbertson and Tompsett [1931] centrifuged the solution half-an-hour
after the precipitation. Pirie [1934] recommended at least two hours in ice
for complete precipitation. Trials here showed that better results were
obtained when the samples were kept overnight in the refrigerator.
(e) Recovery of added organic sulphates.—The results obtained by follow-
ing the technique suggested have been frequently checked by duplicate
analyses with different quantities of serum filtrate and by gravimetric
methods. Further, to test the accuracy and reliability of the method,
minute quantities of benzidine sulphate solution in hydrochloric acid were
F
124
Estimation of Total Sulphate in Blood Serum
added to serum filtrate, which were then estimated as usual. Blank determina-
tions on the serum filtrate alone were made simultaneously. The results
obtained are given in Table VI.
TABLE VI
—
Sample A . . .
Sample B . . .
Mg. S added to
serum filtrate
0
0.002
0.004
0.006
0
0.002
0.004
0.006
Mg. S found
0.0244
0.0265
0.0285
0.0301
0.0253
0.0274
0.0294
0.0313
Excess over
blank
....
0.0021
0.0041
0.0057
...
0.0021
0.0041
0.0060
The figures show that added sulphates could be accounted for satisfactorily
As a result of the above tests, the following method was found to give
quite satisfactory results for total sulphates. Two c.c. serum are diluted with
6 c.c. water, 2 c.c. of 20 per cent trichloracetic acid added, mixed thoroughly
and filtered through a dry ashless filter after fifteen minutes. Two c.c. filtrate
are taken in a 15 c.c. centrifuge tube and hydrolysed with 0.4 c.c. conc,
hydrochloric acid for two hours in a beaker of boiling water. After the
hydrolysis the liquid is evaporated to dryness in vacuum at 100° C. The
residue is dissolved in 2 c.c. of 2 per cent trichloracetic acid and sulphate
precipitated with 5 c.c. of 0.5 per cent benzidine in acetone. After mixing
thoroughly the tube is placed in a wide mouth stoppered bottle and kept in
the refrigerator.
Next day the precipitate is washed and sulphate estimated colorimetri-
cally. The method of Cuthbertson and Tompsett [1931] was found quite
satisfactory for this purpose. The addition of 15 per cent sodium hydroxide
was omitted as recommended by Pirie [1934].
SUMMARY
1. A method had been described for the determination of total sulphate
in blood serum.
N. C.
DAS
GUPTA
125
2. The hydrochloric acid which usually interferes with the estimation of
total sulphates is removed by vacuum evaporation.
3. The optimum acidity required for the quantitative precipitation of
sulphates and for keeping the phosphates in solution has also been studied.
ACKNOWLEDGMENTS
The author is grateful to Dr. F. J. Warth for the interest he has taken
in this work. I am also indebted to Dr. K. C. Sen for his help in the pre-
paration of this paper.
REFERENCES
Cuthbertson, D. P. and Tompsett, S. L. (1931). Biochem. J. 25, 1237.
Denis, W. and Reed, L. (1926-27). J. Biol. Chem. 71, 191.
Fiske, C. H. (1921). J. Biol. Chem. 47, 59.
Hubbard, R. S. (1930). J. Biol Chem. 88, 663.
Pirie, N. W. (1934). Biochem. J. 28, 305.
Wakefield, E. G. (1929). J. Biol. Chem. 81, 713.
DIPETALONEMA DRACUNCULOIDES (COBBOLD, 1870)
BY
M. ANANT NARAYAN RAO, G.M.V.C.
Lecturer in Parasitology, Madras Veterinary College
(Received for publication on 30th March 1938)
[With three text-figures]
THE presence of Dirofilaria repens Railliet and Henry [1911] in the subcuta-
neous tissue of dogs in Bihar and Orissa has been recorded by Panda [1932]
and Dirofilaria immitis [Leidy, 1856] has been recorded in indigenous dogs in
South Bengal by Mitter [1912]. As regards D. immitis, it would appear
from the references cited by Bhalerao [1935], that in other places in India, it
has been recorded only in imported dogs particularly from Burma and Federa-
ted Malay States. Hence Mitter's finding of D. immitis in indigenous dogs
of South Bengal may require confirmation. So far as Madras Presidency
is concerned, these two species of worms have not been found in indigenous
dogs hitherto. The embryos of these two species, however, are found in the
circulating blood.
Rao [1923] and Korke [1924] described Haematozoon lewisi (Filaria re-
condita Grassi) and Microfilaria lewisi, respectively, in the circulating blood
of dogs, but neither of them could find any adult filarid worms in the blood
vessels and other organs at post mortem examination. No worker in India
has so far recorded Dipetalonema dracunculoides [Cobbold, 1870] in dogs. The
present opportunity is, therefore, taken to record its presence in this country.
The embryos of this species are found in the circulating blood and resemble
very closely the microfilariae described by Rao [1923]. The description of
Microfilaria lewisi given by Korke [1924] conforms to that of D. dracuncu-
loides. Korke himself admits this fact and further adds that it is
probable that the microfilariae so frequently observed by Lewis in Calcutta
(and whose measurements as mentioned by Mitter [1912] were shorter than
those of the embryos of D. immitis) are identical with Microfilaria lewisi. It
is, therefore, apparent that both Rao and Korke described the same species of
microfilariae which are actually the embryos of D. dracunculoides, and this is
confirmed by the finding of the adult worms in the peritoneal cavity of an
indigenous dog of the Rajapalayam breed of South India. The author
obtained 18 males and 43 female worms from that dog.
Description of the male worm.—Length 22 to 27 mm.; breadth 0.140 to
0.200 mm. at its middle. Filiform, cuticle smooth, but in some it looked as
if there are a few fine longitudinal straitions. The two extremities gradually
taper, the head end being blunt and the tail end pointed. The tail is coiled
spirally and has three to four coils. The end of the tail bears on it two lateral
digitations giving it a trifid appearance, as in the genus Setaria [Viborg, 1795].
( 127 )
128
Dipetalonema dracunculoides
Fig. 1—Tail end of Male.
Fig. 2—Head end of Female.
M.ANANT
NARAYAN
RAO
129
Fig. 3—Tail end of Female.
There are four pairs of pre-anal and two pairs post-anal papillae. These pa-
pilllae are small and the cloacal opening is guarded by the last pair of pre-anal
and the first pair of post-anal papillae. The second pair of the post-anal
papillae is just in front of the lateral digitations. The spicules are
unequal. The left one is long and is divisible into an anterior wide
and cylindrical part and the posterior narrow and tubular one. It
is approximately 0.310 mm. long. The right spicule, which is smaller
than the other, measures nearly 0.161 mm. in length, is cylindrical and below
its middle it is more or less twisted and then gets attenuated to terminate into
a fine hook or a crochet hook as Railliet and his collaborators [1912] designate
it. The head end is rounded. The mouth is a simple pore and is guarded by
two lateral flattened papillae. There are four sub-median papillae on
the head.
Description of the female.—Length 30 to 55 mm. ; breadth 0.200 to 0.300
mm. at its middle. The head end is as in the male. (Fig. 2.)
The tail end is also as in the male (Fig. 3) but not spirally coiled.
In some of the immature females the tail end shows a tendency to coil spirally
and usually there is a curve representing half a turn and rarely a full one.
The vulva-is situated in the region of the posterior half of the oesophagus,
about 1.5 mm. from the head end. Opisthodelphys. Viviparous. Embryos
unsheathed 200 to 225 × 5µ in length.
It will be thus seen that the description of the worms given above agrees
with that of Dipetalonema dracunculoides [Cobbold, 1870] given by Railliet and
his collaborators [1912].
130
Dipetalonema dracunculoides
REFERENCES
Bhalerao, G. D. (1935). Imp. Coun. Agri. Res. Sci. Mon. No. 6, pp. 365.
Korke, Vishnu T. (1924). Ind. J. Med. Res. 11, 1231-1237.
Mitter, S. N. (1912). Bull. Soc. Path. Exot. 5, 731-732.
Panda, P. (1932). Ind. Vet. J. 8, 241-250.
Railliet, A., Henry, A. and Longeron, M. (1912). Bull. Soc. Path. Exot. 5, 392-395.
Rao, M. A. N. (1923). Imp. Inst. Agri. Res. Bull. No. 144.
POTERIOSTOMUM RATZI (KOTLAN, 1919)
BY
M. ANANT NARAYAN RAO, G.M.V.C.
Lecturer in Parasitology, Madras Veterinary College
(Received for publication on 30th March 1938)
[With two text-figures]
ON examination of a collection of Trichonema obtained last year, two
species of Poteriostomum were found, viz., P. imparidentatum [Quiel, 1919] and
P. ratzi [Kotlan, 1919]. The former has been reported from the Punjab and
United Provinces [Bhalerao, 1935]. The latter species has not been recorded
hitherto in horses in India, and a brief description of it is given below. The
infestation with these worms seems to be small and the writer found about
six of the former and four of the latter species in the large intestines of two
different hackney ponies in Madras. No dimensions of the worms are noted
since there were only three males and one female available for study, and only
the male worm is described below for obvious reasons.
Male.—Mouth collar is marked off from the body by a groove. The ele-
ments of the external leaf crown are slender and many, and the internal leaf
crown is composed of elements of equal size and furnished with pointed ends
(Fig. 1, see page 132). The formula of bursal rays is as follows:—
The main trunk of the dorsal ray is not cleft to its base but only to half
its length and the two lateral branches arise from the undivided portions
close to the place of origin of the externo-dorsal ray. The externo-dorsal
ray originates from a common trunk with the dorsal (Fig. 2, see page 132).
Host.—Hackney pony.
Habitat.—Large intestines.
Place.—Madras.
REFERENCE
Bhalerao, G. D. (1935). Helminth Parasites of the Domesticated Animals in India.
Imp. Coun. Agri. Res. Sci. Mon. No. 6, pp. 365.
( 131 )
G
132
Poteriostomum ratzi
Fig. 1—Head end.
Fig. 2—Bursa of male.
A CHECK- AND HOST-LIST OF IXODOIDEA (TICKS)
OCCURRING IN INDIA
BY
PURNENDU SEN, M.Sc. (CAL.), PH.D. (LOND.), D.I.C.
Offg. Entomologist, Imperial Veterinary Research Institute, Mukteswar
(Received for publication on 18th April 1938)
INTRODUCTION
A PERUSAL of the literature reveals that comparatively little work has
been done to compile a check-list and host-list of the Ixodoidea (ticks), a
highly interesting group of Arthropoda, many of which are of immense impor-
tance to human economy as being responsible for the transmission of various
diseases amongst men and domesticated animals.
The Ixodidae occurring in this country have been very ably listed by
Sharif [1928] who recorded altogether nine genera and forty-five species,
four sub-species and six varieties, but the only reliable account of the Arga-
sidae has been given by Nuttall, Warburton, Cooper and Robinson [1908-
1926].
Since these publications, additional records have accumulated and it
is felt that the data such as that given in the present paper concerning the
distribution and hosts of the ticks (both Argasidae and Ixodidae) are worth
publishing for ready reference of interested workers.
Almost all the new records that have come to light as a result of the
present study are based on collections of the Imperial Veterinary Research
Institute at Mukteswar (India), and the ticks dealt with were for the most
part already determined by Warburton, Sharif and Sen (S. K.), excepting a
few specimens examined by the author.
In arranging the species in the text, only the original references to the
ticks concerned, together with the reference of either Nuttall et al " A
Monograph of Ixodoidea " in case of the Argasidae, or Sharif's paper " A Revi-
sion of the Indian Ixodidae with special reference to the Collection in the
Indian Museum " in case of the Ixodidae are given. The species of the various
Indian ticks that are represented in the collection of the Imperial Veterinary
Research Institute at Mukteswar are marked with asterisks (*) in the host-
parasite list appended. In discussing the distribution and hosts of the Ixo-
doidea only the records of the strictly Indian territories have been taken
into consideration.
( 133 )