NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
144 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, II
Three of the organisms did not produce any appreciable amount of volatile
acid nor did they show any marked increase in volatile acidity after addition of
citric acid, but the streptococcus from butter shows in milk alone a high volatile
acidity, which is further increased by the addition of citric acid to milk. The
number of c.cs. of N/10 NaOH required for neutralizing the distillate from S.
citrovorus culture as recorded by Hammer [1920] are 20.3 without citric acid and
57.3 with citric acid on an average, while for S. paracitrovorus the numbers
recorded by Hammer [1923] are 28.6 in milk without citric acid and 73.9 in milk
with citric acid, the least number of c.c. of N/10 NaOH required for neutralising
the distillate from milk and citric acid culture of S. paracitrovorus being 44.4.
Judged by a comparison with these two sets of results, the streptococcus isolated
by us from butter appears to be a citric acid fermenter more akin to S. citrovorus.
(3) Relation of temperature to growth and activity of the streptococcus from
butter.—10 c.c. tubes of sterile separated milk were inoculated with the organism
and incubated at 21°C., 30°C. and 37°C. and the acidity determined by titration
with N/10 NaOH after 24 hours and 72 hours. The following table gives the
number of c.c. of N/10 NaOH required to neutralise the acid formed ; the figures
represent the average for four tubes.
TABLE II
Temperature of incubation
21°C . . . . . . . .
30°C . . . . . . . .
37°C . . . . . . . .
After 24 hrs.
8.1
9.4
10.2
After 72 hrs.
10.4
11.4
10.1
The optimum temperature for the growth and formation of lactic acid by this
streptococcus from the above table is regarded to be 30°C. because it has given the
highest acidity at this temperature after 72 hours, although the organism shows
nearly equal amount of acid at the other temperatures.
(4) Cream ripening tests.—Cream ripening tests with different organisms were
next carried out to see which of them gives aroma to the butter. Three lots of
cream, each 4 ounces, were heated and held at 100°C. for half an hour and sepa-
rately inoculated after cooling with the following organisms :—
(i) Streptococcus lactis.
(ii) Bulgaricus type from dahi.
(iii) Streptococcus from butter.
After keeping for 48 hours at 21°C. the creams were churned and the butter
separated out from each. The samples of butter were submitted to different
AROMA-PRODUCING LACTIC ACID ORGANISM
145
persons for judging the taste and flavour. As a combined result of the judgments
of several persons, it was found that:
(i) Streptococcus lactis butter had no flavour and was mildly acidic.
This butter stood second in the order of merit according to the
judgment by taste of different persons.
(ii) Bulgaricus type butter had a decided acid flavour and a strong acid
taste. This butter stood third in the order of merit.
(iii) Streptococcus from good butter gave a mildly acid tasting butter.
It had also aroma and flavour. This butter stood first in the order
of merit.
The experiment was repeated three times with identical results.
(5) Large-scale cream-ripening tests.—A large-scale experiment on the produc-
tion of butter from cream was conducted at the Institute of Dairying and Animal
Husbandry at Bangalore in June 1933. The cream used was mixed (morning and
evening). The morning cream, after separation of milk which had been pasteurised,
was kept without any further treatment till the time of inoculation. No effort
was made to prevent any natural souring of the morning cream except that it was
kept immediately after separation of the pasteurised milk in a cold temperature
room. After separation of the evening cream, both were mixed and divided into
lots of 9 lbs. each in separate buckets. The starters for use were prepared in
separated milk from pure cultures. The cream was ripened for 24 hours at the
dairy room temperature, about 22°C., to 25°C., and then transferred to the cold
room. The butters were scored according to the combined judgment of several
persons including the Imperial Dairy Expert, the members of the staff and some
students of the Dairy Institute with the request that they should place the
butters in order of merit; and the final places were determined by adding the
numbers of the order which each butter obtained. The butter with the smallest
number was considered to have obtained the first place. As this common general
judgment tallied with the expert judgment of the Imperial Dairy Expert, no
attempt was made to have any more accurate scoring of the butters. The results
are given in the following Table :
TABLE III
Organism tested
Dahi organism (resembling the Bulgaricus type of
lactic acid organisms).
Streptococcus lactis
.
.
.
.
.
.
.
Culture of the organism (Streptococcus) described in
this paper
The local starter used in the Bangalore Dairy . .
Per cent
acidity
of ripened
cream
0.34
0.43
0.35
0.37
The place
obtained by
the butter
from orga-
nisms in order
of merit
2
3
1
4
E
AROMA-PRODUCING LACTIC ACID ORGANISM
147
collecting the distillate in fractions. To the first fraction (25 c.c.) of the distillate
were added a mixture of the following solutions:
2 c.c. of hydroxylamine hydrochloride (20 per cent solution).
3-5 c.c. of sodium acetate (20 per cent solution), and
1-2 c.c. of nickel chloride (10 per cent solution) and warmed to 80°c.
If the first fraction showed a significant precipitate, the reagents were added
to the second fraction (75 c.c) of the distillate. The quantities of the reagents
given are sufficient for about hundred mgms. diacetyl according to Van Niel
[1927]. The distillate with the reagents was allowed to stand for 24 hours for
complete crystallisation. This can also be accomplished by heating it to one hour
on a water-bath at 80°C. The nickel salt precipitated is then filtered into a weighed
crucible, washed and dried to a constant weight at 105°C.—110°C. and the results
recorded as mgms. of nickel salt equivalent to acetyl-methyl-carbinol+
diacetyl per 200 grms. of milk. The quantity of ferric chloride solution used to
oxidise acetyl-methyl carbinol to diacetyl was first determined by trial distilla-
tions of milk to which were added watery solutions of these substances which were
kindly supplied as samples by a firm of manufacturing chemists in Holland. The
results of the experiment are given in the following table.
TABLE IV
Mgms. of nickel salt equivalent to acetyl-methyl-carbinol and diacetyl
Culture used
Streptococcus under study . . .
Streptococcus under study+Hammer's cul-
ture M. 29.
Streptococcus lactis
.
.
.
.
.
Streptococcus under study . . .
Streptococcus
under
study+Hammer's
culture, M. 29.
Hammer's culture, M. 29 . . . .
Streptococcus under study . . .
No. of days
of
Incubation
4
4
5
5
5
5
7
Milk alone
Traces .
Traces .
Nil
Traces .
Traces .
Traces .
Nil
Milk plus
0 . 5 per cent
citric acid
30
42
Traces
77
57
49
16
From the Table IV, it is clear that in milk they do not produce any detectable
quantities of acetyl-methyl-carbinol+diacetyl, but with the addition of citric acid
to milk, both M. 29 and the organism under investigation produce a considerable
amount of acetyl-methyl-carbinol+diacetyl, while S. lactis does not produce any,
even with citric acid addition to milk. Further, we find that after seven days'
incubation, the amount is much less than that after five days of incubation with
the organism under study. This appears to show that the organism first
148 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, II
forms acetyl-methyl-carbinol-and diacetyl, but afterwards utilises these compounds
for its own use or decomposes them. This would be in accordance with the results
of Michaelin et al [1933]. A fresh experiment was, therefore, carried out and the
products determined in separate flasks containing milk and citric acid which were
inoculated with the organism under study and distilled with steam at different
periods. The results are given in the table below :—
TABLE V
Mgms. of nickel salt equivalent to acetyl-methyl-carbinol and diacetyl in 200
grm, culture
Culture
Streptococcus under study
.
.
.
.
.
Ditto
.
.
.
.
.
Ditto
.
.
.
.
.
Ditto
.
.
.
.
.
Ditto
.
.
.
.
.
No. of days
1
2
3
5
7
Milk+0.5
per cent citric
acid
Mgms.
Nil
7
28
70
18
These results show that the acetyl-methyl-carbinol+diacetyl is produced in
small quantities in the first 48 hours of the culture and that it reaches its maximum
on the fifth day and subsequently declines considerably on the seventh, rather
suddenly, suggesting that it is lost not so much by evaporation as by being assi-
milated and decomposed by the organism itself for its own use. Incidentally, this
behaviour of the organism provides a useful hint that the time to be allowed to
develop the aroma in the ripening of cream cannot be extended beyond a certain
limit which can be ascertained with the cultures of starters used in dairies, by
the above method.
(7) Morphological and biochemical characters.—We next turned our attention
to a study of the morphological and bio-chemical characters of the organism. A
description of these prepared from the results of our observations is as follows :—
Form.—Spherical or slightly longer than broad. Usually arranged in
pairs in milk culture. Chain formation occurred in agar.
Size.—0.6-1.0µ
×0.7-l.2µ.
Motility.—The organisms have not shown motility at any time.
Staining reactions.—Stain readily with aniline stains; gram positive.
Cultural characteristics:—
Whey agar streak.—After 24 hours at 21°C. small non-viscid closely
grouped colonies precipitate in condensation water. Growth
was feeble on Lemco agar.
Agar stab.—Slight growth at the surface, but more along the line of stab.
Facultative anaerobic.
AROMA-PRODUCING LACTIC ACID ORGANISM
149
Purple lactose agar.—Medium changed to yellow after 24 hours' growth
at 21°C. Under low magnification, circular rough-edged, very
minute colonies which did not increase in size after further incuba
tion.
Whey gelatine stab.—Gelatine not liquefied, growth along stab 24 hours,
filiform; little surface growth.
Bouillons.—No growth in plain bouillon. Sediment was observed in
bouillons to which fructose, galactose, glucose, lactose and maltose
had been added, but no growth in bouillons with glycerine, sucrose,
mannitol or raffinose.
Potato growth was not evident.
Plain milk coagulates smoothly in 24 hours at 21°C. No gas production.
Acidity was distinct.
Litmus milk.—Litmus was reduced and decolourised at first, but turned
red afterwards and milk coagulated.
Biochemical features—
Gas production.—No gas produced from any sugars or other fermentable
materials.
Acid production.—Distinct acid production was observed. 10 c.c. milk
titrating on an average to 10.0 c.c. N/10 NaOH.
Oxygen relation.—Facultative anaerobic.
Examination of these characteristics shows that the organism is closely
related to Streptococcus citrovorus (Hammer) from which it differs in the fact that
while S. citrovorus does not produce much acid and does not coagulate milk, this
organism as described above produces acid and coagulates milk within 24 hours
at 21°C. This organism differs also from S. paracitrovorus in producing stronger
total acidity—about 0.9 per cent as compared with 0.4 per cent to 0.7 per cent
total acidity produced by S. paracitrovorus and lesser amount of volatile acidity
than S. paracitrovorus, as has been already observed. Thus, although a citric
acid fermenter itself, the organism described by us differs from the two described
by Hammer in its marked ability to produce a large amount of lactic acid in milk,
we, therefore, propose to call it " Streptococcus lactis aromaticus " Nov. Sp., as it
combines the property of producing lactic acid with the production of diacetyl or
its precursor acetyl-methyl-carbinol in high amount in milk with citric acid.
Finally, we may be allowed to say that our aim in writing this paper is not so
much to claim superiority for the organism described by us, but rather to draw
attention to—
(1) the rarity of the aroma-producing organisms in Indian butters,
(2) the necessity of having the starters used in Indian dairies examined for
the presence of aroma-producing organisms,
150 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, II
(3) the necessity of introducing such organisms where they may not be
already present in the starters if improvement in aroma and flavour
of the butter is desired, and
(4) the fact that we have a culture of such an organism which has been
found useful in improving the aroma and flavour in comparison with
the organisms usually present in many localities in India and with
which a beginning may be made for improving the flavour of butter
till such time as the dairies can develop their own starters containing
the best aroma producers suited to their locality.
SUMMARY
Examination of a large number of samples of milk, cream and butter in India
shows that the organisms producing aroma in butter are comparatively of rare
occurrence in these products. Hence there is a necessity of introducing these in
starters in dairies where they may not be present if the quality of the butter is to
be improved. One such organism isolated by us from a sample of good butter and
its ability to ripen cream properly have been studied, and these tests have shown
that when used as a starter, a butter of better flavour and aroma is obtained as
compared with the lactic acid organisms likely to be present under local conditions
in several Indian dairies.
The cultural and morphological characters show the organism described by
us to be similar to S. citrovorus and S. paracitrovorus, but on account of its
marked ability to produce lactic acid, combined with the property of producing
aroma in butter, we have named it S. lactis aromaticus nov. sp.
REFERENCES
Boekhout and de Vries (1919), Centbl.f. Bakt. II. Abt. 49 : 377-382.
Conn (1889-1900), Bull. Storrs Agric. Expt. Station 4, 2nd and 3rd Ann. Rep. Storrs
Agric. Expt. Stn.
Evans (1918), J. Agric. Res. 13 : 225.
Evans et al (1914), J. Agric. Res. 2 : 167.
Hammer (1920), Iowa Agric. Expt. St. Res. Bull 63
—and
Bailey (1919), ibid, 55.
—and
Baker (1923), ibid, 81.
Hart et al (1914), J. Agric. Res. 2, 193.
Michaelin, Farmer and Hammer (1933), Iowa Agric. Expt. St. Res. Bull. 155.
Orla Jensen (1919), " The lactic acid bacteria."
Schmalfuss (1928), Ztschr. Angen. Chem, 41: 847.
—
and Barthmeyer (1928), Biochem. Ztschri. 176: 282-286
Storch (1890), Nogle Unders gelser over flodens syring. 18 Bertn fra Torsogslab
Abs. in milch Ztg. 19 : 304.
—(1919),
ibid. 102 de Beretu fr—Torsogslab.
S Ø ncke Knudsen, (1931), J. Dairy Res. II. 2.
van Neil (1927), Biocli. Ztschr. 187: 472-478.
—Kluyver
and Der (1929). Bioch Ztschr. 210 : 234-251.
Weigmann, (1890), Milch. Ztg. 19 : 593 ; 944.
IS PIROPLASMA TAYLORI SARWAR A VALID NEW
SPECIES ?
BY
B. L. BHATIA, D.SC., F.Z.S., F.R.M.S.,
Principal, Government College, Hoshiarpur.
(Received for publication on 5th November, 1935.)
I have read with interest the paper by S. M. Sarwar in the June (1935) Num-
ber of this Journal. He has briefly reviewed the previous literature dealing with
records of various Piroplasmidea from goats and sheep both in this country and
elsewhere, and given a fairly adequate description of a form that he came across
in smears made in the course of a post mortem examination of a goat. There is a
vast amount of literature dealing with Piroplasmidea, and good deal of confusion
prevails as regards the nomenclature of various genera and species. It should,
therefore, be the desire of every worker to avoid adding to that confusion, and be-
fore one describes any organism as a new species, he should see that his observa-
tions are correctly made and interpreted, and carefully compared with the exist-
ing descriptions of similar organisms. I have no fault to find with his description
or the microphotographs and the coloured plates that accompany it, but on
ac-count of an error of interpretation he has been led to regard it as new species,
and to place it in a genus to which it does not belong.
GENERAL REMARKS
Before dealing with the specific organism, I would like to make a few general
remarks. The earliest record of piroplasms of goats in India is not of Krishna
Iyer [1932], but of Lingard and Jennings [1904], who studied and figured the piro-
plasms from a large number of domestic animals including the goat. Christophers
is quoted by Dschunkovsky and Urodschevich as having remarked that the para-
sites found in sheep in North and South India differ from each other and from the
forms described from other countries. Towards the conclusion of his paper
Mr. Sarwar observes as follows :
" The generic name of Piroplasma has been retained on account of the in-
admissibility of the three recognised genera Babesia, Nuttalia, and Theileria ".
I am aware of the fact that certain authorities stick to Piroplasma Patton [1895],
as a generic name, instead of Babesia Starcovici [1893], which has priority over
it; and that Nuttalia Franca [1910], is now generally regarded as one of the
synonyms of Babesia and not a separate genus. But I wonder what is the author's
authority for considering Theileria Bettencourt, Franca and Borges [1907], as
inadmissible : Theileria is a well established genus, which is so distinctly marked
off from Babesia, that the two are placed in separate families by du Toit [1918],
( 151 )
PIROPLASMA TAYLORI SARWAR
153
parasites are found in a cell they measure 2 µ by 1.5 µ. Although single parasites
are frequently seen, it is also not uncommon to find two, four, eight and sixteen
elements in one red cell. Another feature is the occurrence of a fairly large num-
ber of parasites in extra-cellular forms. The occurrence of eight and sixteen ele-
ments in a red cell as also the occurrence in fairly large numbers of extra-cellular
forms is quite unusual for a Babesia, and shows that the organism is a Theileria.
But the author rules it out by his remark that " the absence of Koch's blue bodies
in the smears excludes the possibility of this parasite belonging to the genus Thei-
leria ". Now what are the extra-cellular bodies showing multiple division (vide
his plates VI a and VII) *if they are not schizonts or Koch's blue bodies ? No doubt
ordinarily in Theileria Koch's blue bodies are found in endothelial cells of internal
organs, but they have also been observed in peripheral blood in certain species.
The author has compared his form with the three species of Babesia and with
Theileria hirca previously known from goats. Although he mentions Dschunkovs¬
ky and Urodschevich's paper [1924] in his list of References, it is obvious that he
did not consult the paper in the original, as the following misstatements of facts
would clearly show. He mentions (p. 172)* that T. hirci is characterised by the
presence of Koch's blue bodies in the lymphatic glands and internal organs, and
in his table (p. 175)* states that the species is characterised by the presence of
haemoglobinuria. In both these respects, the statements made are quite contrary
to the original description. I quote the relevant portions below :—
" Elements similar to Koch's blue bodies or " Plasmakugeln " were also en-
countered in peripheral blood films. These bodies were round or oval, at times as
large or larger than a blood corpuscle, and contained chromatin granules of vary-
ing size and shape. We were unable to examine the goats ' internal organs with
a view to studying the parasites therein, this being unfortunate in view of the
' blue bodies' above referred to ". Further on, under symptoms they state
" Urine cloudy but no haemoglobinuria ".
I will further quote Dschunkovsky and Urodschevich's description of the
form as seen in the peripheral blood, " the parasites occurring singly, in twos, rare-
ly in threes, in the centre of the corpuscles. The parasites were mostly small and
distinctly ring-shaped, others being bacillary or nail-like, oval or piriform ". . . . . .
. . . . . . " Many extra-celluler parasites were observed, all were rounded. Rod
or nail-like forms are rare. Cross-like forms of parasites occur, each member being
piriform ".
A perusal of the above extracts will show that there is a complete resemblance
between Piroplasma taylori Sarwar and Theileria hirci Dsch. and Urod., as regards
form of the parasite, presence of Koch's blue bodies in the peripheral blood, and in
the absence of haemoglobinuria, and the two must be regarded as identical.
* The Indian Journal of Vety. Science and Anim. Hus., Vol. V, Pt. II.
F
154 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VI, II
Theileria hirci Dsch. and Urod., was described from goats in Siberia in 1924.
The same year Lestoquard recorded Theileria ocis as a parasite of sheep in Algoria,
and found the schizonts in the liver, spleen, kidneys, and bone marrow, and
observed a transient haemoglobinuria on the second day of the disease. Wenyon
[1926] regards this species as identical with T. hirci Dsch. and Urod.
Other forms correctly referable to Babesia and Plasmodium have also been
recorded from goats, but I shall not discuss them here.
REFERENCES
Dschunk ovsky, E., and Urodschevich, V. (1924). Parasitology, 16, 107.
Lestoquard, F. (1924). Bull. Soc. Path. Exot., 17, 122.
Lingard, A., and Jennings, E. (1904). Ind. Med. Gaz., 39, 161.
Mello, F. de, and Paes, S. (1923). C. R. Soc. Biol. Paris, 88, 829.
Reichenow, E. (1929). Lehrbuch der Protozoenkunde, 2, 1026, 1036.
Sarwar, S. M. (1935). Ind. J. Vety. Sci. & Ani. Husb., 5, 171.
Thomson, J. G., and Hall, G. N. (1933),. J Comp. Path. Therap., 46, 218.
Wenyon, C. M. (1926). Protozoology, 2, 1004, 1036.
Yakimoff, W. L. (1931). Arch. Protistenk., 74, 372.
Ind. Jour. Vety. Sci. & Anim. Husb. Vol. VI. Pt.
2.
PLATE IV.
Hoemoproteus (rileyi) n. sp. in Peacock's blood.
(The drawing is not to scale)
HOEMOPROTEUS RILEYI (SP. NOV.) CAUSING A FATAL
DISEASE IN INDIAN PEACOCK
BY
P. G. MALKAN1, B.A. (HONS.), B.SC. (LOND. VET.), M.R.C.V.S.,
Research Officer and Professor of Pathology and Bacteriology, Bihar
Veterinary College, Patna.
[Received for publication on 22nd February, 1936.]
(WITH PLATE IV.)
Several distinct species of pigmented parasites of the red blood corpuscles
belonging to the genera Hoemoproteus and Plasmodium have been recorded from
different species of birds. Although practically nothing in the case of members
belonging to the genus Hoemoproteus, and very little in the case of members of the
genus Plasmodium, is definitely known of the possibility of one and the same spe-
cies occurring in different hosts, recent investigations have tended to prove that
probably many of the several distinct species, primarily created only on the assump-
tion that each parasite is specific to the host in which it occurs, do actually exist.
Reference to the check list given by Wenyon [1926] and the available literature
shows that pigmented parasites of the red blood corpuscles of the peacock (Pavo
cristatus) have not hitherto been described. The object of this note is to place
on record the finding of a halteridium in the peacock and its being apparently the
cause in this bird of a disease which proved fatal. The finding was made during
the course of routine examination of the blood smears submitted by the hospital
to the laboratory.
The peacock was brought to the hospital on the 28th of July, 1935. The
owner related that the bird was suffering for a week from what appeared to him
to be a serious illness, as its mate had died a week ago showing practically the same
symptoms. It was off feed, dull, its head hanging down. The feathers were rough
and the appearance was very poor. It was passing white and slimy faeces. There
was a white sticky discharge from the eyes. Temperature at that time (morning)
was 107°F. The body was covered with lice, visible mucous membranes were
anaemic and the bird was so weak that it was unable to stand.
EXAMINATION OF THE BLOOD
Examination of the blood smears revealed that a large number of the red
blood corpuscles contained intracorpuscular parasites (Plate IV). Such corpuscles,
however, did not show any deformity either of the cytoplasm or of the nucleus.
Among the corpuscles free from parasites slight anisocytosis was observed.
( 155 )