NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
SALMONELLA ENTERITIDIS ISOLATED FROM PIGEONS
239
represent complete, partial and trace agglutination respectively. The homolog-
ous titres of the samples of immune sera, when submitted to test, are given in
Table V.
TABLE V
Homologous agglutinin titres of immune sera
Particulars of anti-serum
Pigeon No. 115 Salmonella .
Do.
do. .
Pigeon No. 127 Salmonella .
Do.
do. .
S. 44. Salmonella enteriti-
dis Gaertner.
S. 97. S. entertidis var.
Dublin. Calf enteritis.
S. 53. S. pullorum . .
S. 52. S. gallinarum .
Antigen
Homologous .
Pigeon No. 127
Homologous .
Pigeon No. 115
Homologous .
Do. .
Do. .
Do. .
Agglutinin titre after
incubation for 24 hours
at 37°C.
Dilutions
1/10.
C
C
C
C
P
P
C
C
1/50.
C
C
C
C
—
C
C
C
1/100.
C
C
C
C
—
C
C
C
1/500.
C
C
C
C
C
C
C
C
1/1,000.
C
C
C
P
C
C
C
C
1/5,000.
C
P
P
T
C
C
P
T
1/10,000.
—
—
—
—
C
P
—
—
Antigen
control
—
—
—
—
—
—
—
—
Remarks
Prezone reaction.
Do.
CROSS AGGLUTINATION TESTS
Pigeon Salmonella Antigen vs. authentic strains of Salmonella anti-sera
Agglutination tests conducted on four different pigeon Salmonella antigens,
whose fermentation reactions were similar, with two samples of anti-sera for
S. enteritidis (S. 44 and S. 97), S. gallinarum and S. pullorum are shown in Table VI.
It indicates that there is some relationship with S. 97 (calf enteritis) and that
the poor agglutinin titres with S. 44, S. pullorum and S. gallinarum immune sera,
are due to the presence, in the sera, of group agglutinins.
240
THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VII, III
TABLE VI
Pigeon Salmonella antigens vs. anti-sera for different Salmonella
Particulars of anti-serum
S. 97. S. enteritidis var. Dublin
Calf.
Do.
do. .
Do.
do. .
Do.
do. .
S. 44. S. enteritidis Gaertner .
Do.
do. .
Do.
do. .
Do.
do. .
S. pullorum from Rabbit No. 162 .
Do.
do. .
Do.
do. .
Do.
do. .
S 52. S. gallinarum from Rabbit
No. 160.
Antigen Salmonella
from
Pigeon No. 115 . .
„ No. 124
. .
„ No. 125 . .
„ No. 127 . .
„ No. 115 . .
„ No. 124 . .
„ No. 125 . .
„ No. 127 . .
„ No. 115 . .
„ No. 124 . .
„ No. 125 . .
„ No. 127 . .
„ No. 115 . .
Agglutination titre after
incubation for 24 hours
at 37°C.
Dilutions of serum
1/10.
C
C
C
C
C
C
C
C
C
C
P
P
C
1/50
C
C
C
C
P
P
—
—
P
P
T
P
P
1/100.
C
C
C
C
—
—
—
—
—
—
—
—
—
1/500.
P
T
T
T
—
—
—
—
—
—
—
—
—
1/1,000.
—
—
—
—
—
—
—
—
—
—
—
—
—
1/5,000.
—
—
—
—
—
—
—
—
—
—
—
—
—
1/10,000.
—
—
—
—
—
—
—
—
—
—
—
—
—
Antigen
control
—
—
—
—
—
—
—
—
—
—
—
—
—
From the above table, it is seen that pigeon Salmonella bears no relation-
ship to S. pullorum and S. gallinarum, the two pathogenic species incriminated
in fowl epidemics, a possibility which has been ruled out by the biochemical
tests.
AGGLUTININ ABSORPTION TEST
Two samples of anti-sera for S. 44 and S. 97 (Table I) were first absorbed by
the Salmonella antigen from pigeon No. 115 independently, by the following
method. Into two centrifuge tubes of 10 c.c. capacity, 2.5 c.c. of the dilution
of the respective serum was made and then 2.5 c.c. of a thick saline suspension
of twenty-four hours old agar surface growth of pigeon No. 115 Salmonella added.
The antigen-antibody mixture was then kept at 55°C. in an electrically heated
water-bath for four hours after which the tubes were centrifuged at 2,000 R. P. M.
for twenty minutes to sediment the bacterial mass. The supernatant fluid was
separated and further agglutination tests were conducted with the absorbed
sera against their homologous antigens and the pigeon Salmonella for evidence
of absorption on the original homologous titres.
SALMONELLA ENTERITIDIS ISOLATED FROM PIGEONS
241
As recorded in Table VII, S. 97 anti-serum when absorbed by pigeon No. 115
Salmonella, lost a considerable amount of its agglutinating anti-body for the specific
antigen and this suggests some similarity between the agglutinogen fraction of
the two. The titre was unaffected in the case of absorption of S. 44.
TABLE VII
Agglutinin absorption tests
Particulars of serum
Anti-serum for S. enteritidis
Gaertner. S. 44, absorbed
by pigeon No. 115, Sal-
monella organism.
Do.
do. .
Anti-serum for S. enteritidis
var. Dublin (calf enteritis)
S. 97, absorbed by pigeon
No. 115, Salmonella orga-
nism.
Do.
do. .
Antigen used
for absorp-
tion
Pigeon No. 115
Salmonella.
Specific . .
Pigeon No. 115
Salmonella.
Specific . .
Agglutinin
titre after
incubation
for
24 hours
at
37° C.
Neg. 1/20 .
1/5,000 .
Neg. 1/20 .
1/1,280
.
Remarks
—
Original
homologous
titre with unabsorbed
serum 1/10,000.
—
Original
homologous
titre with unabsorbed
serum 1/10,000.
CROSS AGGLUTINATION TEST
Pigeon Salmonella anti-serum vs. various authentic types of Salmonella antigens
Having determined the agglutinin titre of pigeon No. 115 organism anti-
serum against the specific antigen, viz., 1/5,000 the same serum was tested against
a number of authentic strains of the Salmonella group, stocked at this Institute
for comparative study and from the titres so obtained, Table VIII indicates that
S. 63 and S. 65 cultures are highly suggestive of the serological relationship with
the Salmonella from the pigeons referred to in this paper. S. 63 and S. 65 were
locally isolated strains of S. enteritidis from animal sources.
242 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VII, III
TABLE VIII
Cross agglutinin titre of pigeon No. 115. Salmonella anti-serum against various
types of authentic Salmonella antigens
No.
S. 44
S. 97
S. 51
S. 63
S. 65
S. 85
S. 86
S. 87
S. 88
S. 84
S. 93
S. 52
S. 53
Antigen
Type of organism
S. enteritidis Gaertner . .
S. enteritidis var. Dublin . .
Paracoli enteritidis . . .
S. enteritidis from the Pathology
Department of this Institute.
S. enteritidis, abortion material
from brood mare.
S. enteritidis var. Danzig .
S. enteritidis var. Dublin
S. enteritidis var. Rostock (Kauff-
mann). S. enteritidis var. Moscow . .
S. enteritidis Limerick . .
S. anatum
.
.
.
.
S. gallinarum
.
.
.
.
S. pullorun
.
.
.
.
Agglutinin titre after incubation for 24 hours at 37°C.
Dilutions of the serum
1/10
T
P
C
C
C
C
P
T
C
C
C
...
P
1/50
...
...
P
C
C
T
T
...
T
C
P
...
...
1/100
...
...
...
C
C
...
...
...
...
C
T
...
...
1/500
...
...
...
C
C
...
...
...
...
P
...
...
...
1/1,000
...
...
...
C
C
...
...
...
...
...
...
...
1/5,000
...
...
...
P
C
...
...
...
...
...
...
...
1/10,000
...
...
...
...
P
...
...
...
...
...
...
...
Antigen
control
...
...
...
...
...
...
...
...
...
...
...
SUMMARY AND CONCLUSIONS
1. In a batch of pigeons inoculated with experimental pigeon-pox virus at
this Institute, deaths from a septicæmic disease occurred and from the heart-
blood of such pigeons, a gram negative bacterium was isolated.
2. The cultures from the diseased pigeons were, on their cultural and patho-
genic merits, subjected to a detailed study.
3. Agglutination, cross agglutination and agglutinin absorption tests con-
ducted with authentic types of the genus Salmonella suggest a marked serological
relationship with two stock strains of S. enteritidis isolated from animals at this
Institute.
REFERENCES
Bergey, D. H. (1934). Manual of Determinative Bacteriology. pp. 368-381.
Gaertner, A. (1888), referred to by Kelser, Manual of Veterinary Bacteriology.
Kelser, R. A. (1933). Manual of Veterinary Bacteriology. pp. 196-208.
White, P. B. (1929). A System of Bacteriology. 4, pp. 86-153.
ON A PIROPLASM OF THE INDIAN CAT
(FELIS DOMESTICUS)*
BY
M. Y. MANGRULKAR, M.Sc. M.R.C.V.S., D.T.V.M. (EDIN.)
Assistant Pathologist, Imperial Veterinary Research Institute, Mukteswar
(Received for publication on 14th April 1937)
(With Plate XVI)
ON the 17th March 1936, the carcase of one of a number of cats obtained from
Bareilly (U. P.) flor experimental work at the Imperial Veterinary Research In-
stitute, was received for autopsy in the post-mortem room. The animal was in a
poor condition; otherwise it showed no external signs of disease. Apart from a
general somewhat anaemic condition, the internal organs showed little of interest
superficially. Slight congestion of the kidneys, however, was noticed. The bowels,
too, showed hyperaemic patches. No evidence of icterus or haemoglobinuria was
seen at autopsy. From the helminthological point of view, the carcase was full of
interest as it harboured the following different parasites in varying numbers :—
Lungs—Paragonimus westermanni.
Liver—Opisthorchis felineus.
Stomach—Chlamydonema praeputiale.
Bowels—Echinochasmus perfoliatus.
Mesocestoides lineatus.
Rictularia cahirensis.
Taenia taeniaeformis.
Toxocara mystax.
No ectoparasites were found.
In view of the rich parasitic fauna, the author expected that examination of
blood would reveal some protozoan parasites or micro-filariae, and hence several
smears from the peripheral blood were prepared and a sample of heart-blood
collected. The smears were stained by Leishman's method and microscopically
examined. A careful search for micro-filariae proved negative, but the effort was
rewarded by the detection of non-pigmented protozoan parasites in the red blood
corpuscles. Judging from the small number of erythrocytes which showed the
*Paper presented to the 24th Annual Meeting of the Indian Science Congress, 1937.
( 243 )
M
244 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VII, III
parasites, the infection was by no means of a severe degree ; so that many fields had
to be examined before it was conclusively ascertained that the parasite was a piro-
plasm. The accompanying diagram (Plate XVI) drawn with the aid of the camera
lucida shows a representative group of infected red blood corpuscles collected
from different fields.
The individual parasite was small, being on an average 1.5 µ the actual
size varying from less than one µ to 2.50 µ.. Morphologically it resembled to some
extent the one described by Davis [1929] as Babesia felis from the Sudanese wild
cat (Felis ocreata). Small and large, rounded or ovoid forms, sometimes
vacuolated (ring-forms), were common, one or more (up to four) being present in
one red blood corpuscle. Amoeboid and dividing forms (Plate XVI, a) were
very rare. Not infrequently typical pear-shaped parasites were seen. These were
either single or, as was more often the case, in pairs ; sometimes the pair was
markedly divergent as in Babesia bovis ; rarely three or four parasites were seen
in one red blood corpuscle. In no instance, however, a cruciform arrangement
suggestive of division into four, as seen by Davis and by Wenyon and Hamerton
[1930] was noticed. The chromatin had no fixed place in the parasite but was
inclined to be peripherally situated in the rounded forms and towards the blunt
end in the pear-forms. Anaemic changes were observed in the blood smears.
Realising that the material was not fresh, blood smears were made from time
to time from the other living and apparently healthy cats, and a careful search was
made for piroplasms in their blood smears and for ectoparasites on, their bodies.
The result was negative in every case. But the presence of intracorpuscular
bodies resembling Anaplasma marginale was detected in the blood smears of all
the cats. The finding of these bodies is in conformity with that of Davis [1929]
who doubts if they " bear any relation to the piroplasm " and adds " whether they
are actual parasites or merely Jolly bodies it is difficult to determine ".
Lingard and Jennings in 1904 mentioned the occurrence of piroplasms in
various species of animals including lizards, fowls, cats, camels and elephants.
They even went to the extent of recording this parasite from a man. These obser
vations have not been confirmed and for certain reasons their article is open to
much criticism. From their coloured plates and description of some of the para-
sites it seems highly probable that the authors were, in some cases, dealing with
what were really not piroplasms ; indeed, in the human case the parasites appear
to be Plasmodium vivax. Unfortunately their " preliminary note " was never
modified, as they had proposed to do. No description or illustration of their
feline piroplasm is available and there appears, therefore, enough justification to
believe with Davis [1929] that until the latter's paper appeared in 1929 no piro-
plasm had " been found in any member of the Felidae ".
Ind. Jour. Vety. Sci. & Anim. Husb., Vol. VII, Pt. III ]
PLATE XVI.
Representative group of infected red blood
corpuseles collected from different fields.
ON A PIROPLASM OF THE INDIAN CAT
245
In 1930, Wenyon and Hamerton described from the Bay Lynx (Felis rufa)
a piroplasm which " resembles closely " Babesia felis of Davis. Subsequently
Carpano [1934] recorded a new piroplasm from the Puma (Felis concor) resembling
B. gibsoni, and asserted that it too was transmitted from the local felines (presum-
ably including F. domesticus and F. ocreata), which were carriers, by the tick
Rhipicephalus simus. He proposed the name Babesiella felis for his parasite and
would have the parasite of Davis called Nuttallia felis, perhaps because it produces
cross-forms. But Wenyon points out: " the difference in size and in the number
of daughter individuals produced are not sufficient grounds for the recognition of a
new genus ". It is evident, however, that Carpano was dealing with a parasite
entirely different from the one described by the author and by Wenyon and
Hamerton.
There appears to have been no further publication on feline piroplasms. In
the present case, as already pointed out, a material difference from B. felis of Davis
is shown by the absence of cross-forms, division being into two daughter parasites.
The occurrence of cross-forms is corroborated by Wenyon and Hamerton in their
description of the parasite from F. rufa. They leave little doubt as to the mode
of division of the parasite into four daughter individuals, which is similar to that
in B. equi. They admit, however, that even in their heavily infected case " the
cross-form are few in number, but smaller forms showing division into two daughter
individuals were fairly frequently seen ". From this it appears that in the Bay
lynx the parasite divides usually into two, the cross-shaped arrangement resulting
from a departure from this normal mode of division.
Since in the Indian cat no evidence of division into four, or the presence of
cross-forms, was encountered, the rare occurrence of four parasites in one eryth-
rocyte may have arisen much as it sometimes does in Babesia bigeminum or
B. canis. Indeed, the author has seen, although not commonly, infected red blood
cells (Plate XVI, b) showing coupled pear-shaped parasites one or both of
which are suggestive of stages preparatory to further division.
From the description and illustrations of B. felis of Davis and from the suggest-
ed resemblance of the piroplasm of Carpano with B. gibsoni, it would appear
that the two parasites are similar but entirely different from those described from
F. rufa by Wenyon and Hamerton, and from the Indian cat in this paper. It is,
however, not clear why, in spite of the evidence of division, " fairly frequently ",
of the parasite from F. rufa into double forms, Wenyon and Hamerton have found
it comparable to B. felis [Davis, 1929] wherein no such forms have been described
or illustrated. The division into four as described by Wenyon and Hamerton
was entirely wanting in the Indian cat, and therefore, the author believes his
parasite to be different from those hitherto described but refrains, in the absence of
suitable material to carry out further studies by inoculation work, from referring
it to a new species.
M 2
246 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VII, III
ACKNOWLEDGMENT
The author is indebted to Mr. F. Ware, C.I.E., F.R.C.V.S., F.N.I., I.V.S.,
Director, Imperial Veterinary Research Institute, for the interest shown in this
case, to Dr. H. N. Ray, Ph.D., Systematic Protozoologist, for helpful suggestions
and criticism, and to Mr. H. D. Srivastava, M.Sc., Offg. Helminthologist, for
identification of the helminth parasites.
REFERENCES
Carpano, M. (1934). Abstract in Vet. Bull. 6, 584.
Davis, L. J. (1929). On a Piroplasm of the Sudanese Wild Cat. Trans. Roy. Soc.
Trop. Med. Hyg. 22, 523.
Lingard A., and Jennings E. (1904). A Preliminary Note on a Piroplasmosis
found in Man and in some of the lower Animals. Ind.
Med. Gaz. 39, 161.
Wenyon, C. M. (1926). Protozoology 2, 1009.
Wenyon C. M., and Hamerton, A. E. (1930). Trans. Roy. Soc. Med. Hyg. 24, 7.
DETERMINATION OF FATS IN BIOLOGICAL MATERIAL
II.—THE ALKALI HYDROLYSIS METHOD*
BY
P. A. SESHAN, B.A.
Animal Nutrition Section, Imperial Veterinary Research Institute, Izatnagar
(Received for publication on 16th February, 1937)
THE alkali-hydrolysis method originally developed by Liebermann
[Liebermann and Szekeley, 1898] finds very common adoption in the technique of
fat-determination in biological material. Other modifications of this method
such as that of Kumagao and Suto [1908], have for their object only a thorough
breaking down of the tissues to enable the ether or other extracting solvent to
have a ready access to the fat molecules of the substance. It should also be
added that this method is considered particularly useful when it is specially desired
to avoid the changes or losses in fat that generally occur on drying the material.
During recent years there seems to be a comparative paucity of reported work
on the testing of fat-determination methods. In the first paper of this series
[Seshan, 1935] only a few references regarding the use of alkali hydrolysis method
were cited. Offering as they do an adverse comment on the use of this method, at
least so far as ragi straw and bullock faeces are concerned, the findings reported
in that paper [Seshan, 1935] make it necessary to examine in greater detail some
more instances in point.
Sperry [1926-27] working on lipid excretion tested his control diets for their
freedom from lipids by this alkali-hydrolysis method, the faecal lipids also being
estimated by the same process. In brief, the method consisted in digesting 10
grms. of material for twenty-four hours with alkali over a boiling water bath, acidi-
fying the digest with sulphuric acid, extracting with ether and taking up the
resulting extract in petroleum ether for a subsequent drying to constant weight.
It should also be noted that for a complete removal of the volatile fatty acids in the
acidified digest, this was distilled to dryness under reduced pressure over a steam
bath. In a similar experiment on the sterol metabolism of rats, Eckstein and
Treadwell [1935] have, as a preliminary to the digitonin precipitation, separated
the total lipids from the control diet as well as from the resulting tissues and faecal
matter by digesting them with boiling 7.5 per cent alcoholic NaOH for two
hours. Tidwell and Holt [1936] observe that a number of methods are available
for the estimation of total lipids in faeces which depend on a primary saponi¬
* For Part I, please see Vol. 5, P. 355 of this Journal.
( 247 )
248 THE INDIAN JOURNAL OF VETERINARY SCIENCE AND ANIMAL HUSBANDRY [VII, III
fication of the material. Reference may also be made to the recent work of
Horwitt, Cowgill and Mendel [1936] on a method of determination of the fat-
content of spinach leaves. They attribute their failure to obtain results by directly
saponifying vegetable tissues to a probable resistance offered by the cellulose
structures of the plant.
It is apparent from the brief review given above that none of these workers
considered the possibility of formation of a fresh quantity of fatty acids in the
alkali digest of the material. Sufficient evidence has, however, been adduced in
the previous paper to indicate such formation, under the conditions obtaining in
the usual digestion with hot alkali, of samples of ragi straw and faeces. As this
technique of fat-determination is of common use, it has been considered desirable
to carry out further investigations to test its reliability and the results obtained
are given in this paper.
EXPERIMENTAL
Repetitions of the alkali hydrolysis with ragi straw and faeces having yielded
confirmatory results, a study of its effect on pure carbohydrates was undertaken.
The experiments reported here were all conducted on pure specimens of cotton-
wool, filter-paper, starch and dextrin as representing some types. It was thought
advisable at the outset to determine the ether-extract content of these substances.
Weighed quantities of the four samples in paper thimbles were subjected to a
continuous extraction for eighteen hours in Soxhlet tubes. To test the completion
of the extraction it was continued for another thirty-six hours. The results ob-
tained were as follows :—
TABLE I
Control experiments with pure carbohydrates
Solvent
Petroleum ether B. P.
50°-60°C. . . .
Ditto . .
Ethyl-ether . . .
Ditto . .
Percentage extraction
Period
Hrs.
18
54
18
54
Cotton-wool
0.025
0.050
0.222
0.192
Filter-
paper
0.025
Nil
0.335
0.285
Starch
Nil
0.050
0.119
0.149
Dextrin
0.050
0.075
0.189
0.189
The actual weights used for the extraction were about 2 grms. and the results
were then calculated to a 100 grms. basis. So an error of one milligram in weigh¬
ment would make a difference of 0.05 per cent. Although the extracts obtained
DETERMINATION OF FATS IN BIOLOGICAL MATERIAL
249
with petroleum ether show that the materials used were practically free from fat,
the extraction with ethyl-ether yielded slightly different results. It is,
however, sufficiently satisfactory for the purpose of this experiment to find that a
continuation of the extraction over three times the period did not yield further
quantities of extract.
Having thus determined the limits of the possible errors that might result
from the impurity already present in the pure substances, they were subjected to
the usual alkali hydrolysis treatment. The modified technique described in the
first communication [Seshan, 1935] was followed. The method in brief was as
follows :—
The substance, weighed out into a conical flask, was hydrolysed in aqueous
20 per cent alkali for twenty-four hours over a water-bath. The alkaline digest
was then transferred to a distillation flask and rendered decidedly acid to litmus
with sulphuric acid. The volatile acids, thus liberated, were steam-distilled and
titrated against standard alkali. The mother liquid was rendered alkaline and
retransferred to a porcelain evaporating basin. When the material was nearly
dry, it was acidified with dilute hydrochloric acid and dried thoroughly over a water-
bath, being powdered at the same time. The powdered material was then ex-
tracted in a Soxhlet. It might be mentioned here that the essential condition of
this treatment with alkali, which alone matters for the purpose of this particular
investigation, remains the same in this method as in those of others.
In this connection it is of interest to note that, in carrying out a volatile acid
distillation, the condensation delivery-tube should dip well inside the receiving
liquid, either water or standard alkali. By collecting the distillates in an open
beaker in droplets, 500 c.c. of distillate gave varying titrations, with phenol¬
phthalein as indicator, ranging from 0.5 to 3.0 c.c. N/10 alkali. This fact was
repeatedly verified in distillations with pure solutions and hydrolysates as well, as
will be seen from the data given in Table II.
TABLE II
Acidity given by 500 c.c. of distillate
Number of test
1
2
3
Blank mother-liquor with no
carbohydrate in it
c.c. N/10
1.0
1.0
2.9
Blank mother liquor with un¬
hydrolysed pure carbohydrates
c.c. N/10
1.05
1.9
1.7