NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
TRANSMISSION OF FOWL SPIROCHAETOSIS THROUGH
AGENTS OTHER THAN ARGAS PERSICUS
BY
H. R. KAPUR, M. R. C. V. S.
Imperial Veterinary Research Institute, Mukteswar-Kumaun
(Received for publication on 1 April 1940)
INTRODUCTION
T HE earliest record of transmission of fowl spirochaetosis through
arthropods is that of Marchoux and Salimbeni [1903], who incriminated A.
persicus as the vector of the disease. The findings of Marchoux and Salimbeni
were later confirmed by Neufeld and Prowazek [1907]. Ficker and Rosenblat
[1907] succeeded in transmitting the disease through the bite of infected
A. miniatus but failed to do so through the progeny of these infected ticks.
They concluded that all members of the Argas family, except A. miniatus,
could transmit infection through the egg. Balfour [1909] suspected lice of the
Menopon genus as probable transmitters of the disease. He failed, however,
to transmit the disease to a healthy chick through the lice harboured by a bird
showing acute infection. In 1914, this worker found two undoubted spiro¬
chaetes in the eggs of Hyalomma aegyptium, although he believed that the ticks
concerned could not have fed on any bird affected with spirochaetosis.
Fulleborn and Mayer [1908] and Shellack [1908] found that the disease could
be transmitted through A. reflexus and Ornithodoros moubata, the incubation
period being eight to eighteen days. The ticks were infective up to the sixty-
fourth day. This opinion was simultaneously supported by Brumpt [1908].
Knowles, Das Gupta and Basu [1932] confirmed that infection is hereditarily
transmitted in the female line of A. persicus. They were of opinion, however,
that poultry mites—Dermanyssus gallinae—were of no significance in the trans-
mission of the disease, although they harboured actively motile spirochaetes.
The same conclusion was deduced in the case of bed bugs and leeches which
harboured spirochaetes after feeding on infected birds but failed to transmit
the infection. In Palestine, Theodor's experiments [1932] indicated that
spirochaetosis can occasionally be transmitted through 0. coniceps. Zuelzer
[1936], in an outbreak in fowls in the area of the Baltic Sea, failed to find
A. persicus, and suspected that Culex mosquitoes transmitted fowl spirochaeto-
sis. This worker succeeded in identifying fowls' blood in Culex species collec-
ted from poultry houses. Hungerford and Hart [1937] described three
outbreaks in which D. gallinae was suspected as the vector. A. persicus was
not found and spraying with kerosene emulsion to destroy D. gallinae reduced
the incidence.
Shellack [1912] obtained successful results in six out of thirteen cases by
smearing infective material on the unbroken skin under the wing, and recorded
an incubation period of five to twelve days. When the material was smeared
on the comb, three cases out of fifteen were positive, the incubation period
being six to seven days.
( 354 )
356
Transmission of Fowl Spirochaetosis
breast. The areas were thoroughly examined with a hand lens for the pre-
sence of any abrasions and then painted with citrated virulent blood.
EXPERIMENTS
Ninety O. papillipes in the third nymphal stage were fed upon an infected
fowl. On moulting into the fourth nymphal stage after ten days, the ticks
were divided into three batches, each batch being fed separately on a healthy
fowl (Table I). Forty O. savignyi, in their third nymphal stage, were first
given an infective feed and on reaching their next moult after twelve days were
fed, fifteen on one fowl and twenty-five on the other. Transmission experi-
ments with A. persicus were carried out in a similar manner. The results
were as follows :—
TABLE I
Species of ticks
O. papillipes
}
O. savignyi
}
A. persicus
Infect-
ed
fowl
No.
1245
59
61
Number
of clean
ticks
fed
90
40
30
Healthy
fowl No.
229
265
227
250
266
287
Number
of infec-
tive ticks
fed
36
20
30
15
25
19
Interval
between
feeds
Days
10
12
14
14
12
14
Results
Negative
Negative
Positive
on
the
6th day
Fowls Nos. 250 and 266 were tested for immunity thirty days and Nos.
265, 227 and 2529 forty days after the infected ticks had fed and all were
found susceptible.
The experiment was repeated with a second batch of seven fowls and the
results shown in Table II obtained. All the ticks in this experiment received
their infective feed at the third nymphal stage and were fed on healthy fowls
at the fifth stage.
H.
R.
KAPUR
357
TABLE II
Species of ticks
O. papillipes
}
O. savignyi
}
A. persicus
Infect-
ed
fowl
No.
62
58
61
Number
of clean
ticks
fed
80
50
10
Healthy
fowl No.
273
272
271
270
268
264
288
Number
of
infective
ticks fed
18
22
33
19
13
16
8
Interval
between
feeds
Days
28
29
30
28
29
30
30
Results
Negative
Negative
Positive
on
the
6th day.
Fowls that had infective O. papillipes fed on them were tested with a dose
of virulent blood on the fiftieth day and those of the O. savignyi group
on the fifty-first day. The fowls tested developed spirochaetosis in 24 to
72 hours. It is concluded, therefore, that O. papillipes and O. savignyi
do not transmit the disease under ordinary conditions.
As for A. (S.) albopicta, 12 mosquitoes of this species were fed after being
starved for three days, first on an infected fowl and three days later ten of these
were fed on a healthy fowl. No reaction was observed for 39 days. When the
immunity test was carried out, this fowl was found susceptible.
The succeeding set of experiments was carried out to determine whether
infection takes place through the unbroken skin, using virulent blood on the
comb (Table III).
TABLE III
Fowl No.
57
58
59
61
62
Control No. 60
Results
Negative
Positive
Do.
Do.
Do.
Negative
Incubation period
Days.
..
6
5
5
6
Tested for immunity
after 30 days and
found susceptible.
358
Transmission of Fowl Spirochaetosis
From the foregoing table it is seen that in four out of five cases the disease
was transmitted by application of virulent blood over the combs.
The breasts of another six healthy fowls were then painted with citrated
virulent blood. The fowls showed no reaction for thirty-one days. They were
then given 1 c.c. of virulent blood subcutaneously and all were found immune.
TABLE IV
Fowl No.
120
121
122
123
124
125
Results
Negative
Do.
Do.
Do.
Do.
Do.
Reaction to immu¬
nity test
Immune
Do.
Do.
Do.
Do.
Do.
In order to confirm the above findings the experiment was repeated on a
fresh batch of six fowls using in this case a larger quantity of blood. Curiously,
in all cases except one in this batch a virulent disease was produced (Table V).
TABLE V
Fowl No.
208
209
210
211
213
214
Control 212
Incubation
period
Days
2
2
3
2
..
5
..
Results
Positive
Do.
Do.
Do.
Immunity test—
28th day—sus-
ceptible
Positive
Immunity
test—
28th day—suscep-
tible
H. R.
KAPUR
359
These latter experiments, as opposed to the former, indicate that the
organism of fowl spirochaetosis is capable of passing through the unbroken
skin with the production of the disease in an acute form.
Two possible explanations may be advanced for the negative results
summarized in Table IV : firstly, the birds were immune prior to their being
brought under experiment and secondly, the infection produced was so mild,
owing to a larger dilution of blood, that no clinical syndrome capable of detec-
tion under the microscope was induced. This probably explains their sub-
sequent immunity. These birds, along with others maintained for strain
propagation, were selected at random from one batch of the main stock.
Since all those remaining proved susceptible, the possibility of those used
in this experiment being immune appears to be remote. The feasibility of
the second explanation is, however, supported by Marchoux and Couvy
[1913], who evolved a method for counting the number of spirochaetes suffi-
cient to produce infection or immunity. These workers found that when given
subcutaneously 20,000 spirochaetes produce infection, 1,700 produce immunity
and 700 produce neither infection nor immunity.
While conducting these experiments, a very interesting observation was
made in the case of those fowls that were given infection either through the
bites of infected A. persicus or by blood inoculation. In most of these cases
there was no appreciable rise in the temperature synchronizing with the ap-
pearance of spirochaetes in the peripheral blood, though, in fact, in some in-
stances the blood was teeming with the parasites. The data collected in this
respect from the charts of sixteen infected fowls are detailed in Table VI.
TABLE VI
Fowl No.
206
208
213
227
229
230
*248
249
265
266
287
289
*260
*210
*211
*214
Normal range of temperature
°F
105.6-107
105.4-106.7
105.8-107.2
105.0-108.0
106.4-108.8
105.4-107.4
105.4-108.2
106.2-107.2
105.8-107.4
105.8-107.6
105.8-108.4
106.0-108.2
106.4-107.8
106.6-107.6
105.4-106.8
105.0-108.2
Temperature when spirochaetes
appeared
°F
105.4
106.4
106.4
105.4
106.8
107.0
109.4
106.4
106.8
106.2
107.0
107.8
108.6
109.2
108.0
109.2
360
Transmission of Fowl Spirochaetosis
Literature on avian spirochaetosis invariably lays stress on the high
temperature recorded in this disease, whereas it would appear from the above
records, that in only five (marked *) out of 16 fowls was a distinct rise in tem-
perature noted.
SUMMARY
A series of experiments was carried out to see if fowl spirochaetosis could
be transmitted through the agency of Ornithodoros papillipes and O. savignyi.
Ticks were infected in their third nymphal stage and used on healthy fowls
in their fourth and fifth nymphal stages. About 159 infective O. papillipes
were fed on six healthy fowls and 88 O. savignyi on five healthy fowls and the
result was negative in each case, whereas two controls on which only 27 infec-
tive Argas persicus were fed showed a severe reaction after an incubation
period of six days.
In another series of experiments, mosquitoes belonging to the species
Aedes (Stegomyia) albopiota were used. These could be made to feed on fowls
readily, but failed to transmit the disease.
A third series of experiments carried out on three batches of fowls showed
that the disease could be successfully transmitted to healthy fowls by smearing
the infective material either on the unbroken skin of the comb or the breast.
Incubation period in such cases was two to six days.
Pyrexia was found to be an inconstant symptom in the experimentally
induced disease.
ACKNOWLEDGEMENTS
In carrying out these experiments much help and encouragement was
given by Mr. J. R. Haddow. I have also to thank Mrs. J. B. Polding for her
translation of German and French literature and Mr. S. K. Sen for his guidance,
REFERENCES
Balfour, A. (1909). J. Trop. Med. & Hyg. 12, 285
*—
(1914). Trans. XVIIth Int. Cong. Med. Lond. Sect. 21, Part 2, p. 275
(Cited by Knowles, Das Gupta & Basu, Ind. Med. Res. Mem. 22, 1932.)
Brumpt, E. (1908). Bull. Soc. Path. Exot. 1, 577
Ficker, M. and St. Rosenblat (1907). Hygienische Rundschau 17, 1114
*Fulleborn, F. and Mayer, M. (1908). Arch. f. Schiffs. Trop. Hyg. 22, 31. (Cited by
Knowles, Das Gupta and Basu, Ind. Med. Res. Mem. 22, 1932)
Hungerford, T. G. and Hart, L. (1937). Agric. Gaz. N. S. W. 48, 591
Knowles, R., Das Gupta, B. M. and Basu, B. C. (1932). Ind. Med. Res. Mem. 22
Marchoux, E. and Couvy, L. (1913). Ann. Inst. Pasteur 27, 450
Marchoux, E. and Salimbeni, A. (1903). Ann. Inst. Pasteur 17, 569
Neufeld, F. and Prowazek, S. V. (1907). Arb. a. d. Kais. Gesundheitsamte 25, 494
Shellack, C. (1908). Centralblatt f. bakt. 1 Abt. 46, 486
—
(1912). Arb. a. d. Kais. Gesundheitsamte 40, 78
Theodor, O. (1932). Z. Parasitenk. 5, 69
Zuelzer, M. (1936). J. Trop. Med. & Hyg. 39, 204
Note.—Papers marked with an asterisk (*) have not been seen in original but only
in reviews or references.
ANTI-OXIDANTS OF GHEE
BY
S. V. GOVINDARAJAN
AND
B. N. BANERJEE
Department of Biochemistry, Indian Institute of Science, Bangalore
(Received for publication on 8 June 1940)
(With two text-figures)
T HE deterioration of oils and fats generally involves a process of oxida-
tion and the conditions governing these reactions have been extensively
studied [Dastur and Banerjee, 1936]. It has been found that exposure of
fats to heat [Bevis, 1923], light [Greenbank and Holm, 1930], air (oxidation)
[Holm, Greenbank and Deysher, 1927], presence of free acids [Holm and Green-
bank, 1924] and other factors [Davies, 1928, Pennington and Hepburn, 1912,
King et al., 1933] are conducive to rapid deterioration. Since the changes
involved necessitate absorption of oxygen by the fat, attention has been
principally directed to the study of the problem from this standpoint. The
work of Holm, Greenbank and Deysher [1927], French, Olcott and Mattill
[1935], and others [Mattill and Crawford, 1930] show that fats exposed to
oxygen exhibit the typical characteristics of an autocatalytic oxidation,
viz. a latent or induction period of variable length during which the oxygen
absorbed is very small, followed by a period of rapidly accelerating oxygen
absorption. The length of the induction period is determined by the presence
of promoters or inhibitors of oxidation, and as is now well recognised, even
very small amounts of these inhibitors help to retard oxidation [Moureu and
Dufraisse, 1927].
The onset of rancidity usually coincides with or follows shortly after the
end of the induction period. Remarkable variations in the resistance of vita-
min A in butter-fat to destruction by heat have been noticed [Steenbock,
Boutwell and Kent, 1918, and Osborne and Mendel, 1920] and the resistance
of ghee to rancidity and vitamin A destruction by heat and oxygen has been
attributed to the presence of different amounts of the protective substances.
Numerous methods have been used to determine the length of the induc-
tion period and to follow the progress of the underlying reactions. Variations
in the amount of organic peroxides have been considered significant [Taffel
and Revis, 1931, and Wheeler, 1932]. Aldehyde or other products of oxida-
tion develop colour in the presence of Kreis [1902] and Fellenberg [1924]
reagents and with limitations such colour reactions are used to determine
rancidity. More recently [Lea, 1934] the aldehyde value has been determined
by determining the aldehyde-bisulphite compound. The exact significance
of these and other tests is still uncertain, and doubts have been raised as re-
gards the utility of the Kreis value, the peroxide value, etc. [Barnicoat,
1931, and Coe and LeClerc, 1934]. However, the organoleptic tests depend-
ing upon taste and smell are still the final criteria for rancidity.
The only direct method in addition to those mentioned above to study
the oxidative reaction is to follow oxygen absorption by the fat. This method
should serve well, because the main purpose of this work is to determine the
factors influencing the prolongation of the induction period in butter-fat.
( 361 )
362
Anti-oxidants of Ghee
Methods of study.—The oxygen absorption method used by French,
Olcott and Mattill [1935] has been followed with suitable modifications.
Erlenmeyer flasks (capacity 110 ml.) containing the test fats were immersed
in a constant temperature oil bath, and each one was connected by a well
fitting rubber bung to an open mercury manometer. Dry oxygen was passed
into the flask through a side tube, allowing the air in the flask to escape by a
tube having a stop cock. At the end of 15 minutes, the oxygen stream was
discontinued and the stop cocks on both the inlet and outlet tubes were closed.
Any pressure difference that might have been set up was equalized by momen-
tarily opening the outlet stop cock. The readings of the two levels of the
mercury manometer were taken initially at half-hour intervals. During periods
following the commencement of absorption, readings were taken every 15
minutes, and subsequently, at much longer intervals. The pressure changes
due to temperature variations in the oil bath rarely amounted to 1 mm.
and hence the absorption of even 0.5 ml. of oxygen resulted in a noticeable fall
in pressure (circa 4 mm.). The time of the beginning of the oxygen absorp-
tion was taken to start when a fall in pressure of 5 mm. took place. The rate
of absorption of oxygen during and subsequent to the induction period was
followed by noting the drop in pressure for suitable lengths of time. Slight
changes in temperature and atmospheric pressure as measured by pressure
changes in a flask containing no fat in the course of the experiments were not
appreciable and were neglected.
A possible disadvantage of this method as mentioned by Milas [1930]
is that the rate of oxygen absorption is influenced by large pressure changes.
For a great part of this work, attention was directed towards ascertaining the
induction period of fat oxidation, and hence any error involved by small
pressure changes was insignificant.
Influence of temperature on rate of oxidation.—Oxygen absorption methods
are time consuming unless high temperatures are used. The choice of a suit-
able high temperature was made after a study of the rates of oxygen absorption
at three different temperatures, viz. 75°, 85° and 95°C, using 10 gm. of a
sample of ghee in each case. Fig. 1 shows the relation between temperature
FIG. 1. Oxygen absorption of Kathiawar ghee at different temperatures
S. V. GOVINDARAJAN AND B. N. BANERJEE
363
and oxygen absorption in the fat. It is seen that at the higher temperatures
the rate of absorption is greater for a 10°C rise in temperature than at the
lower temperature, while the induction period is also considerably shortened.
At 95°C this period is reduced to a short measurable period, and hence this
temperature was chosen as being suitable.
Influence of volume of fat on the rate of oxidation.—The process of oxidation
involves the absorption of oxygen by the fat molecules and hence the amount
absorbed and also the rate of absorption would be controlled by the area of
surface exposed to oxidation. When there is proper stirring of the liquid
substrate, it might be expected that new fat molecules are continually exposed
to oxygen. Under the conditions of this experiment the liquid could
be occasionally stirred by shaking the vessel. It was considered that the high
temperature used in the reaction and the use of only small quantities of liquid
would overcome the disadvantage due to lack of continuous stirring. To
test this point 5, 10, 15 and 20 gm. quantities of ghee were placed in flasks
under oxygen and the absorption of oxygen measured by the fall in pressure.
Fig. 2 shows that in the two cases (5 and 10 gm.) the quantity of oxygen
absorbed up to a certain time was almost directly proportional to the time.
With larger quantities absorption of oxygen was too rapid for accurate measure-
ment. The end of the induction period was taken to correspond to a fall in
pressure of 5 mm. of mercury, indicating the absorption of 0.6 ml. of oxygen.
In most of the subsequent work either 5 or 10 gm. quantities of ghee were
used, because with these amounts the oxygen absorption was found to be fairly
uniform.
FIG. 2. Oxygen absorption of a ghee sample (Kathiawar)
Study of the induction period in ghee from different sources.—The resistance
of ghee towards rancidity is known to vary within wide limits depending
upon the origin, and the methods of separation of the fat and the clarification
of the butter. The ghee prepared from some of the imported cow breeds,
such as the Jersey, is recognised as being able to maintain its quality for long
364
Anti-oxidants of Ghee
periods of time. Defective methods of fat separation, such as insufficient
coagulation of curdy matter and high acidity, are known to cause rapid deterio
ration of the fat. It was considered of value to determine the induction period
from the oxygen absorption of a variety of ghee samples with a view of as
certaining the conditions which affect this period.
Table I gives the
acid value, the peroxide value and the vitamin values of fats at the start of
the experiment along with their induction period. The acidity is expressed
as percentage of oleic acid. The peroxide values were determined according
to Lea [1931] and expressed in terms of ml. of 0. 002N sodium thiosulphate per
gram of fat. Vitamin A was measured in blue units per gram of fat given by
the unsaponifiable matter with antimony trichloride in chloroform solution.
Carotene was determined by measuring the yellow colour of the unsaponifiable
matter in chloroform solution. The vitamin A and carotene values are express
ed in Lovibond Tintometer units per gram of fat.
TABLE I
Induction period of ghee from different sources
(Temperature 95°C)
Source
Jersey
.
.
.
.
Gir
.
.
.
.
Imperial Dairy . .
Sindhi
.
.
.
.
Kathiawar [Agmark] .
Gujarat . . .
Tiruppur . . .
Induction
period
hr.
8.5
6.0
5.0
5.5
2.0
1.5
1.0
Acidity
olcic
acid
per cent
0.57
0.32
0.38
0.56
0.95
2.77
3.53
Peroxide
value ml.
0.002N
thiosulphate
solution per
gram of fat
3.1
3.9
1.8
1.0
1.2
9.9
11.2
Vitamin values
Lovibond
Blue
units
Yellow
units
per gram of fat
45.1
35.2
27.5
40.7
8.8
19.8
4.4
24.0
1.0
2.4
2.9
0.4
0.3
0.5
It can be observed that the samples with high initial vitamin values have
low acid and peroxide values, and that .their induction periods are much greater
than the samples showing high acid values.
Effect of anti-oxidants on the induction period.—In an extensive series of
studies Olcott and Mattill [1936] have ascertained the protective action of
various compounds on the oxidation of lard. As a result, substances were
classed into three types, viz. (1) the acid type inhibitors, (2) the unsaponifiable
matter obtained from various vegetables and vegetable oils and (3) the phenolic
type including hydroquinone, α-naphthol, pyrogallol, catechol and others
S. V. GOVINDARAJAN AND B. N. BANERJEE
365
Hydroquinone in particular has been widely studied as an anti-oxidant of fats
[Olcott, 1934, and Wagner and Brier, 1931] and has been used to protect the
vitamin A of fish liver oils. Huston et al. [1928] have demonstrated by feeding
experiments that hydroquinone has a protective value on the vitamin A con-
tent of milk fats. Holm, Corbet, and Hartzler [1936] have found that mix-
tures of lecithin and hydroquinone have superior anti-oxidative properties to
either of them separately, and Evans [1935] has shown that lecithin exhibits a
retarding effect on the formation of peroxides in fats when the oxidation is
catalysed by cobalt oleate.
It is thus seen that substances which help to retard the oxidation of fats
have a protective action on vitamin A and also retard the formation of
peroxides.
Mattill and his coworkers [1930, 1931, 1934] demonstrated that the un-
saponifiable lipoid fractions of vegetables and vegetable oils contain compounds
which are active anti-oxidants in lard. They proposed the name inhibitol for
these compounds to indicate their function as inhibitors, and also the
occurrence in them of hydroxyl groups upon which their inhibiting action
was found to depend.
Whether the unsaponifiable matter obtained from vegetable oils had any
anti-oxidative action towards ghee was studied by adding different amounts
to ghee. The unsaponifiable matter obtained after saponification of the fat
in the usual manner was dissolved in petrol ether. After addition to the ghee
samples under test the solvent was completely evaporated before starting
the experiment. Cotton seed, coconut and gingelly (til) oils were used as
sources of the unsaponifiable matter, and the prolongation of the induction
period (Table II) indicated that the added substances were capable also of
protecting ghee from oxidation.
TABLE II
Protective action of the inhibitols of oils and ghee on Kathiawar ghee (10 gm.)
(Temperature 95°C)
Unsaponifiable fraction from
Gingelly oil
Gingelly oil
Gingelly oil
Cotton seed oil
Cotton seed oil
Cotton seed oil
Coconut oil
Coconut oil
Coconut oil
Gir ghee
Gir ghee
Jersey ghee
Jersey ghee
Sindhi ghee
Sindhi ghee
Amount of
unsaponifiable
fraction from
volume of fat
(ml.)
10
20
30
10
20
30
10
20
30
10
30
10
30
10
30
Induction
period
(hr.)
20.0
26.0
38.0
15.0
17.5
22.0
9.5
12.0
17.0
6.5
8.0
3.5
4.5
4.0
6.0
C