NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
17
examined in order to ascertain the percentage of children with enlarged
spleens.
In the examination of the films the variety of parasite met with in each
slide should be noted, in order that the proportion of each variety present in the
village and district may be ascertained. The number of "large infections"
should also be noted, as if there are a good number of heavy infections there
will be more likelihood of finding infected anopheles.
Having ascertained the endemic index, the next thing is to search for
adult anopheles in the houses, out-houses and stables, and to find out by
dissection—
(1) What species of anopheles are carrying malaria at the time.
(2) The percentage of infection with sporozoites of the malaria-carrying
species.
It will generally be found that only two or three species of anopheles are
present in sufficient numbers in the houses to be easily caught, and in order to
ascertain the two points noted above, at least one hundred anopheles of each
of these species should be dissected.
It may happen that a fairly high percentage of infection (5-10 per cent.)
will be found in one species, and no infection at all in another, but before it can
be definitely stated that only one species of anopheles is carrying malaria in
any place, at least 300 or 400 of any other species than the one in which infection
was found should be examined.
I have already described* the method of catching adult anopheles and
the best places in which to look for them, so it is unnecessary to do so here.
I may mention, however, that in places where anopheles are plentiful, a muslin
net will sometimes be found useful.
We have proved that it makes no differenće in the sporozoite rate whether
the anopheles in a village are caught in the stables and out-houses, or in the
houses where people are actually living, and sporozoites are as likely to be found
in mosquitoes caught in stables as in those caught in inhabited rooms. This is
a point of some importance, because it is far easier to catch anopheles in stables
and empty houses than in inhabited rooms.
If desired, the anopheles which are caught may be kept in bottles until
they have got rid of their blood, and they may then be examined for zygotes,
but there is no particular object in determining the zygote rate as it gives
us little help in determining the liability to infection, for some at least of the
mosquitoes which are infected with zygotes will die before they reach the stage
when sporozoites are in the glands.
* " The Causation and Prevention of Malarial Fevers ; a statement of the results of researches, drawn
up for the use of Assistant Surgeons and Hospital Assistants," G. C. Press, Simla.
D
Having determined the percentage of infected anopheles, we can turn to the
simpler but very important questions of
—
(i) The exact identification of all the species of anopheles that are
present in the village.
(ii) An estimation of the abundance of anopheles as a whole in the houses
and the relative abundance of each species.
(iii) An examination of breeding grounds.
The breeding grounds of each species should be ascertained separately,
and special attention should be paid to the breeding grounds of the species
which we have found infected. Every pool, stream and collection of water
of any kind within a radius of half a mile of the village should be thoroughly
searched for larvæ.
Very important points to ascertain are
(a) the extent of the breeding grounds as a whole and the extent of the
breeding grounds of each species ; and
(b) the distance of the nearest breeding grounds from the village and the
distance of the nearest breeding grounds from the village of the
infected species.
When all the points noted above have been determined, we may be said to
have an accurate knowledge of the conditions determining the prevalence of
malaria in the village or town under examination, and are able to compare
our results with those obtained from the examination of other villages where the
conditions may be different.
We are also in a position to formulate a definite system of prophylaxis
for the place we have examined, for if there is one thing more certain than
another with regard to the prophylaxis of malaria, it is that no general system
of prophylaxis, which can be applied to any and every place, will ever meet with
success. Each place must be studied by itself on the lines laid down above
and independently of every other place.
It will be profitable, I think, to conclude this section with an example from
one of the places we have examined, in order to show the kind of information
which can be obtained from an investigation on these lines.
I shall select, as an example, the village of Ennur, which is situated on the
coast about ten miles from Madras. The village was formerly used as a health
resort by Europeans, but has been deserted by them for many years past on account
of the fact that it is scarcely possible to pass even one night there without
getting fever. It contains a number of European bungalows, now falling into
decay.
is
A
19
The following were the results of our malarial survey of this place.
I.—The determination of the source of infection and the Endemic Index.
TABLE I.—SHOWING THE ENDEMICITY OF MALARIA AT ENNUR.
Date of Investigation.
Number of children
examined,
Percentage of children with
enlarged spleen.
Percentage of children with
malarial parasites in their
blood (endemic index).
February 1902.
40
95
55
Thus in February, which is considered almost the healthiest time of the
year, 55 per cent. of the native children have malaria parasites in their blood and
they form therefore the source of infection at Ennur.
II.
—
The variety of the infection.
TABLE II.—VARIETY OF PARASITES FOUND.
Number of infected
children.
Percentage infected with
Quartan parasites only.
Percentage infected with
simple Tertian parasites only.
Percentae with a mixed
infection of simple Tertian
and Quartan.
22
8 1
5
14
No malignant Tertian parasites were found at this place.
I I I . — T h e c a r r i e r o f t h e i n f e c t i o n .
Only two species of anopheles were found in the houses, viz., A. Rossii
(Giles), and A. Culicifacies (Giles).
The results of our dissections were as follow.
TABLE SHOWING INFECTION OF ANOPHELES AT ENNUR.
Species of anopheles.
Number dissected.
Number in which sporozoites
were found.
Percentage of infected
anopheles.
A. Culicifacies .
69
6
87
A. Rossii
240
n i l .
nil.
The carrier of infection at Ennur is therefore A. Culicifacies, and the high
infection rate of this species indicates great liability to infection of any one
residing at this place. As a matter of fact, this liability was also shown in
another way by the fact that four out of the five servants who accompanied us
developed malarial fever between 14 and 18 days after we had left the place.
In the blood of three of these servants Quartan parasites were found, and in
that of the fourth simple Tertian parasites.
D2
20
IV.—Conditions determining the prevalence and liability to infection.
Species of anopheles present and their abundance.—A. Rossii and
A. Culicifacies were present in the houses, A. Rossii being in great abundance
and A. Culicifacies moderately so. A third species not present in the houses
and of which only the larvæ could be found was A. Fuliginosus (Giles).
Nature, extent and distance of breeding places.— The breeding places were
very extensive and surrounded the village. The nearest were within ten to
twenty yards of the houses.
A. Culicifacies was found to be breeding almost exclusively in the "borrow
pits " by the side of the railway and in the tanks in the compounds of the
European bungalows.
A. Rossii was found to be breeding chiefly in the rice fields and in the pits
dug for irrigating purposes.
The condition of the European bungalows :—
(i) Each bungalow has a main source of infection within fifty yards of
it, viz., the infected children in the village.
(ii) Each bungalow has an additional source of infection within ten to
twenty yards of it, viz., the servants' children who live in the
compounds and who were also found to be infected with parasites.
(iii) Each bungalow has its own tank in which anopheles are breeding.
Under conditions such as these, unless very careful precautions were
taken to avoid being bitten, it would be almost impossible for any one living in
the bungalows to escape getting malaria.
The example given above refers to the month of February, but it would not
be difficult to carry out a similar investigation, showing the variations in mala--
rial endemicity, in the species of anopheles and in their infection, and in the
other conditions noted above, month by month throughout a period of a year, and
such an investigation would yield valuable results.
V.—Methods for the study of mosquitoes.
The study of mosquitoes—and especially the study of mosquitoes of the
anopheles genus—is inseparably associated with the investigation of malaria,
and as comparatively little is at present known regarding even the identifica-
tion of the various species of this genus, a great deal of study might be
profitably spent on this subject. It has hitherto been considered sufficient
to study the anopheles genus more or less as a whole, so that an exact identi-
fication of each species was not of great importance, but at the present time
general statements regarding the characters and habits of the genus anopheles
as a whole are of little or no value, and it is as necessary to study the different
species separately as it is to study separately the different varieties of the
2I
parasites of malaria. In order to do this it is necessary to be able to identify
each species correctly, and it is also of great advantage to be able to identify the
larva of each species without the necessity of breeding out each batch of larvæ
that have been caught.
The following are the chief points to be aimed at in a study of anopheles :—
(i) An exact and detailed description of the male and female adults of
each species of such a nature as to prevent the possibility of
confusing one species with another.
(ii) An exact description of the eggs and larvæ of each species with
the points of difference between the eggs and larvæ of different
species carefully noted.
(iii) An account of the breeding places, habits, prevalence and life
history of each species separately.
(iv) An account of the pathological significance of each species
separately.
The methods of collecting adult anopheles and their larvæ I have already
described elsewhere,* and the method of mounting adult
anopheles for examination is probably well known to all.
† I t i s u n n e c e s s a r y , t h e r e f o r e , t o r e f e r t o t h e s e p o i n t s h e r e .
To keep mosquitoes permanently in India is a very
difficult matter on account of the fact that the speci-
mens either become very quickly mildewed, or destroyed
by insects. For this reason I have devised the "mos-
quito tube" shown in the drawing (Fig. 6), and mos-
quitoes kept in these tubes remain in good preservation
for almost any length of time.
For the identification of adult mosquitoes, an examina-
tion under a low power of the microscope ½ or ⅔inch
objective) is almost a necessity, especially for the identi-
fication of closely allied species.
For this purpose the mosquitoes, which have
a l - r e a d y b e e n m o u n t e d o n c a r d d i s c s i n t h e u s u a l m a n n e r ,
are fixed by a pin thrust through the edge of the card
disc to a flat piece of cork about an inch square.
A. Disc carrying the mosquito. This disc is pinned to the cork in the mouth of the tube.
B. Perforated cardboard disc pinned to the cork D in order to keep the bag of napthalene C in place.
C. Muslin bag containing napthalene.
D. Cork firmly fixed in bottom of the tube.
* See "The Causation and Prevention of Malarial Fevers; a statement of the results of recent
researches." G. C. Press, Simla.
† For the methods of mounting mosquitoes, see "A Handbook of the Gnats or Mosquitoes," by
Lieu-tenant-Colonel Giles, I.M.S., 2nd edit. Bale Sons and Danielsson, London, 1902 ; or "A Monograph of
the Culicidæ of the World, "by F. V. Theobald, F.E.S. British Museum (Natural History), London, 1901.
22
This cork, carrying the mosquito to be examined, is placed on the microscope-
stage and can be moved about on the stage at will.
The angle at which the pin is fixed in the cork should be such that the
part of the mosquito which is being examined is always as nearly as possible
parallel to the microscope stage.
It is best to begin with an examination of the palpi and to work back-
wards, noting every marking and character that presents itself, and the position
of the disc carrying the mosquito will of course have to be frequently altered
by removing the pin from the cork and readjusting it at a different angle. The
palpi, the wings, and the legs are the structures to which chief attention should
be directed, and diagrammatic drawings should be made showing clearly every
marking on these structures. In addition, it is important to note the characters
of the scales on the head, thorax, and abdomen.
In order to ascertain the arrangement of the transverse veins of the wings,
and the detailed shape of the wing scales (an important point to note), one or
more wings must be mounted separately on a slide and covered by a cover slip.
The wing can then be examined if necessary under a higher power of the
microscope. The tips of the male legs, especially the fore legs, and the male
genitalia should be mounted on slides in xylol balsam. The characters of the
fore ungues in the male and the male genitalia are of use in identification.
In order to obtain eggs for examination, a few female anopheles that have
been caught in a village should be transferred to a glass jar similar to those
already described for use in feeding experiments. The jar should be inverted
over a small tin of water on which a piece of paper is floating for the mosquitoes
to rest upon. If this is done in the early evening, by the next morning one or two
of the anopheles will almost certainly have laid their eggs. Some of the eggs
will almost always be found to have been deposited on the pieces of damp paper
floating on the water in the tin, so that all that one has to do is to remove the
paper carrying the eggs—which can be easily seen by the naked eye—and to
place it on the microscope stage over the hole in the centre of the stage. The in-
dividual eggs can then be examined by reflected light with a ½ or ⅔ inch objective.
The differences that can be made out between the eggs of different species.
will be referred to later. For the measurement of the eggs and other structures
in connection with mosquitoes an eye piece micrometer will be found useful.
The characters necessary for the identification of larvæ can be made out
either by examining fresh larvæ or in permanently mounted specimens.
In order to examine a larva when it is alive, it is caught in a small spoon—
care being taken not to injure it—and transferred with a small drop of water on
to a glass slide. A cover-glass is then dropped very gently on to it. This has
the effect of preventing the larva from continually wriggling out of the field of
the microscope, and if carefully done it does not break any of the larval hairs.
23
or injure it in any way, so that at end of the examination the larva can be
returned to the bottle and allowed to develop, in order that by an examination of
the adult insect we may test the correctness of our observations on the larva.
After some practice it is of course possible to make out all the structures in
the intervals of rest which follow each effort to wriggle off the slide, without the ne-
cessity of applying a cover-glass, but as the resting intervals are usually very brief,
it is best at first to examine the larvæ imprisoned by the method just described.
Permanent specimens of larvæ may be obtained either (1) by mounting
them simply in a solution of formalin in hollow ground glass slides—the deeper
the hollow of the slide the better,—applying a cover-glass and ringing it with
thick Canada balsam ; or (2) by the following more permanent method:—
(a) Kill the larvæ by immersing them in strong formalin solution, and
allow them to remain in this solution for I2 hours. This partially
fixes them without causing any shrinking of the larvæ.
(b) Complete the fixing and dehydration by immersing the larvæ for ten
or fifteen minutes in absolute alcohol.
(c) Remove the alcohol by immersing the larvæ for half an hour or more
in oil of cloves.
(d) Clear with xylol and mount on an ordinary flat slide under a cover
in Canada balsam.
During the above process great care must be taken to handle the larvæ
very carefully so as not to break any of the finer hairs. They are best removed
from one dish to another by lifting them up very gently on the edge of a
piece of stiff paper.
The general anatomy of the larva is well described in Dr. Nuttall's paper
in the Journal of Hygiene, " The Structure and Biology of Anopheles"—Journal
of Hygiene, Volume I , No. 1, Jan. 1901, and need not be referred to here. I
shall note therefore only the structures we have found of use in the identifica-
tion of the larvæ of different species.*
These structures are the following :-
(1) The "frontal hairs" (Figure 7, I-A and II). These are four fine hairs
which are attached to the dorsal surface of the head anteriorly. They may
be called the external and the median frontal hairs. The external hairs are
placed one at each corner of the dorsal chitinous end of the head and exactly
overhang the prominent so called whorl organs or " shaving brushes." When
the whorl organs are pushed out the external (corner) hairs are difficult to see,
but when the whorl organs are drawn in (as often happens permanently when
larvæ are killed with formalin solution), they stand out prominently.
* The initiation of the work on the differentiation of eggs and larvæ and the chief part of the results
obtained are due to Dr. Christophers.
The drawings of larvæ reproduced here are copied from "A Monograph of the Anopheles of India," by
Capt. S. P. James and Capt. W. Glen-Liston. Thacker, Spink and Co., Calcutta (in the press).
24
The median hairs arise close together near the middle line and project
forward in front of the head. They are not difficult to see with a ½ or ¼ inch
objective. A very important difference between the larvæ of different species
depends upon the fact that the characters of these external and median frontal
hairs differ in different species.
Thus in some species both the external and median hairs are simple and
entirely unbranched (see figure 7, Larva of A. Rossii), in other species both pairs
of hairs are slightly branched (e.g., A. Superpictus, Grassi), in others the
median hairs are slightly branched or frayed, while the external are very much
branched ("like a small tree," Grassi, e.g. A. Fuliginosus), and again the
median may be simple or slightly branched and the external undergo dicho-
tomous branching so that it forms a very prominent and distinct "cocade"
(A. Nigerrimus, A. Barbirostris).
Further details regarding these hairs are given under the descriptions of
the different anopheles of India.
Fig. 7.—The larval characters of A. Rossii.
I. A. Frontal hairs.
II. Frontallhairs enlarged.
B. Antenna.
C. Palmate hairs,
D. Whorl organs or "shaving brushes."
III. Palmate hair enlarged,
E. External hair on antenna.
25
(2) The "palmate hairs." (Figure 7, C.)
If the dorsal surface of the abdomen of the larva be examined at about the
third or fourth segment, a pair of prominent small conical or fan-shaped hairs will
be seen. The hairs are arranged in pairs—one pair to each segment of the abdo-
men—and their function is to keep the larva floating in a horizontal position at
the surface of the water. Each palmate hair consists of a stalk, to the free end
of which is attached a conical bundle of about fifteen to eighteen hairs,
arranged round the stalk, like the petals of a flower of which a portion of the
circle is incomplete. At the surface of the water these leaflets spread out and
form a series of little cup-shaped organs which keep the larva afloat.
Both the number and the shape of the palmate hairs of different species of
larvæ vary considerably, so that in this fact we have a second means of dis--
tinguishing between the larvæ of different species of mosquitoes.
In some species, for example, the larvæ are provided with a pair of palmate
hairs on the thorax and on all the segments of the abdomen except the last two,
e.g., A. Fluviatilis, A. Culicifacies.
In other species there are no palmate hairs at all on the thorax and only
rudimentary ones on the first and second abdominal segments, the true palmate
hairs being present from only the third to the seventh segments of the abdomen
(e.g., A. Rossii, A. Stephensii). In other species again, the first three abdo--
minal segments are devoid of true palmate hairs (rudimentary ones may be
present on the third segment), e.g., A. Turkhudi. As regards the shape of the
palmate hairs, considerable differences exist, which are useful points of distinction
between larvæ.
Thus, in A. Rossii larvæ each leaflet of the palmate hair is provided with a
very long terminal filament (see Fig. 7), in the larva of A. Fluviatilis and
A. Theobaldi, on the contrary, the terminal filament is so short as to form only
short spine.
A reference to the figures of the different larvæ will show other differences
in the shape of the palmate hairs by which the larvæ can be recognised.
(3) The Antennœ. (Fig. 7, B.)
These structures are of course very easily recognised, and in the larvæ of the
majority of Indian species the characters of the antennæ differ but little. A
very small hair (E, Fig. 7) should be noted on the outer side of the antenna
in the majority of Indian species. Two of the Indian species, however (viz.,
A. Nigerrimus and A. Barbirostris), are easily distinguished from all other larvæ
by the fact that their larvæ possess a stout branching hair on the inner side of
the antenna, quite distinct from the small external hair referred to above.
(4) The pattern on the head.
Although the pattern on the dorsal surface of the head of anopheles larvæ
26
is formed by dots of pigment only, and is therefore liable to considerable varia--
tion, yet in some species the head pattern is sufficiently constant to be of
assistance in the identification of larvæ. The inverted triangular area enclosing
four dots of pigment on the dorsal surface of the head of the larva of A. Rossii,
for example, is fairly characteristic, as is also the complete absence of any
pattern on the head of A. Stephensii larvæ. The usual markings on the heads
of these and other larvæ are given in the diagrams.
The above are the chief characteristics by which we have differentiated the
larvæ of the Indian species of anopheles. Further work would doubtless show
that other structures might be used for the same purpose, such for example as
the spines on the maxillæ or the varying length of the caudal papillæ. It
would also be interesting to discover the characters which distinguish the male
and female larvæ from each other if in this stage of the mosquito's existence
any marked difference exists.