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Journal of Experimental Medicine

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THE

R E L A T I O N OF M O S Q U I T O E S A N D F L I E S T O T H E
EPIDEMIOLOGY OF ACUTE POLIOMYELITIS.

BY HIDEYO NOGUCHI, M.D., AND ROKUSABURO KUDO, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research.)(Received for publication, March 15, 1917.)
Of several hypotheses concerning the m o d e of conveyance of poliomyelitis, t h a t of direct contact, supported b y Flexner, appears tobe the only one which stands on a substantial experimental basis.l. *Although in all epidemics instances have been reported in which moret h a n two members of a family were victims of the disease, as a rule
only one out of several children in a family is attacked. This peculiarfeature of the epidemiology of poliomyelitis seemed to support thetheory t h a t the disease m a y be inoculated b y the bite of an infected
insect r a t h e r t h a n t r a n s m i t t e d directly from one individual toanother.
Typical examples of insect transmission of disease are found in yellow fever,malaria, spirochetoses, trypanosomiases, and other tropical fevers. Rosenan
and Brue# in 1912 announced their success in transmitting poliomyelitis froman infected to a normal Macacus monkey by means of the bites of stable-flies(Stomoxys calcitrans). Their experiment consisted in allowing many thousandsof the flies to feed first on an infected monkey and immediately afterwards ona normal monkey. The transmission was mechanical, but not in the sense of
an intermediary host. Anderson and Frost4 confirmed Rosenau's finding in at Flexner, S., The mode of infection and etiology of epidemic poliomyelitis,
Am. J. Dis. Child., 1915, ix, 353.
Flexner, The nature, manner of conveyance and means of prevention of
infantile paralysis, J. Am. Med. Assn., 1916, lxvii, 279.
a Rosenau, M. J., and Brues, C. T., Some experimental observations uponmonkeys concerning the transmission of poliomyelitis through the agency of
Stomoxys calcitrans, Tr. XVlh Internat. Cong. Hyg. and Demography, Washington, 1913, i, 616.
4 Anderson, J. F., and Frost, W. It., Transmission of poliomyelitis by meansof the stable fly (Stomoxys calcitrans), Pub. Healtk Rep., U. S. Mar. Hosp.
Sero., 1912, xxvii, 332.
49

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EPIDEMIOLOGY OF ACUTE POLIOMYELITIS

limited number of instances, and then later failed to obtain further positive results. Howard and Clarks were unable to transmit the infection from an infected to a normal monkey by the bites of Stornoxys calcitrans. The peculiarity
of conveyance of the infection is far from being solved by the theory of transmission by these blood-sucking flies.
The most plausible hypothesis so far advanced seems to be that offered by
Amoss and Taylor, 6 who, having experimentally demonstrated the existenceof a protective principle in the secretion of the nasal mucous membranes of norreal individuals, consider that the incidence of infection may to a considerable
extent depend upon the condition of these mucous membranes; that is, as longas they remain intact, the virus will be destroyed before it can become established in the individual. The disturbance of this natural protective mechanism
exposes t o infection, but not the presence or absence of the virus alone. They
were able to show that the protective substance in question was more generally
and abundantly present in adults than in young children.
T h e sudden disappearance of poliomyelific cases with the returnof cold weather is often used as an argument in favor of insect transmission of the disease. Flies and mosquitoes have constantly beenu n d e r suspicion. While the elimination of these insects'from ourhouseholds is desirable as a m a t t e r of routine cleanliness, yet witho u t a systematic experimental investigation t h e y should not be acc e p t e d as a factor in the epidemiology of poliomyelitis. Indeed,i t would be unwise if, through a misapprehension of the facts, thea t t e n t i o n of health officers and laity alike were to be diverted fromthe real source of danger.

Scope and Mode of Experiments.
T h e question whether a given v a r i e t y of insect plays a p a r t in
t h e spread of poliomyelitis is within reach of exact determination,s i n e . these insects are amenable to artificial propagation t h r o u g h
m a n y generations. During the past several months, we have beenable to collect a n d propagate in tanks in the laboratories a quant i t y of Culex pipiens from sewer waters near Jersey City 7 and in
5 Howard, C. W., and Clark, P. F., Experiments on insect transmission of
the virus of poliomyelitis, dr. Exp. Med., 1912, xvi, 850.

6 Amoss, H. L., and Taylor, E., Neutralization of the virus of poliomyelitis
by nasal washings, Y. Exp. Med., 1917, xxv, 507.
We are greatly indebted to Dr. Headlee, chief entomologist of the New Jersey Agricultural Experiment Station, at New Brunswick, and to Dr. Chidester

HIDEYO NOGUCHI AND ROKUSABURO KUDO

51

Queens County, Long Island. It was our intention to study several
well known genera of mosquitoes at the ~ m e time, but owing to thelateness of the season, none of the Aedes or Anopheles species wereavailable. The only variety which we were able to obtain was Culex
pipiens. Experiments were also performed to determine whether thecommon house-fly or the bluebottle fly is able to take up thepoliomyelific virus and make it increase within its body.
There are at least two totally different ways of attacking the
problem. The first method is to feed young growing larvae withthe active poliomyelitic virus by putting it in the poRuted waterin which they are being grown. When the adult mosquitoes have
hatched out, they m a y be allowed to feed on a normal Macacusmonkey. The second procedure is that ordinarily practised by investigators and consists in allowing the mosquitoes to feed first on
an infected animal and then, within a certain length of time, on anormal animal.
The experiments with non-biting flies had to be carried out by
still another method. They were allowed to feed on the nervous
tissues containing an enormous amount of the poliomyditic virus
until they pupated. Part of the pupa~ were allowed to hatch. Boththe pupa~ and the imagos were then crushed together and the emulsion was filtered through a Berkefeld filter. The clear filtrate wasintroduced intracerebraUy and subsequent events were observed."

Experiments with Mosquitoes Hatched in Polluted Water Experimentally Contaminated with the Virus of Poliomyelitis.
To twelve jars, covered with a wire net cage and containing mosquito larvae of various ages in ordinary stagnant water, were added
varying amounts of the brain or cord emulsions or their filtrates
derived from two monkeys that died of experimental poliomyelitis.
The virus was introduced every 24 hours for a period of 10 days.
The temperature oi the laboratory was kept at about 70°F. and theof Rutgers College, New Brunswick, for their assistance and instruction as tothe propagation of mosquitoes in the laboratory. We wish to express our gratitude also to Mr. Winchell of Rutgers College, who assisted us in collecting thespecimens in the field.

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E P I D E M I O L O G Y O]? ACUTE P O L I O M Y E L I T I S

relative humidity at about 40 per cent. Numbers of adult mosquitoes, both male and female, hatched out from the jars everyday. They were carefully collected and used for transmission experiments on normal monkeys. The rate of hatching from day to
day is shown in Table I.
TA B LE I.
Date.

Female.

Male.

21
12
11

20
9

1916

Oct. 31
Nov. 1
"
2
" 3
"
5
"
{i
"
7
"
8
"
9
Total . . . . . . . . . . . . . . . . . .

17

2
8

10
15
30
25
13

12
8
18
9
18

154

104

The first transmission experiment was performed with mosquitoes
on two young Mavavus rhesus monkeys. As the mosquitoes attack
the monkeys only during the night, the experiment was begun atdusk and discontinued the next morning. The results were as follows: Of 150 females and 60 males put into the cage, 114 females
were fully engorged with the blood, 23 fenmles were apparently notengorged, and 52 males were still living. Some of the mosquitoes
had been killed by the monkeys.
Close observation was maintained on the monkeys for 21 days,
but no sign of poliomyelitis developed nor has any been noticed up
to the present time.
Some of the engorged females sooner or later laid eggs and perished,
while some died without oviposition. A number of female mosquitoes hatched out of the egg boats and also from the virus-containing water were kept alive for about 3 weeks and then allowed
to bite two young Mavacus monkeys. There were about 40 wellengorged specimens when examined the following morning. The
monkeys showed no symptoms of poliomyelitis.

H I D E ¥ O NOGUCHI AND R O K I I S A B U R 0 K U D O

53These experiments demonstrate that the mosquitoes raised inwater containing the virus of poliomyelitis in large quantities cannot transmit the infection by their bites to normal Macacus monkeys.
There is no indication that the virus enters and multiplies in the
body of mosquitoes, even if 3 weeks are allowed to elapse between
the time of hatching and that of the biting experiment. The offspring of these females are not capable of transmitting the disease
to the monkey.

Experiments to Determine Whether the Females Well Engorged with
the Blood of Infected Monkeys Will Ever Be Capable of Transmitting
the Infection by Their Bites to Normal Macacus Monkeys.
In order to decide this point, eight groups of mosquitoes, someobtained from the vicinity of Jersey City, and some from Queens
County, Long Island, were allowed to feed on several Macacus monkeys inoculated for this purpose. To insure ourselves of including
different stages of the infection, feedings were made every 24 hours
after the inoculation of the virus into the monkeys and were extended
into the paralytic stage, which of course varied in different animals
from 6 to 9 days, according to the activity of the virus and the quantity injected. The protocols of these feeding experiments show
the time which elapsed between the feedings and the transmission
experiments. The term "feeding mosquitoes" is used merely todenote that these mosquitoes have sucked blood from an infectedmonkey.
Four out of the eight groups were tested on November 20; thatis, 1 to 2 weeks after the time of feeding. The remaining four groups
were allowed to bite after having been kept from 2 to 3 weeks. It
must be mentioned that the engorged females do not survive for any
length of time after oviposition, and comparatively few specimens
remained alive in the second series of groups.
For the purpose of transmission two young Macacus monkeys
were employed for each of the two experiments (Groups 1 to 4 and
5 to 8). We also inoculated 2 cc. of sterile normal horse serum
into the intrathecal space in order to derange the protective mechanism which the meninges provide against the penetration of the virus

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EPIDEMIOLOGY OF ACUTE POLIOMYELITIS

into the central nervous s y s t e m j n experimental poliomyelitis in
m o n k e y s . I t m a y be recalled that F]exner and Amoss 8 d e m o n s t r a t e d
t h a t a fraction of the virus, intravenously introduced, will readily
localize in the central nervous s y s t e m when the meninges are p r e viously disturbed b y the inoculation of various substances, a l t h o u g h
the m o n k e y m a y b e a r a multiple of such doses with i m p u n i t y if there
has been no previous meningeal injury,.

Group 1. Feeding Mosquitoes.--Nov. 13, 1916. Adult mosquitoes hatchedin the laboratory from the larvae collected in Queens were allowed to feed on
Macacus rhesus 1 on the 4th day of experimental poliomyelitis. 30 mosquitoes
were well engorged when examined the next morning. Used for experiments
on Nov. 20, or in 7 days.
Group g. Feeding Mosquitoes.--Nov. 15, 1916. Another lot of adult mosquitoes hatched in the laboratory from the same material were fed on the samemonkey (No. 1) on the 6th day of the disease. The animal showed partial
paralysis of the limbs. 23 well engorged females were obtained. Used forexperiments on Nov. 20, or in 5 days.
Group 3. Feeding Mosquitoes.--Nov. 16, 1916. Another lot of adults froma similar source were fed on the same monkey (No 1) on the 7th day of the disease. The animal was completely paralyzed. 17 engorged females were collected. Used for experiments on Nov. 20, or in 4 days.
Group 4. Feeding Mosquitoes.--Nov. 7, 1916. A lot of adults hatched in
the laboratory from the larv,e from Queens were fed on Macacus rhesus 2 on the
9th day of the disease. 25 engorged females were obtained. Used for experiments on Nov. 20, or in 13 days.
Group 5. Feeding Mosquitoes.--Dec. 4, 1916. A lot of adult mosquitoes
hatched in the laboratory from the larvm secured from a Jersey City suburb were
fed on two Macacus rhesus monkeys, Nos. 3 and 4, on the 7th day of experimental
poliomyelitis. 13 engorged females were obtained. Used for experiments on
Dec. 19, or in 15 days.
Group 6. Feeding Mosquitoes.--Dec. I, 1916. A lot of adult mosquitoes
hatched in the laboratory from the materials gathered in the neighborhood ofJersey City were fed on Macacus rhesus 5 on the 3rd day of experimental poliomyelitis. 34 engorged females were obtained. Used for experiments on Dec.
15, or in 14 days.
Group 7. Feeding Mosquitoes.--Nov. 24, 1916. A lot of adult mosquitoes
hatched in the laboratory from the material from Queens were fed on Macacus
rhesus 6 on the 2nd day of the disease. 45 engorged females were obtained.
Used for experiments on Dec. 15, or in 21 days.
s Flexner, S., and Amoss, H. L., The relation of the meninges and choroid
plexus to poliomyelific infection, Y. Exp. Med., 1917, xxv, 525.

H I D E ¥ O NOGUCHI AND ROKUSABUR0 K U D 0

55Group 8. Feeding Mosquitoes.--Nov. 25, 1916. A lot of adult mosquitoes
hatched in the laboratory from the material from Queens were fed on Macacus
rhesus 7 on the 5th day of the disease. The animal had not yet become paralyzed. 20 engorged females were collected. Used for experiments on Dec. 15,
or in 20 days.

The results of the experiments were negative, in spite of the factthat many females (about fifty) attacked the monkeys and became
fully engorged with the blood sucked from them.
As in the earlier series of experiments, many of these engorgedfemales oviposited and gave rise to a new generation of larwe. It
was easy to propagate them in the laboratory for an indefinite period. These offspring were in turn allowed to bite a normal monkey, but so far no positive transmission of the infection by theirbites has resulted.

Experiments with Non-Biting Flies Reared in the Laboratory with an
Abundant Quantity of Poliomyelitic Virus.
Non-biting adult flies r~ay mechanically carry the virus of poliomyelitis just as they do various pathogenic microorganisms2 But
no experimental evidence has been adduced to prove whether thevirus can penetrate into the interior of the fly larwe when the latterare fed on the poliomyelitic material, or, if it does, whether it is capable of multiplication within the body cavity of the larwe. The
question here is not the same as that of blood-sucking flies or mosquitoes, which may be the intermediary hosts as well as the directtransmitters of the virus by their bites. But in the case of non-bitingflies, though the virus may multiply in their bodies, its distribution
must depend upon their well known habits of gathering about foodor persons while they regurgitate, resuck, and excrete. In this way
the question of the non-biting flies as intermediary hosts of the virus
would be quite as important as that of the biting varieties.
In the present experiment, a number of young larwe of house-flies(Musca domestica) and of bluebottle flies (Calliphora vomitoria) werefed with the brain tissue of a Macacus rhesus, which had died of ex9 Flexner, S., and Clark, P. F., Contamination of the fly with poliomyelitis
virus. Tenth note, J. Am. Meal. Assn., 1911, lvi, 1717.

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E P I D E M I O L O G Y OF A C U T E POLIO~fYEUTIS

perimental poliomyelitis with typical s y m p t o m s a n d pathologicalfindings. T h e a c t i v i t y of the m a t e r i a l used was established in as u b s e q u e n t experiment.
Group /.--Nov. 23, 1916. About 50 house-fly larvae were put on several
slices of the fresh poliomyelitic monkey brain (No. 1). The larvae eagerly penetrated into the nervous tissue as if trying to shield themselves from the light.
At room temperature the tissue underwent a putrefactive decomposition within
24 hours, so the larvae had to be placed on a fresh lot of the virus-containing
brain. When left in the decomposed mass of tissue, many larvae died. Afterbeing fed on the poliomyelific brain for about 6 days, the larva~ became pupae.
After pupation, they were left with the brain tissue for several days longer and thenwashed from the tissue and removed to the refrigerator.
Group 2.--No,¢. 28, 1916. Another lot of house-fly larva~ were similarly fed
with poliomyelitic brain emulsion (No. 1) for 6 days. Pupation took place on
Dec. 4. The pupae were washed and removed to the refrigerator on Dec. 9.
Group 3.--Nov. 23, 1916. 25 larvae of bluebottle flies were fed on several
slices of brain tissue from a Macacus rkesus which had died of poliomyelitis.
The larva~ attacked the tissue vigorously and within 24 hours seemed to haveconsumed a great deal of it. Unlike the house-fly larvae, they feed on deadflesh or meat during their larval stage, but it was,necessary to put them on freshmaterial every 24 hours in order to protect them from the deadly effect of thedecomposed nervous tissue. Pupation occurred on Dec. 7. After several days,
the pupae were washed and removed to the refrigerator.
F o r the purpose of obtaining a d u l t flies from the infected pupae,a dozen specimens f r o m each lot were p u t in a Petri dish a n d left
a t 80 ° F. I m a g o s h a t c h e d within a b o u t 12 days. These a n d thepupae were used for the e x p e r i m e n t described below.

Preparation of Filtrate of the Infected Pupce and Imagos of theHouse-Fly and the Bluebottle Fly.
70 house-fly pupae, Groups 1 and 22:}12"
imagos, " 1 "Weight 1.36 gin.
15 bluebottle fly pupae, Group
6 "" imagos, "

~:}

,,

0.97 "

These were m~ed together and thoroughly ground up in a mortar, 22 cc. of 0.9
per cent saline solution being added. The milky emulsion thus obtained wasbriefly centrifuged at a low speed to separate the coarse particles from the fluid.
The latter was then passed through a sterile Berkefeld filter V, the result beinga clear and sterile filtrate.

HIDEYO NOGUCHI AND

ROKUSABURO

KUDO

57

Animal Inoculation.--A Macacus rhesus was inoculated intracerebrally with
3 cc. of the above filtrate on Dec. 16, 1916. The result was negative, no symptoms suggestive of poliomyelitis having been observed.

The foregoing experiment indicates that it is improbable that thevirus of poliomyelitis is taken up by fly larva~ and multiplies therein.
Not only does the virus fall to multiply, but probably it is rapidly
destroyed within the body of the insects. The notion that these
non-biting flies m a y act as intermediary hosts or a virus reservoir
is not justified by the evidence brought out in the present study.
SUM~ffARY.

1. Culex pipiens raised from the larval stage in water experimentally contaminated with an abundance of poliomyelitic virus were
found to be incapable of causing the infection when allowed in large
numbers to bite normal Macacus monkeys.
2. Culex pipiens which were fed on infected poloirnyelific monkeys
during different stages of the disease were found to be incapable oftransmitting the infection when allowed in large numbers to bite
normal Macacus monkeys. A previous disturbance of the meninges
by an injection of horse serum into the intrathecal space did not alter
the result, which was negative.
3. The offspring of the mosquitoes which were either reared in theinfected tanks or fed on infected monkeys were found to be entirely
harmless when allowed to feed in large numbers on a normal monkey. There was no hereditary transmission of the virus from one
generation to another.
4. No trace of the virus of poliomyelitis was demonstrable in thefiltrate of an emulsion of adult flies and pupm of the common housefly and bluebottle fly which were reared in the laboratory on slices,
emulsion, or filtrate of monkey brain containing the poliomyelificvirus. The intracerebral injection of the filtrate produced nouoliomvelitic infection in the normal monkey.