The Insect Bite Reaction

THE INSECT BITE REACTION II EVALUATION OF THE ALLERGIC REACTION*

EVELYN M. ROCKWELL, M.D. AND PATRICIA JOHNSON, B.S.

Exposure to the injection or deposition of foreign material by insects in the act of feeding is a common experience. That the substance injected may be a primary irritant is exemplified by the pain and reaction that follows the first sting by bees or wasps. Allergic reactions to the bites of insects should be expected since re-

peated parenteral injection of a foreign protein may produce the anaphylactic state. Asthma, generalized urticaria, and anaphylactic reactions from bee and wasp stings have been reported by a number of observers (1). Thus it is evident that both toxic and allergic reactions follow bee and wasp stings. Our study concerns the mosquito bite reaction. The marked swelling with pain which occurs after the first sting of a bee does not follow the first bite of a mosquito (2, 3). However, both immediate wheals and late papular reactions commonly follow mosquito bites. Vesicles, bullae, extensive edema and erythema

resembling ceflulitis, and lymphadenitis have been observed by us and others (4—9). Brown et al (10) report Arthus' phenomena from mosquito bites. Asthma or anaphylactic shock with death have not been reported. Thus reactions to mosquito bites are less severe than follow bee stings. It is our belief, however, that severe local reactions to mosquito bites are more common than the literature indicates. That both early and late reactions may develop with repeated mosquito bites has been demonstrated (2). Likewise decreased reactions, particularly of the delayed type, occur with repeated bites or injections of aqueous extracts of mos-

quitoes. Passive transfer of the immediate reaction has been demonstrated (9, 10). Seaman et al (U) attempted to produce protection in rabbits against mosquitoes by three weekly subcutaneous injections of 5 cc. of 1: 100 crude suspension of mosquitoes. They succeeded in producing sensitization rather than protection. Thus, it has been demonstrated that reactions to mosquito bites can be allergic and based on specific sensitization. Gordon and Crewe (12), however, state that the late reaction is due to irrita-

tion. As evidence, they state that the biopsies of 24 hour reactions to Aedes aegypti in guinea pigs not previously bitten show distinct hemorrhages and swelling caused by separation of the collagenous fibers. * From the Department of Dermatology and Syphilology of the College of Medicine of the University of Cincinnati.

This investigation was supported by a research grant from the Division of Research Grants and Fellowships of the National Institutes of Health, United States Public Health Service.

Parts of the study were done through the cooperation of Dr. W. V. King, In Charge, United States Department of Agriculture, Agricultural Research Administration, Bureau of Entomology and Plant Quarantine, Orlando, Florida. Read by title at the Twelfth Annual Meeting of The Society for Investigative Dermatology, Atlantic City, N. J., June 7 & 8, 1951. 137

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It is our present purpose: (1) to determine if the reactions are always the result of sensitization or if irritation also plays a role (2) to further evaluate the types of allergic reactions that occur (3) to study cross sensitization among various species of mosquitoes, as it is important to know whether increasing injections of an extract of any one species of mosquito will desensitize to all species in the environment of the patient (4) to study aqueous mosquito extracts since extracts are of value in diagnosis and treatment of severe sensitivity reactions to mosquito bites as well as in further research concerning the mechanisms of the allergic reaction. MATERIAL AND METHODS

The common mosquito in the area around Cincinnati is Culex pipiens. Our work with mosquitoes, however, has been confined principally to two species, Aedes aegypti and Anopheles quadrimaculatus. Patients whose mosquito bite reactions were studied included newborn babies, workers at the United States Department of Agriculture, Agricultural Research Administration, Bureau of Entomology and Plant Quarantine, Orlando, Florida who had known exposures to specific species of insects, a group of persons who gave no history of severe reactions to mosquito bites, and patients with severe mosquito bite reactions. Our extracts have been sent to a group of allergists throughout the country for evaluation. Their records, however, are not available and are not included in this report. In order to attempt to standardize the bite reaction, the insects were always allowed to fill. Tracings of several bite reactions were made when comparing the reactions in sensitive patients before and after a medication. This was necessary because it was observed that two simultaneous bite reactions on the same individual on similar areas often vary. All intradermal tests were done by the injection of approximately 0.02 cc. of extract w-ith a sharp 26 or 27 needle. 0.25 cc. of extract was used for patch tests. Animal studies were done to observe the reactions in animals not previously exposed to mosquitoes and to attempt the development of anaphyactic shock with mosquito extract. The toxicity of dust extract, mosquito extract, and bee venom on tissue cultures of embryo chicken hearts was compared. The United States Department of Agriculture, Agricnltural Research Administration, Bureau of Entomology and Plant Quarantine, Orlando, Florida furnished supplies of dead mosquitoes for the preparation of extracts. These dead and dry insects were put in the icebox when received and the extracts prepared as soon as possible. Our 1:10 stock extracts of Aedes aegypti and Anopheles quadrimaculatus for skin testing and desensitizing patients were prepared as follows: One grain of dried mosquitoes was added to 5 cc. of freshly made Coca's solution. This mixture was ground in a ball mill for 1—2 hours and then allowed to extract in the icebox for 1 day with intermittent shaking. The mixture was then filtered, the p11 adjusted to approximately 7.0 (mosquitoes are acid), and Seitz filtered. An equal part of sterile glycerine was added as a preservative and the extract cultured for sterility. Extracts without the addition of a preservative were prepared for tissue culture, animal work, and patch tests.

INSECT BITE REACTION

139

Preliminary work on the preparation of extracts was done as follows. The heat sensitivity was tested by heating Aedes aegypti extract to 100°C. for 1 hour and comparing the skin test reactions to the heated with the same concentration of non-heated extract. It was found

that heating diminished the diameter of the resulting wheal in sensitive patients about 50%. Benson (6) found his extracts to be thermostable when a few drops of acetic acid were added. It has been reported that preliminary defatting is necessary to prepare potent mosquito extracts (2). We prepared 3 A. aegypti extracts as follows for comparison. 1. The mosquitoes were first defatted with diethyl ether. The insoluble portion was separated by filtration, dried, and extracted in saline. One gram of mosquitoes (weight before defatting) was added to 100 cc. saline. 2. One gram mosquitoes was extracted in 100 cc. Coca's solution. 3. One gram mosquitoes was extracted in 100 cc. saline. The method was otherwise the same as used for preparing the stock extracts except that 1:10,000 Merthiolate was added and no glycerine. All factors such as amount of grinding and time of extracting were kept constant in the preparation of the above 3 extracts. When used in the same concentrations for skin testing, the 3 extracts were found to produce identical u-heals in sensitive individuals. (See figure 1.) Thus, it can be concluded that preliminary defatting is unnecessary. Perhaps when grinding is done by a less efficient method than the ball mill, preliminary defatting results in better wetting of the particles by the aqueous extracting fluid. Extracts for comparison of various portions of freshly killed mosquitoes were prepared by grinding the following in fine sand, extracting in the icebox for 1 day in saline containing 1:10,000 Merthiolate, neutralizing and Seitz filtering for sterility: 1. Salivary glands from 10 Anopheles quadrimaculatus per cc. saline 2. Abdomens from 10 Anopheles quadrimaculatus per cc. saline 3. Legs and wings from 10 Anopheles quadrimaculatus per cc. saline 4. Probosci from 20 A. aegypti per cc. saline 5. Legs and wings from 20 A. aegypti per cc. saline 6. Abdomens from 20 A. aegypti per cc. saline 7. Thoraces from 20 A. aegypti per cc. saline

8. 20 adult female A. aegypti per cc. saline 9. 20 adult male A. aegypti per cc. saline

Comparison of extracts 1, 2, and 3 by skin tests in sensitive individuals revealed that the largest reaction was obtained with the extract of Anopheles abdomens and very little reaction resulted with the legs and wings. (See figure 2) Comparison of extracts 4, 5, 6, and 7 by the same technic revealed that the largest reaction was again obtained with the abdomens and the smallest with the legs and wings. (See figure 3) Comparison of extracts 8 and 9 showed no difference between the female and the male mosquitoes (6). (See figure 4) Because the legs and wings and probosci of mosquitoes weigh less than their abdomens and thoraces, we prepared a w/v extract containing 10 mgs. A. aegypti legs and wings per 10 cc. saline (a 1:1,000 concentration). This was found to

produce wheals in sensitive individuals identical with those produced by a 1:1,000 extract of whole A. aegypti. One sensitive patient who reacted with an immediate wheal to 1:1,000,000 extract of whole A. aegypti reacted similarly to a 1:1,000,000 Aedes legs and wings. These tests confirm the earlier work showing

that the antigen or antigens are distributed throughout the entire body of the mosquito (13, 14).

As will be pointed out later, the mosquito extracts are toxic. Benson (6) has shown that the active fraction can be precipitated with cold alcohol, cold acetone,

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or ammonium sulfate. We prepared extracts containing the acetone precipitate and the acetone filtrate in the hope that the allergens and toxins might thus be separated. The final concentrations were based on the original weight of mosquitoes. Comparison of skin tests using 1: 1,000 Aedes extract, 1:1,000 Aedes

FIG. 1. Comparison of skin test reactions. A. 1:1000 A. aegypti extracted in Coca's solution B. 1:1000 A. aegypti extracted in saline C. 1:1000 A. aegypti defatted and extracted in saline (2 tests) D. Control.

acetone ppt., and 1:1,000 Aedes acetone filtrate revealed that most of the material causing both the immediate and the late reactions was precipitated with 88% acetone. RESULTS

Toxicity study Aedes aegypti extract was compared with a saline extract of dust and 'Lyovac'

Bee Venom Solution* for toxicity in tissue culture. Eight-day chick embryo *

Sharp & Dohme's product—i cc. represents 10 bee stings.

INSECT BITE REACTION

141

heart tissue was cultured in hanging drop slides with a chick embryo extract and chick plasma clot. Various dilutions of the extracts were added directly to the clot, and outgrowth readings were made at 48 hours. Aedes inhibitory concentration was between 1:40 and 1:80 of the total clot. Dust inhibited out-

Fin. 2. Comparison of skin test reactions. A. Legs and wings B. Abdomens C. Salivary glands.

growth between 1:24 and 1:40; Bee Venom between 1:6400 and 1: 12800 of the prepared solution. These results parallel the clinical observation that bee stings are more toxic than mosquito bites. Animal studies

Rabbits used daily for feeding a colony of Aedes aegypti showed only slight erythema following A. aegypti bites. Two rabbits not previously used for feeding

mosquitoes reacted with definite immediate and late 2—4 mm. papules. The caretaker of the mosquito colony stated that this reaction gradually disappears.

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Thus a resistance to the primary irritation develops. Guinea pigs not previously exposed to mosquitoes also reacted with immediate and late 1—4 mm. papules

following Aedes aegypti bites. These observations indicate that reactions to mosquito bites due to primary irritation are possible and confirm Gordon and

d• Tell

•

.'

Crewe's observations.

FIG. 3. Comparison of skin test reactions. A. Probosci B. Legs and wings C. Abdomens D. Thoraces.

Animal experiments were started in an attempt to develop anaphylaxis in guinea pigs with mosquito extract. Our results are reported to exemplify that the extracts are toxic. Two guinea pigs were given 4.5 cc. of 1:1000 A. aegypti extract I. P. and two, 4.5 cc. 1: 100, Twelve days later these 4 pigs and 4 controls were given 0.2 cc. 1: 100 A. aegypti extract IC. The controls as well as the supposedly sensitized pigs developed anaphylactoid symptoms consisting of rubbing the nose, sneezing, and dyspnea. One control and one sensitized pig died.

INSECT BITE EEACTION

143

Eight guinea pigs given 3 cc. of 1:10 Anopheles extract I.P. died in 24 hours, as did a pig given 3 cc. of 1: 10 concentration of the acetone ppt. of the Anopheles

extract. A pig given 3 cc. of a 1: 10 concentration of the acetone filtrate, however, survived.

A

B

Fro. 4. Comparison of skin test reactions. A. Female mosquitoes B. Male mosquitoes C. Female mosquitoes.

Sprent et al (15) has demonstrated that a substance causing anaphylactoid symptoms in worm-free guinea pigs is associated with the tissues of Ascaris lumbricoides. He attributed this to non-specific protein shock—an effect \vhich is possibly common to many invertebrate proteins. He succeeded in causing anaphylactic shock in sensitized animals with deproteinized ascaris material. It is also possible that the toxicity of our extracts is due to protein breakdown products since mosquitoes undoubtedly have enzymes and the grinding in the preparation of the extracts was done at room temperature. A suitable technic for producing anaphylaxis with mosquito extract may

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offer a method for comparison of various species of mosquitoes. Plans are being made for employing the Schultz-Dale Technic. Bite reactions in newborn babies

A. aegypti fed from the forearm of 10 newborn babies. In 4 a 1—2 mm. petechial reaction was noted immediately. Late readings were possible in 9 cases, and 6 of these showed 1—2 mm. red papules.

Studies in individuals with known exposures to specific species of mosquitoes

At the U. S. Department of Agriculture, Agricultural Research Administration, Bureau of Entomology and Plant Quarantine, Orlando, Florida, a group of entomologists and technicians with known reactions to A. aegypti and Anopheles quadrimaculatus were tested with A. aegypti and Anopheles quadrimaculatus extracts. It was felt that this group offered a good opportunity for evaluating the extracts. Four gave histories of reactions which vary with the species of mosquito. See Table 1. The skin test reactions on the whole correlate with the histories. Patient 7, however, who had no history of late reactions to mosquito bites developed a 24 hour reaction to the extracts.

Six entomologists and technicians with known exposures to A. aegypti, Anopheles quadrimaculatus and the saltmarsh mosquitoes were tested with actual insect bites. The results are summarized in Table 2. Patient 4 reacts to the two species of Aedes to which he has been exposed and gives no reaction to Anopheles quadrimaculatus to which he has not been exposed. Patient 2 shows varying reactions to the 4 species of mosquitoes. Thus, differences between the mosquitoes is illustrated. Study of persons with mild or no mosquito bite reactions

Fifty seven persons of varying ages, most of whom had spent the greater part of their life in Cincinnati, were tested with an A. aegypti bite and intradermal tests with the following extracts: 1:1,000 A. aegypti, 1:10,000 A. aegypti, 1:10,000 Anopheles quadrimaculatus, dust, and control (1% glycerine in saline). Thirty eight of these persons were also tested with an Anopheles quadrimaculatus

bite. There were 13 one plus reactions to the dust extract but none to the control. Both immediate and late reactions were recorded in all cases. (See Table 3) It will be noted that: 1. There is little correlation between the reactions to A. aegypti and Anopheles quadrimaculatus bites. 2. Reactions to A. aegypti were more common than to the Anopheles bites. Entomologists (16) state that the tribe Aedini are often included with the tribe Culicini (which includes the

Culex, found in the Cincinnati area). 3. The bite reaction decreased with increasing age (3). 4. The immediate reactions to insect bites do not correspond exactly with the whealing response to the extracts, but there is good correlation. 5. Late papules resulted from the injection of the extracts when none occurred after the mosquito bites. Whether these reactions to the extracts are due to primary irritation or whether the allergenic content of 0.02 cc. of the mosquit> extract is greater than in the material deposited by 1 mosquito is not known.

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INSECT BITE REACTION

Studies of severe reactions to mosquito bites

S. M., a 4 year old white boy, was seen because of lichen urticatus which occurred after the family obtained a dog. He gave a history of episodes of "severe swelling" in the summer. TABLE 1 Results of skin tests with mosquito extracts RESULTS OF SKIN TESTS HISTORY

Aedes aegypti Quaitus Mixturet 1mm.

1

I

1mm.

reaction to A. aegypti. Slight

imm. reaction to Anoph. 2

Late

Late

1mm.

Late

+

Immediate wheal. No difference be- ++ tween mosquitoes. Immediate 1 cm. when!. No difference between species. Severe immediate reaction to all mosquitoes.

1mm.

+

+

+

+

+++

7 1mm. reaction to A. aegypti and

+

Anoph. No reaction to saltmarsh mosquitoes.

+++++

*

++

Mosquito bites last 2—3 days. No +

+

3 4

5 No reaction to saltmarsh mosquitoes. Slight reaction to A. aegypti.

+

6 No reaction to saltmarsh mosquitoes. Slight wheal for a few minutes when bitten by A. aegypti and Anoph.

8

Late reaction to A. aegypti. Slight reaction to Anoph.

9

species difference. 10 11 12

Mosquito bites usually last 3—7 days. No species difference. Aedes and Anoph. reactions last 1—2 days and are about equal.

—

+' —.

——+ + —

+ Immediate wheal to all mosquito ++ —

+

+

++ +

—

++

bites.

* 1:1000 dilution of extract. All other extracts 1:10,000 dilutions.

Equal mixture of 1:10,000 Aedes aegypti and Anopheles quadrimaculatus extracts. (Obtained from Arlington Chemical Co.) Entomologists or technicians at the Bureau of Entomology in Orlando, Florida. A. aegypti = Aedes aegypti Anoph. = Anopheles quadrimaculatus Family history revealed that a sibling also had lichen urticatus and the father developed severe poison ivy every summer. An A. aegypti was allowed to bite the bo with a resulting immediate 1 cm. wheal and an 18 hour reaction 5 inches in diameter. Results of skin tests were as follows: There were no reactions to bedbug, dust, and chigger extracts: and no immediate reactions to A. aegypti and rat flea extracts. The late reaction to 1:100,000 rat flea extract was 1 cm. in diameter; and thelate reaction to 1:1,000,000 A. aegvpti extract was

Bitten once or twice a month by A. aegypti. Ex-

1

= Aedes

= Wlteal

w.

e. = Erythema

Anoph. = Anopheles qundrimaculatus pap. = Papule

A.

Code:

6

5

4

3

I

posed constantly to Anoph. Exposed for 3—4 months to the saltmarsh mosquitoes. Exposed constantly for 5 yrs. to A. aegypti. Bitten for 2 mos. in the summer of 1950 by the salt-marsh mosquitoes but not exposed for 6 mos. Bitten intermittently 6 mos. of the yr. for 5 yrs. by Anoph. Heavy exposure to A. aegypti for the last month. Possibly a brief exposure to Anoph. over 1 yr. ago. Heavy exposure to A. aegypti until 1 mo. previously. Heavy intermittent expnsure to the saltmarsh mosquitoes.No previous exposure to Anoph. Exposed intermittentlyto A. aegypti and Anoph. Exposed every summer to the salt-marsh mosquitoes. Constantly exposedto A. aegypti. Exposed6 mos. previously to Anoph, and the saltmarsh mosquitoes.

HISTORY

PT.NO.

2

TABLE 2

0

mm. pap. 1

0

Late

2 mm. pap.

0

em. Small pap. 1.8 em. e. 1 em. 3 enm. w. pap. w.

em. e.

.95

1

.

pap.

2 mm.

0

Early

A. AROYPTI

w.

0

0

Late

pap.

1 em. 2 em.

0

0

Early

A. TAeNIORHYNrHIJS

Comparison of bile reectioe with exposure

0

1.2 em. w. 2.3 em. e.

w.

2 em.

0

Early

0

mm. pap. 1

0

0

Late

A. SOLLICeTA\5

w.

em.

0

3 em. v.

0

e.

2 mm.

1

0

Early

0

pap.

2+ mm

0

0

mm. pap. 1

0

Late

_____

ANOPII.

6

80

100

48

0

75

25

5

5

23

1

1

12

w.

nag.

= negative = wheaT pap. = papule

Code:

1

100

2

neg.

neg.

Early

Aedes

6

6

mac.

nag.

pap.

neg.

Late

'IV.

pap.

mac.

nag.

neg.

neg.

nag. mm. w.

cm.

nag.

w.

mm.

4w.

2 mac.

1

1—5

2 mae.

nag.

2—i

w.

1—2

9

1-1 mm. w.

3

4

4 nag. 12 cm. w. 2 nag.

pap.

mac.

6

1

pap.

nag.

2 pap.

1

pap.

8

mac.

3 9

4

8

nag.

pap.

mac. 11

1

mac. 2 pap. 1

1

mac.

1 mac. 22 pap.

nag. pap. pap. 2 3

pap.

Late 2 rieg. 3 pap.

1:000Aedes

+ to +++

+

nag.

—

+

+

15 neg.

1

3 neg.

neg.

neg.

2 rieg. 2 mac.

neg.

neg. pap. pap.

Early

4 neg. 1 — + 2 neg. 3 — +

4

3 2 2

Late

mac. = macule Aedes = Aedas aegypti Anoph. = Anophelas quadrimaculatus

w.

+ mm.

w.

+ mm.

+

mm.

w.

Anoph.

Early

REACTIONTO BITES

to 5 mm. pap.

w.

ito 5 mm. w. ito 5 mm.

80

OLD

40 yE YRS

5

NO. PrS.

% I'TS.

TABLE 3

5 7

1 3

+

++

nag. + to

nag.

—

+

+

neg.

—

15

8

+

neg.

1 —

4

neg.

neg.

neg.

Early

pap.

mac.

nag.

mac.

lOpap.

1

2 pap. 1 nag.

1

1

mac.

21

1 nag. 1 mac.

nag. pap. pap.

2 3

3

neg. pap. pap.

2

Late

1:10,000 Aedes ______________________

REACTIONTO EXTRACTS - ______________

Comparison of mosquito bile and skin test reactions

6 6

+

—

+

neg.

neg.

nag.

—

16 neg.

7

neg.

neg.

neg.

neg.

Early

pap.

3 nag. 9

1 nag. 3 pap.

mac.

19

2 nag. 3 pap. 1 nag. 1 pap. 2 neg. 3 pap. 1 mac. 4 pap. 3 neg. 1 mac.

Late

1:10,000 Anoph.

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5 mm. in diameter. These late reactions are larger than any in the group summarized in Tables 1 end 3. Suggestive evidence of the presence of blocking antibodies in the serum of a man completely desensitized to mosquitoes (the caretaker of a colony of mosquitoes) was obtained by partially blocking the late reaction in this patient. (See Table 4) Control serum was obtained from a man who gave moderate early and late reactions to mosquito bites. Both sera were heated to 56° for 3 hours to destroy the roagin. In these tests 0.02 cc. was injected

ID.

Two siblings, aged 6 and 9, gave histories of severe mosquito bite reactions. One also gave a history of severe reactions to chigger bites. Family history revealed that many members of the maternal side of the family bad eczema of the hand. One sibling gave an immediate wheal to 1:100,000 A. aegypti extract but not to 1:1,000,000 extract. The other reacted only to 1:10,000 A. aegypti extract. During the summer of 1950, both were put on a twice weekly program of hyposensitization starting with 0.05 cc. of 1:1,000,000 A. aegypti extract administered S. C. and working up to a maintenance dose of 0.8 cc. of 1:10,000 concentration. On one occasion no injections were given during a period of 10 days and one

TABLE 4 Blocking entibodzes in octively sensitized skin EXTRACT

1:100,000 Aedes aegypti extract Equal parts 1:50,000 Aedes aegypti extract and C serum* Equal parts 1:50,000 Aedes aegypti extract and B serumf Equal parts saline and B serum 1:1,000,000 Aedes aegypti extract Equal parts 1:500,000 Aedes aegypti extract and C serum Equal parts 1:500,000 Aedes aegypti extract and B serum

LATE REACTION

12 mm. papule 1 cm. papule 3 mm. papule Negative 5 mm. papule 4 mm. papule 1 mm. papule

* C Serum = Serum from control patient t B Serum = Serum from desensitized patient severe reaction occurred consisting of edema of half the face. However, as long as injections were administered twice weekly, the children developed only small papules from mosquito

bites. This demonstrates that A. aegypti extract can be used to reduce reactions to Culex bites. Benson (4, 6) found that a relatively small number of injections of mosquito extract desensitized 5 of 6 patients. J. J., a 5 year old white boy, gave a history of intermittent areas of induration particularly in the late summer and fall for the past 4 years. lie was seen in November, 1948 with a diagnosis of questionable scabies, and in June, 1949 with a diagnosis of lichen urticatus. He was admitted to Cincinnati General Hospital in September, 1949 with a red hot swollen ear. The patient was first seen by the authors on August 15, 1950 with a rectal temperature of 101°F., swelling under the left eye, and indurated lesions 2k inches in diameter. Some of the lesions had central vesicles. Anopheles quadrimaculatus bites at this time produced a late papule 1 inch in diameter. The patient was seen again on September 12, 1951 with a rectal temperature of 100°F., a P.H. of 142/mm., and 4 inch diameter indurated lesions with central vesicles. At this time the reaction was reproduced by the bites of Anopheles quadrimaculatus. (See Figure 5) The child was again seen in November, 1950, with similar lesions. Other findings included a 10% eosinophilia and generalized lvmphadenopathv which have

persisted for 6 months. His stool was repeatedly negative for ova and parasites. The Allergy Clinic has obtained positive reactions to intradermal tests with dust, banana, lettuce, white potatoes, and wool. Family history reveals that two siblings have been treated for worms, and that an uncle develops hives from certain foods.

INSECT BITE REACTION

149

His reactions to mosquito extracts administered intradermally were: positive immediate reactions to 1:1,000,000 A. aegypti, and to 1:100,000 Anopheles quadrimaculatus; positive

late reaction to 1:100,000 Aedes aegypti. He also reacted to patch tests of both 1:10 A. aegypti and Anopheles extracts with vesicles, edema, and erythema. 1:100 Aedes extract administered at both poles by iontophoresis (kma per sq. in.) for 15 mm. resulted in late follieular papules at the positive pole. The child was admitted to Cincinnati General Hospital in February, 1951. He had a lung infiltrate which was possibly Loeffler's pneumonia and cleared with no therapy. On his first day of admission, at which time he had not been exposed to mosquito bites for over 2 months the effect of 6 A. aegypti bites on his pulse rate and WBC was studied. There was no

significant change. Sodium bicarbonate was then administered until his urine became al-

FIG. 5. Heaction to Anophcles quadrimaculatus. kaline, but there was no change in his mosquito hite reactions. 75 mg. of Cortone were in-

jected 1 hour before A. aegypti bites, and 30 minutes later an additional 25 mg. were injected. The immediate, 6hour, and 24 hour reactions were not altered. On this admission it was noted that the immediate reaction to A. aegypti was slightly larger than the reaction to Anophclcs quadrimaculatns, with the reverse true of the late reactions. Heactions to both mosquitoes were smaller than had been noted 2—3 months previously. Apparently some spontaneous hyposcnsitization occurred during the months of no exposure to mosquito bites. J. L. is now on a program of hyposensitization with the insect extracts. After 6 weeks of twice weekly injections his late reaction to A. acgypti was smaller than in February, 1951. Serum from the above patient was used to attempt passive transfer of sensitivity to A. aegypti bites to 10 persons with no reaction or only 1 mm. immediate reactions. The technic consisted of the lID. injection of 0.1 cc. of J. L.'5 serum in 2 places and control serum (obtained from a person with no reaction to A. acgypti bites) in 1 place. Twenty four hours later

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A. aegypti were allowed to fill from3 spots: one injected with J. L's serum, one injected with

control serum, and an nninjected area. In only one of the 10 persons was passive transfer successfol. It was repeated fonr times on this patient with consistently positive results in 24 hours but only questionahle rosults in 72 hours. S. 0., a 9 year old white boy, gave a history of papulo-vesicular reactions to the mosqnitoes around Cincinnati. Family history reveals that the father has hay fever and a sibling has bad recurrent nrticaria. Bites of A. aegypti and Anopheles quadrimaculatns resulted in immediate wheals to both, with a larger reaction following the Aedes bite. Tbe late reactioii to A. aegpti was a 6mm. papule with vesicles, that to Anopheles, a 2 mm. papnle. Reactions to intradermal tests with the extracts were: an immediate wheal following 1:1000 and 1:10,000 A. aegypti, a late papule with vesicles from 1:100(1 Aedes, a late 6 mm. papule from 1:1,000,000 A. Aegypti, no immediate reaction and only a small papule from 1:10,000 Anoph-

des quadrimaculatus. Patch tests with 1:10 and 1:100 Aedes extract were positive with erythema and vesicles, and a patch test with 1:10 Anopheles quadrimaculatus n-as only slightly positive with papules hut no vesicles. Patch tests in 7 controls (persons who do not develop vesicles from mosquito bites) with 1:10 Aedes extract were negative. V. A., a 28 year old colored female with subacute disseminated lupus erythematosus, gave a history of severe reactions to mosouito bites and hives from bedbug bites. No other allergic history n-as elicited. She reacted to an Aedes bite with an immediate wheal one inch in diameter and swelling of the entire arm below- the elbow- 6 hours later. Her reaction to Anopheles quadrimaeulatus nas a 2 mm. inimediate wheal and a smaller late reaction than followed the Aedes bite. There was an immediate and late reaction to 1:100,000 Aedes extract. When receiving 60 mg. Cortone per day, the immediate reaction to A. aegypti was

unchanged, hut the late reaction became less pronounced. Three other patients n-ho were given Cortone for various reasons gave only mild mosquito bite reactions before therapy was started. While receiving Cortone their mosquito bite reactions did not change significantly. Tno other patients, however, did show- decreased mosquito bite reactions while receiving ACTH.

V. A. n-as given 0.5% Decapryn by iontophoresis for 15 mi ( ma per sq. in.). The

mosquito bite reactions in this area were not significantly different than in a control area.

Similar results n-crc obtained in other patients. Oral antihistamines (Pyrihenzamine, Benadryl, Neo-antergan) in doses of 50 mgs. four times a day were each administered to three patients, n-ho gave immediate wheals and about 5 mm. late papules to A. aegypti bites. There was no objective change in the bite reactions, although decreased pruritus n-as noted. \T A. was given 1:1000 A. aegypti extract by iontophoresis ( ma per sq. in.) for 15 mm. both at the positive and the negative poles n-ith saline at alternate poles. Follicular papules n-crc noted 2 days later in the area where A. aegypti extract n-as given at the positive pole. 1:100 A. aegypti extract was then similarly administered. Immediate follicular wheals and late papules were noted n-here A. aegypti extract n-as given at both the positive and negative poles. Similar studies were done on another patient who reacted to A. aegypti wIth 3 inches of sn-elling about 6 hours after the bites. In this ease, the immediate follicular n-heals

were more prominent at the positive pole and the late reaction more pronounced at the negative polo. Lenkoeyte cultures with the addition of mosquito extract were done as a test for bacterial type hypersensitivity through the cooperation of Dr. II. Blatt (17, 18). Two different samples of leulcocytos from both V. A. and 4. L. were necrotized by A. aegypti extract in a concentration that did not neerotize the WBC of controls, i.e., patients with mild mosquito bite reactions. Histopatholoçjy

Figure 6 represents biopsies of A. aegypti bite reactions in n 52 year old white

male with moderate early and late reactions. The biopsy 30 minutes after the

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Fin. 6. Moderate mosquito bite reactions. H and E. X 160. A. 30 mm. reaction B. 6 hour reaction C. 2 day reaction D. 5 day rcactioo.

bite shows edema of the upper eorium and the beginning of a perivaseular infiltrate of polymorphonuelear leukoeytes, eosinophils, lymphocytes, and plasma Cells. In 6 hours there is persistence of the edema and an increased perivaseular

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FIG. 7. Mild and severe mosquito bite reactions H and F. A. 1 day reaction X 160 B. 4 day reaction >< 160 C. 3 day reaction X 100 D. 3 day reaction X 650.

infiltrate. The 2 day biopsy shows persistence of the edema but decreased infiltrate which consists of an increased percentage of lymphocytes and histiocytes.

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In 5 days there is a decrease of the edema and infiltrate with a further increase in the proportion of lymphocytes and histiocytes. Figure 7a represents a minimal 24 hour reaction to A. aegypti. It shows the point of insertion of the proboscis and a perivascular infiltrate composed mainly of lymphocytes with an occasional eosinophil and polymorphonuclear leukocyte The density of the infiltrate is much less than in Figure 6. Figure 7b is the biopsy of a 4 day mosquito bite in a patient who develops a late papulovesicular reaction. There is marked subepidermal edema (with vesicle formation in other sections), and a dense perivascular infiltrate composed mainly of lymphocytes with an occasional eosinophil. Figures 7c and 7d represent the biopsy of a 3 day reaction to Anopheles quadrimaculatus in patient J. L. The infiltrate is dense, extends deep into the subcutaneous fat, and is composed of eosinophils (10, 19), polymorphonuclear leukocytes, lymphocytes, plasma cells, and histiocytes. Thus, the severe reactions differ from mild reactions in that the infiltrate is denser, more extensive, and contains a greater proportion of eosinophils and polymorphonuclear leukocytes. This suggests that the mechanism of the development of severe and mild bite reactions is similar with a quantitative difference in degree of sensitivity. DISCUSSION

The allergic reactions that develop following mosquito bites are a complex problem which is far from solved. We have observed that immediate and late reactions to mosquito bites may be due to primary irritation. These reactions due to irritation are small and gradually disappear with regular exposure. The immediate and late reactions that develop in humans following exposure appear, however, to be allergic in nature. The usual pattern is believed to be: no reaction —+ late reaction —* immediate and late reaction —* disappearance of late reaction —* disappearance of immediate reaction (2, 20, 21). This pattern is not always followed (2). One of the authors (P. J.), e.g., gave oniy an immediate reaction to A. aegypti at the onset of our work but now develops both an immediate and late reaction. There also is apparently a cumulative effect as one of the authors (E. R.) develops a late reaction to A. aegypti only when bitten by many mosquitoes. That most people with exposure to mosquitoes develop a bite reaction suggests that allergic reactions are ubiquitous. Only an occasional person, however, develops a severe reaction resembling cellulitis with or without vesicles. A high percentage of these latter patients (4, 6, 10) give a personal and/or family history of other allergies. Three types of allergic reactions to mosquito bites occur clinically: urticarial,

tuberculin, and eczematoid. These reactions are duplicated with patch and intradermal tests using crude aqueous mosquito extracts. Passive transfer con-

firms the urticarial type hypersensitivity. Hecht was able to demonstrate passive transfer antibodies in one, but not in all, of his patients with severe reactions to mosquito bites and therefore concluded that not all persons with severe reactions have circulating antibodies. Our experience suggests that ade-

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quate receptors for passive transfer of sensitivity to mosquitoes are difficult to

find. Many persons do not react to mosquito bites because they have been hyposensitized by exposure. Perhaps a blocking mechanism is developed which prevents passive transfer of sensitivity.

The necrotizing of leukocytes from hyper-reactive patients with mosquito extract in a concentration that does not necrotize controls is supportive evidence for the tuberculin type hypersensitivity. The occurrence of vesicles following mosquito bites and intradermal and patch tests with mosquito extracts, and the presence of spongiosis and vesicles in biopsies of mosquito bites support the view that eczematoid reactions follow mosquito bites.

Our work with blocking antibodies is inconclusive. We attempted their demonstration on actively sensitized skin. In only one case the wheal from the non-specific irritation due to the serum disappeared quickly enough to allow us to read the immediate skin test reaction and in this case, there was no blocking. Suggestive evidence of blocking the late reactions was pointed out. Blocking antibody studies should be done on passively sensitized skin, but unfortunately, the one adequate receptor for passive transfer of mosquito sensitivity that we have found is no longer available. Benson (6) found that extracts and bites of 3 different species of mosquitoes

were interchangeable. Hecht (8, 9), Mellanby (20), and Heilesen (2) cited persons with differences in reactions to different species of mosquitoes. We saw numerous examples of this. On the other hand, some of our patients who presumably had never been previously bitten by A. aegypti and Anopheles quadrimaculatus reacted to these species; and our A. aegypti extract proved adequate for hyposensitization to the mosquitoes around Cincinnati. A possible explana-

tion of this conflicting data is that multiple allergens are involved, some of which are species specific.

That two of the patients with severe reactions to mosquito bites had been diagnosed by dermatologists as having lichen urticatus suggests that in some instances there is a relationship between lichen urticatus and insect bite reactions. Further studies of patients with lichen urticatus are being done. That the crude aqueous mosquito extracts are toxic is demonstrated by the animal experiments. In our experience, however, an immediate and/or delayed reaction to 1:100,000 or 1:1,000,000 mosquito extract administered I.D. or a positive patch test to 1:10 or 1:100 extract is suggestive evidence of hypersensitivity to mosquito bites. That the I.D. administration of aqueous mosquito extract should exactly duplicate the reactions to mosquito bites is not to be expected, as the mosquito proboscis is inserted deeper than just below the dermis. SUMMARY

1. Small mosquito bite reactions can result from irritation alone. 2. Three types of allergic reactions follow mosquito bites: urticarial, tuberculin, and eczematous.

3. A high percentage of persons with severe mosquito bite reactions give personal and/or family histories of other hypersensitivity reactions.

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4. There is evidence that multiple allergens are involved in the mosquito bite reaction.

5. Extracts of various portions of the mosquito produce similar reactions in sensitive individuals. 6. Our aqueous mosquito extracts are toxic in high concentration, but weaker concentrations are of value in the diagnosis and treatment of severe mosquito bite reactions. 7. Adrenocorticotropic hormone in 2 patients decreased bite reactions more than Cortone in 5 patients. Antihistamines and alkalinization did not significantly alter the bite reaction. REFERENCES 1. BRowN, E. A.: Insects and allergy, Ann. Allergy, 2: 303, 1944. 2. HEILESEN, B.: Studies on mosquito bites, Acta Allergol., 2: 245, 1949. 3. MARTINI, E., citation by Hecht, 0.: Uber Insektenstiche, Dermat. Wchnschr., 88: 839, 1929.

4. BENSON, R. L.: Diagnosis of hypersensitiveness to the bee and to the mosquito, Arch. mt. Med., 64: 1306, 1939. 5. BODE, H. G.: Zur Kenntnis Atypischer Muckenstichreaktionen, Dermat. Wchnschr., 96: 7, 1933.

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