The Journal of Asthma Research. Vol. 13. No. 4, July. 1976

Fire Ant Sensitivity?

FRANK K. JAMES, JR . , COLONEL, M.C., U.S. ARMY*

Hypersensitivity to stings of winged Hymenoptera have been described since antiquity. Severe and sometimes fatal reactions to these stings have been described in this country for well over 150 years. At the present time, i t is estimated that over 40 deaths per year are being recorded in the United States. Deaths from Hymenoptera stings have been described now in almost every state in the Union.

Fire ants, representing a small group of the Myrmicinae, are of considerable medical and economic importance. The two imported species of fire ants, Solenopsis invicta and Solenopsis richteri, entered the Gulf Coast area of the United States from South America approximately 35 and 55 years ago respectively. Since their entry into this country, they have rapidly migrated through seven states in the Gulf Coast area with a projected further spread in the next 30-40 years to involve almost the entire Atlantic and Pacific seaboard and via principal waterways to the midwest.

Damage to crops and land value loss in the Gulf Coast area has totalled millions of dollars per year, and the monetary loss from a medical standpoint is incalculable but also is felt conservatively to total several million dollars per year.

Although formic acid was isolated over 300 years ago, specific studies of importance in Formicidae (ants) have been undertaken during the past two decades. These substances known as pheromones are considered key elements in regulating social reactions occurring in ant colonies. They are classified as attractants, repellants such as formic acid, and venoms. Pheromones act as trail substances which are extremely important for blind ants, as warning substances for numerous ant species, and as defensive compounds.

Ant venoms have been studied in a comparatively small group involving the Myrmicinae. One type, as typified by the Australian bull ant, Myrmica gelosa, contains a proteinaceous venom which has a t least eight constituents of which histamine constitutes 2% of the dried secretions. In addition, hyalurina- dase, kinins, and a powerful phospholipase-A have been isolated. This venom closely resembles wasp venom.

By comparison, fire ant venom is hypoproteinaceous but has instead a series of 2,6-dialkylpiperidines in which there are cast variations (queens vs. workers), in addition to variations within a genus. This venom has numerous other unique characteristics including bacteriocidal activities against Staph aureus and E. coli, hemolytic, necrotic, and paralytic properties.

t Presented December, 1975, a t the meeting of the Entomological Society of America. * Assistant Chief, Department of Medicine, Brooke Army Medical Center, Fort Sam Houston,

Texas 78234.

179

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180 FRANK K . JAMES, JR.

Fire ant venom, in addition to the potential of triggering systemic reactions, usually causes a predictable sequence of events in the skin of man. This occurs in approximately 70% of individuals stung. The reaction following a sting starts with a classic wheal and flare usually occurring within a few minutes, followed by vesicle formation within 4-24 hours, and finally a sterile pustule. Complications from secondarily infected pustules have resulted in major surgical and medical problems requiring long term supportive care.

Extensive studies with the venom of various species of Solenopsis have yielded considerable information in this field. The two imported species are felt to inflict a far more painful sting, and their venoms are thought to represent what are considered to be significant evolutionary advances. This has resulted in a series of changes culminating in the more advanced venom being noted in those species felt to be most effective in their stinging action.

It has become evident during the past few years that studies of the venom of winged Hymenoptera have yielded more specific information on testing Hymenoptera-sensitive patients when compared to the previously available mixed stinging (whole body) insect antigen. It seemed appropriate, therefore, to study the fire ant venom, a truly unique toxin, causing a gamut of reactions in man, including fatal and/or severe anaphylactic reactions. It also presented a unique opportunity to study both the whole venom and its component parts.

Materials and Methods Venom was collected from the workers of both imported species by the

method of milking described by Blum. The abdomen was dissected free and held by a remnant of the thorax. The abdomen was then gently stroked with a .5 lambda pipette affixed to a cork. Venom would promptly appear a t the tip of the extended stinger. Approximately .015 lambda was contained in each drop (thought to be the average stinging dose), and i t was possible to elicit 4- 5 drops of venom from each invicta worker.

Raw fire an t venom was processed by adding a solution of 50% glycerin and normal saline and serially passing this mixture through millipore filters ultimately preparing a 1:400 volume per volume dilution. Each batch of venom was carefully checked for sterility. It was then diluted with standard aqueous allergy diluent to achieve appropriate skin testing concentrations. In addition, two of the synthesized alkaloid fractions, specifically the 2,gdisubstituted C13 and C15 piperidines representing the predominant alkaloids in the im- ported species were utilized in the test. Other agents were the whole body ex- tract of a mixture of Solenopsis invicta and richteri and their separate whole body extracts. The mixed stinging insect antigen (winged Hymenoptera) and standard diluent were likewise used in the studies. Each of these preparations was diluted with standard aqueous allergy diluent to achieve appropriate skin testing strengths.

The study group in San Antonio studied 25 fire-ant-sensitive patients who had a history of a systemic (anaphylactic) reaction. Hypersensitivity in this group of patients was documented by a clearcut history of a systemic reaction, previous positive skin testing, the patient’s recollection of the sting and

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FIRE ANT SENSITIVITY 18 1

progression of reactions, and the progression of skin responses following the sting.

A positive skin test was defined as a definite wheal and flare occurring within 20 minutes following the skin test in which the wheal was a t least twice the diameter of the control test done with diluent only. A wheal had to be 5 mm or greater in diameter to be considered positive. All of the extracts had been prepared a few days prior to testing of the first patient utilized in the study and were stored a t 56°C without apparent loss of potency. The skin testing material was utilized in the initial study over a period of approximately eight months following preparation. The results of these skin tests are shown

TABLE I Fire Ant Skin Testing

~~

ID Dilutions Scratch

Whole Body Extract Fire Ant Mix MSI S. invicta S. richteri

S. invicta aqueous S. invicta whole

S. richteri whole

Venoms

venom

venom A1 kaloids

Trans-2 Cis-2 Diluent

1:lOO 2 5

1:lOOO 5

11 8 6

1:40,000 4

13

1 : 10,000 8 1 2 9

1 : 400,000 4 6

1: 100,000 8 2

10 3

1:4 million 3 -

1:100,000 - - - -

1:4 million - -

4 4 4

Neg 6 3

- 1 : 4000

7 2

3 1 4 11 5

12 17 19

6 6 3

3

1 -

2 1 1

TABLE I1 Fire Ant Skin Testing

Scratch Controls ID Dilutions

1:1000 1:10,000 - 3

1 2 2

1 :40,000 1:400,000 1 1 2

- -

-

-

Whole Body Extract Fire Ant Mix MSI S. invicta S. richteri

S. invicta aqueous S. invicta whole

S. richteri whole

Venoms

venom

venom Alkaloids

Trans-2 Cis-2 Diluent

1: 100 3 3 3 4

1 : 4000 4 3

1: 100,000 - - - -

1:4 million - -

1:100,000 - - - -

1:4 million - -

11 2

10 9

13

4 3 2

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182 FRANK K . JAMES, J R .

TABLE 111 Comparison Between 96 RAST & S k i n Test Whole Body Extract & Venom of Solenopsis Inuicta

Whole Bodv Extract Venom

'7 RAST Skin test % RAST Skin test

J . N. C. J. C. M. C. W. G. S. G. R. J . R. K . T. K. M. M. J. M. E . M. B. 0. T. P. w . s. N. W. G. W. K . L. R. P.

13.5 3.1 3.6

32.8 23.1 10.7 8.5

10.8 4.8 5.2 3.8 3.8 8.6 4.1 5.0 5.0 3.5 5.4

13/18 positive

10,000 1,000

100,000 1: 100,000 1:100,000

10,000 1: 10,000

1: 100,000 1: 10,000

1,000 1:10,000

1,000 1: 10,000

1:1,000 10,000

1,000,000 10,000

1.000

38.0 8.2

14.9 51.5 51.6 19.9 40.4 40.0 10.9 38.2 17.4 15.7 40.2 13.7 17.6 27.1 13.3 23.5

16/18 positive

400,000 I :40,000

40,000 400,000 400,000 40,000

1:4 million 1:4 million

1:40,000 40,000

400,000 40,000 40,000 40,000 40,000 40,000

1 : 40,000 1: 400.000

RAST is expressed as 1 of total radioactivity added to each tube. WBE controls, Mean = 3.1 + S.D. of 0.5; two S.D. = + 1 = 4.1 (upper limits of normal). Venom controls, Mean = 8.0 * S.D. 1.6; two S.D. = f 3.2 = 11.2 (upper limits of normal). Total radioactivity count = 182,676.

in Tables I, 11, and 111. In addition to the standard skin testing technique, 18 of the most highly sensitive patients were also evaluated utilizing the RAST technique. In this, both the fire ant venom and whole body extracts were utilized as antigen, combined with the patient's sera and 1-125 anti IgE. These were compared with their skin test sensitivities previously noted, and are depicted in Table 111. It should be emphasized in the latter study that 13 of 18 patients studied with whole body extract and 16 of 18 patients studied with venom had significantly elevated RAST findings as manifest by two standard deviations above the mean.

Finally, passive transfer was successfully accomplished utilizing sera from three highly sensitive patients who had negative histories for systemic disease and who had been screened for serology and hepatitis-associated antigens. Their sera were cultured for study. Three nonatopic, non-fire-ant-sensitive volunteers were utilized in the study. Three sites on the skin on the nonatopic volunteers were challenged with 0.5 cc of the 1:400 volume per volume concentration of the venom of both invicta and richteri, the whole body extract of the imported fire ants, and the trans-stereoisomer (C15) of the synthesized alkaloid. The results of these studies gave positive wheal and flare reactions on the arms in which the unheated sera was utilized, whereas the corresponding skin testing surface where heated sera was utilized revealed negative skin

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FIRE ANT SENSITIVITY 183

tests. These tests strongly suggest reagenic activity in all three materials utilized.

Results The results of the skin testing technique utilizing whole body extract,

venom, alkaloids, and the RAST technique indicate that there are definitive and accurate methods for defining fire-ant-sensitive patients. These results concur with other groups who have studied patients utilizing standard skin testing technique of commercially available whole body extract of fire ants.

Our findings raise further intriguing questions since fire ant venom is hypoproteinaceous and has not been identified as having active enzymatic components such as hyaluronidase and phospholipase. Because the inoculum of the fire ant sting is approximately 1/100th to 1/1000th of the average honey bee sting, i t implies not only that the venom is potent, but represents a com- plete departure from any other known animal venom affecting man. Further studies are in process.

References 1. BARNARD, J. H. Studies of 400 Hymenoptera sting deaths in the United States, J. Allergy Clin.

2. LOFGREN, C. S., BANKS, W. A,, AND GLANCEY, B. M. Biology and control of imported fire ants,

3. BLUM, M. S., WALKER, J. R., CALLAHAN, P. S., AND NOVAK, A. F. Chemical, insecticidal,

4. CARO, M. R., DERBES, V. J. , AND JUNG, R. Skin responses to the sting of the imported fire ant

5. BRAND, J. M., BLUM, M. S., AND BARLIN, M. R. Fire an t venoms: intraspecific and inter-

6. BRAND, J. M., BLUM, M. S., AND Ross, H. H. Biochemical evolution in fire ant venoms,

7. BRAND, J. M., BLUM, M. S., FALES, H. M., AND MACCONNELL, J. G. Fire an t venoms: Compara-

8. LOCKEY, R. F. Systemic reactions to stinging ants, J. Allergy Clin. Immunol. 54:132, 1974. 9. RHOADES, R. B. , SCHAFER, W. L., SCHMID, W. H., WUBBENA, P. F., DOZIER, R. M.,

TOWNES, A. w., AND WtTTIG, H. J. Hypersensitivity to the imported fire ant: A report of 49 cases, J. Allergy Clin. Immunol. 56234, 1975.

Immunol. 52: 259, 1973.

Ann. Reu. Entomol. 20:1, 1975.

and antibiotic properties of fire an t venom, Science 128r306, 1958.

(Solenopsis saeuissima), Arch. Dermatol. 75:475, 1957.

specific variation among castes and individuals, Toricon I Z:325, 1973.

Insect Biochern. 3:45, 1973.

tive analyses of alkaloidal components, Toricon 10:259, 1972.

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