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Insect allergy—Preliminary studies of the cockroach
insect allergy-Preliminary of the cockroach
studies
Harry
M.D.,
S. Bernton,
M.D., and Halla
Brown,
Washington,
D. C.
F
or many years the role of insects as sources of both injectant and inhalant allergens has received much attention. l-4 A survey of these studies, however, reveals that the allergenic potential of the cockroach, a member of the family Bluttidae Orthoptera, has not been fully recognized. There are about 55 species of cockroaches in the United States, of which only seven or eight constitute a menace to indoor sanitation. Our studies were limited to the American cockroach, Periplaneta americana, and to the Oriental cockroach, Bktta orientalis.
MATERIALS Preparation
AND METHODS of extracts
Batches of live insects were stored in a deep freeze for 15 hours. The frozen bodies were crushed by mortar and pestle, extracted with ether, and dried at room temperature. Ten grams of the defatted residue was homogenized in a Waring Blendor in 100 ml. of Coca’s solution. The fine suspension was stored in the refrigerator for 1 week with frequent shaking. The extract was sterilized by Seitz filtration. Dialytic
fractions
Aliquots of the crude extracts of the two species of cockroaches were dialyzed in cellophane tubes against 3 volumes of distilled water, changed after 24, 48, and 72 hours, respectively. The combined dialysates from each species were lyophylized and redissolved in the original volume of 0.4 per cent phenol in From the Allergy Clinics of Freedmen’s Hospital, Providence Washington University Hospital. This study was supported by a grant from the John A. Hartford Presented at the Twentieth Annual Meeting of the American Feb. 13, 1964. Received for publication April 21, 1964.
506
Hospital,
and
Foundation. Academy
of
George Allergy,
Allergy
Volume Number
35 6
Table
I. Analyses of cockroach extracts Total nitrogen (w./ml.J
Preparation American Original Dialyzed Dialysate
roach 1: 10 extract residue
Oriental roach Original 1: 10 extract Dialyzed residue Dialysate
to cockroach
PNlJ
per
507
milliliter
1.08 0.10 0.84
52,000 7,700 28,000
1.27 0.059 1.03
52,000 4,400 29,000
order to restore the phenol lost from the Coca’s solution during the lyophylization procedure. The residue was dialyzed an additional 24 hours against running water and then dialyzed 24 hours against an equal volume of double-strength Coca’s solution. The volume change of the residue was insignificant. Table I records the total nitrogen and protein nitrogen units of the extracts of the two species of cockroach. EXPERIMENTAL Survey of skin
sensitivity
to cockroach
In our survey to determine the incidence of skin sensitivity to the cockroach, two groups were employed for testing. Group 1 consisted of 253 normal and apparently healthy persons, selected from the lowest social and economic strata of our population. Group 2 consisted of 114 patients, classified as allergic, who reported to our clinics for treatment. Thus, a total of 367 individuals was tested. For practical reasons, only the intracutaneous test was performed on sixty-four subjects in the younger age group (to age 16). A solution of the American roach was administered to fortyfour children and that of the Oriental to twenty others. As a result of many exploratory tests, a volume of 0.025 ml. of a dilution of 1 :lO,OOO was found adequate for this purpose. Readings were taken in 15 minutes. The remaining 303 subjects were tested cutaneously with extracts of both the American and Oriental cockroaches. Solutions of the extracts were adjusted in normal saline solution so that each milliliter contained 50,000 PNU. Two or three drops of these concentrations and of a negative control solution were applied to scratch marks on the anterior surface of the lower arm. Vigorous typical reactions with wheals, pseudopodia, and extensive areas of areola developed in the positive reactors within one-half hour of observation. Table II records the incidence of skin response of group 1 to cockroach extracts. The results show that 7.5 per cent of the members of this group give a positive skin response to cockroach extract. The results recorded in Table III indicate that thirty-two of the 114 allergic patients give a positive skin response to at least one of the cockroach extracts. This is an incidence of 28 per cent, as compared with an incidence of 7.5 per cent in the normal group. Group 1, our normal and control group, consisted of 253 subjects, 234 of
Age group (years) 5-10
11-19 20-30 31-40 41-50 51 and over Total
Table
111.Summary
of skin responses to cockroach extmct
in ~~roup S---nllergic
group gruup (wars)
Age
5-10 11-19
20-30 31-40 41-50 51 and over Total
Yegaticp
reactors
5 II 21 !I 15
23
Positive
reactors
3 2
6
11
6 4
whom reacted negatively, and nineteen, or 7.5 per cent, positively (Table II). One hundred and seventy-four of the nonreactors, or about three-quarters of the total, were individuals in t,he age groups 11 to 19 and 31 to 50. Most of the subjects were men. Practically every age, from 12 to 61 years, was represented in the nineteen positive reactors to the allergen of the cockroach. The members of group 2 were selected at random from patients who came to our clinics for treatment of allergic disorders. All of them had previously been subjected to the usua.1 routine tests. Of the 114 allergic patients, eighty-two reacted negatively and thirty-two, or 28 per cent, reacted positively to the allergen of the cockroach. Table III indicates the distribution of t,he reactors and nonreactors according to age. The age group 31 to 40 contained the majority of reactors (eleven). It is noteworthy that group 1, the normal group, registered 7.5 per cent of positive reactors as compared with 28 per cent of the allergic group 2. Of the fifty-one positive reactors found in groups 1 and 2, forty-three had been tested with extracts of both the American and Oriental cockroaches with the following results: thirty-two reacted to both species of cockroaches, eight reacted only to the American, and three only to the Orient,al. In the positive reactors among the children, three were tested intracutaneously with thtx extract of the Oriental cockroach and 5 with that of the American cockroach. A supplementary study was conducted with the original extracts ( 1 :lO), their dialyzed residues, and their dialysates. The total nitrogen and protein nitrogen content of these preparations is recorded in Table I.
Volume Number
Allergy
35 6
to cockroach
509
Fifteen of the positive reactors were tested cutaneously with each of the three designated solutions, derived from both the American and Oriental cockroaches. The former were placed on the right lower arm, and the latter on the left lower arm. Four other positive reactors were tested with the American cockroach reagents. A negative control solution was also placed on a fourth scratch site. It is noteworthy that the skin reactions resulting from the extract of the dialyzed residue have been of smaller dimension than those of the original extract, save in three test subjects. In only one instance, however, did the dialyzed residue fail to react; and in two cases the dialysate yielded positive reactions. Passive
transfer
studies
Blood sera were obtained from twenty-three positive reactors for passive transfer studies. The tests were performed in triplicate. Three recipients were inoculated intracutaneously with 0.05 ml. of serum from each of four pa.tients. As a negative control, 0.05 ml. of serum was injected intracutaneously in the other arm. This serum was obtained from an individual who reacted negatively to the cockroach allergen. The sensitized sites were challenged after an interval of 48 hours with 0.025 ml. of extract. Twenty-two of the twenty-three specimens examined showed the. presence of a. skin-sensitizing antibody. The challenging dose in four tests was 0.025 ml. of the extract of dialyzed residue containing 7,700 PNU per milliliter. In fourteen tests, a dilution containing 7’70 PNU per milliliter was used. A quantitative determination was made in four sera., using the extract in tenfold dilutions from 1:lOO to l:lOO,OOO. Two sites reacted positively when challenged with the extract 1 :lO,OOO (86 PNU per milliliter) and two other sites reacted with the extract 1 :lOO,OOO(8.6 PNU per milliliter). Sensitizing
capacity
of the allergen
It is noteworthy that two recipients developed marked redness and diffuse swelling of the arm which had been previously used for the passive transfer tests. The itching was intense. Swelling gradually subsided during the course of 7 days and was followed by a profuse scaling. No other part of the body was involved. Symptoms usually associated with a constitutional reaction were absent. The two sensitized subjects had each received seven intracutaneous injections of 0.025 ml. of the extract of the dialyzed residue, totaling 1,347 protein nitrogen units. To determine their suitability for passive transfer study, the entire group had been tested 12 days previously by the cutaneous method. Two minims of an extract of the American cockroach and one minim of the Oriental cockroach (each of 50,000 PNU per milliliter) were applied to scratch marks with negative results. Food
infestation
It must be emphasized that the cockroaches from which our extracts were prepared were laboratory born and bred. Their diet has been a constant and plentiful one, consisting of water and small bars of Purina Dog Chow.
Table IV. Tests indicating
the tkermostrrhility .Vormnl
Subjects
Unheated
1. Sensitiw to cockroach A. dialyzed residur of Amrwicnn zl
C: G: kHk. R.
Dialyzed L. R. C. G. w. M.
C. Dialyzed C. H.
2’. Nonwnsitive A. Dialyzed
R.
J. c. M. R. Dialyzed
residue
0
0
C.
Heated for
at 60” 1 how
C’.
4+ 4+ 4+
4+ 4+
a+ 4+
I:
3+ 3+ 3+
4+ 3+ 3+
0
2i
3+
0
0
:
:
Ei
0 0
a
0 0
0 :
0 0 0 0 0 0 0
0 0
0 0
0 0
PNr/ml.)
to cockroach residue of American 0
roach
PNl?/ml.)
(770
PNlr/ml.)
(77
thY+R. ’ J: c. s.
:0 0
residue
at 100” 1 how
4+ 4+ 4+
0
3+
residue
Heat& for
allergen
PNF/ml.)
(77
(7.7
cockroach
P.~~‘/ml.J
4+
0 0 0
J. E. E. D.
(770
i 0
L. c. R. M. G. D.
C. lklyzed
roach
control
4+ ‘I+ 4+ -I+
4+ 3+ 3+ residue
salt
solution,
of
(7.7
PiVO/ml.) 0 0
Extracts were prepared from a fresh supply of Purina Dog Chow bars and from bars which had been partially consumed by the insects. The latter were removed from the cages and presented the appearance of a honeycomb, in sharp contrast with the solid bars of the original material. The designation of chow I is applied to the fresh material and chow II to the partially consumed bars. The food materials were ground to powder form and washed in ether. Twenty grams of each dried lot was a,dd&l to 100 ml. of Coca’s solution and extraction continued for 6 days in the refrigerator. Sterilization was effected by Seitz filtration. As a preliminary step, nine cockroach-sensitive subjects were tested cutaneously with the powdered materials, moistened with N/20 NaOH. The results were negative. Then followed a series of exploratory tests with the extracts in various dilutions: 1:50, 1:500, 1:5,000, 1 :lO,OOO and 1:50,000. Ten cockroach sensitive and two nonsensitive individuals were available, and ninety-eight intracutaneous tests with the varying dilutions and negative controls were performed. It was noted that the dilutions of 1:5,000 and 1 :lO,OOO gave the more decisive results. Thus, with the dilution of 1~5,000, twenty tests with chow I extract of the original material yielded eight positive, three weakly positive, and nine negative reactions, whereas chow II extract yielded fifteen positive, three
Volume 35 Number 6
Allergy
to cockroach
511
weakly positive, and two negative reactions. With the dilution of 1 :lO,OOO,chow I extract gave three positive and seven negative reactions; chow II extra& gave seven positive and three negative reactions. The variations in the reactions may be explained by the irritant qualities of the extracts and especially by the different degrees of the allergen-sensitivity of the individual tested. The results indicate that the chow II extract, prepared from the partially consumed Purina Dog Chow bars, contains an allergenic ingredient. This initial study with its important implications will be extended to overcome the difficulties encountered. Effect of heat
on the allergen
The association of allergen with food supply raises the inevitable question concerning the effect of heat upon the allergen. Accordingly, one sample of the extract of dialyzed residue of the American roach was heated for 1 hour at 100’ C. and another sample was heated for 1. hour at 60° C. The unheated extracts were diluted 1 JO, 1 :lOO, and 1 :l,OOO. Five of our group of cockroach-sensitive subjects were each tested intracutaneously with 0.025 ml. of these dilutions. Table IV shows the results? which indicate that there has been no impairment of the allergenicity of the extracts as a result of heating. Interestingly enough, subject C. H. reacted positively with 0.025 ml. of the three dilutions, each containing 7.7 PNU per milliliter. Another subject, L. C., who presented ma#rkedly positive reactions, developed hives all over his body immediately after the test. Eleven nonsensitive subjects reacted negatively with the three preparations to which our sensitive group had responded positively. DISCUSSION
We are now confronted with the problem of determining the route or routes by which man is sensitized to cockroaches. The experience of the past with insect allergy affords some guidance. The role of the Hymenoptera as sources of injectant allergen has long been recognized. 5 Another concept, that of inhalant allergen, has been developed by the observation of Figley,G Parlato,? and Kern8 with the May fly, sand fly, and mushroom fly, respectively. The following conclusion of Feinberg and his co-workers1 is noteworthy: “Recent findings have indicated that dust from disintegrated insects may constitute a common type of antigen responsible for asthma and hay fever.” Equally challenging is the assertion of Perlman” : “Insects as inhalant allergens fulfill the postulates set down for pollinating plants causing hay fever and asthma.” We must now determine the applicability of all of these observations to cockroaches. A survey, therefore, of their habits and habitats is essential for formulating judgment. In the United States, there are thirty-five species, of which seven or eight can be regarded as domiciliary pests.s Their migratory tendencies in search of food and water present a constant problem to food handling establishments such as dairies, food and meat packing plants, mess halls, and restaurants. Even the kitchens of modern apartment houses do not escape infestation. Cockroaches are omniverous. They eat almost everything that man eats, wears, or otherwise uses. To allergists, the most significant phase of their activity
is tllc: contamination of food not adcclllatcly protc~ctc~cl.This c*ontaminal ion (*onsists of feces, vomitus, and dead insects.“’ Of equal interest are the man!- rc~fet~~es in the litcraturc? to bodil?- conta.ct with cockroaches.” A case in point is that of a 56-Scar-old woman (mployec~ of the Bureau of Entomology. After 2 years of such employmant, she obscr\-cd that, contact with only one species of cockroach, I,eucoljha8e~~,~)ZCI~~WC~C, causc~d itching of her hands. an. eruption of papules also appeared on the dorsal aspect of her hands and arms. The lesions, ho’wcvcr, did not extend aho\-c the elbows. This cast of contact dermatit,is denotes an allergic reaction of occupational origin. There are also records of cockroaches crawling OVPL the faces of sleeping persons and even biting some of tl1em.l” They arc attracted by the odor or taste of food or drink on the mouths and lips of their victims. Bodily contact is inevitable in the slum apartments of our metropolitan arclas. In New York (.‘ity,‘:’ for example, the living habits of certain economic groups have a decided influence upon the extent of cockroach infestation. Accordingly, contaminated food, dishes, and surfaces upon which food is prepared provide other routes for the transfer of allergen. In our limited experience to date, we have encountered four patients who have failed to react to the test allergens routinely used but hare reacted solcl:to the cockroach allergen. From the evidence at hand, we may conclude that in some instances symptoms a,ttributable to food allergy 1na.v be due to the food contaminant. In a recent paper Lopes, Masquelier, and eTensenld state : “They have studied allergies of various origins, in particular the insidious and frequent allergies of provoked by food-contaminating agents, whether chemical (derivatives salicylic acid, terpene, . . .) or biological (moths, shell fish, fungi). ” Because of its rarity, the incrimination of inscct,s in allergic reactions makes this paper noteworthy. Our work with the cockroach paves the way to determine the allergenic potentials of other insects associated with food infestation. The present era is, indeed, a counterpart of that of mold allergp of 35 years ago. SUMMARY
AND
CONCLUSION
1. Seven and five-tenths per cent of 253 normal persons show positive skin tests with extracts of cockroaches, as compared with 28 per cent of an unselected group of 114 allergic patients. 2. Skin-sensitizing antibodies are present in the blood sera of positive reactors. 3. Cockroach allergen has the capacity to sensitize. 4. Cockroach allergen has the capacity to provoke constitutional reactions in reactors. 5. Cockroaches contaminate food substa,nces with an allergen. 6. Cockroach allergen is thermostable. 7. The possibility that the cockroach allergen may bc of the ingestant or injectant variety must be given critical consideration. The advisability of adding cockroach allergen to the routine tests of allergic patients is clearly indicated.
Volume Number
Allergy
35 6
to cockroach
513
Desensitization with cockroach extract as the sole therapeutic measure, or as an adjunct, may reduce the number of refractory allergic patients, especially of those living in an insect-infested environment. This is the challenge. We express our thanks to Mr. Donald Clemmer, Director of the Department of Corrections of the District of Columbia Government, to Mr. John W. Beard, Superintendent of the Workhouse Division, and to their respective staffs for their cooperation in permitting inmates to volunteer for these studies. We are grateful to Mr. R. L. Walker and Mr. J. H. Fales, of the Entomology Research Division, Agricultural Research Service for the supply of insects essential for this investigation and to Mr. William V. Eisenberg, Chief, Microanalytical Branch of the Bureau of Biological and Physical Sciences of the Food and Drug Administration, for his guidance and counsel. The technical assistance of Miss Anita Louise (‘arter and of Mr. Peter Basa is also acknowledged. REFERENCES
1. Feinberg, Allergens, 2. Perlman,
2. 1961. 3. Wiseman, 4. 5. 6. 7. 8. Y lb:
11. 12. 13. 14.
A. J. F.:
R.,
Feinberg,
S. M.,
and
Pinto,
C. B.:
Asthma
and
Rhinitis
From
ALLERGY 27: 5,1956. Insect
Allergens:
Their
Interrelationship
and
Differences,
J.
Insect
ALLERGY 32:
R. D., Woodin, W. G ,.,. Miller, H. C., and Myers, M. A.: Insect Allergy as a Possible Cause of Inhalant Sensltlvitv. J. ALLERGY 30: 3. 1959. Perlman, F.: Insects as Inhalant Allergens, J. ALLERGY'~~: 4, 1958. Waddell, L. A.: Egyptian Civilization, London, 1930, Lazac Co. Figley, K. D.: Asthma Due to the Mayfly, Am. J. M. SC. 178: 338, 1929. Parlato, S. J.: The Sand Fly (Caddis Fly) as an Exciting Cause of Allergic Coryza and Asthma. II. Its Relative Frequency, J. ALLERGY 1: 307, 1930. Kern, R. A.: Asthma Due to Sensitization to a Mushroom Fly (Aphiochaeta agarici), J. ALLERGY 9: 6C4, 1938. United States Department of Agriculture: Cockroaches, Leaflet No. 430, 1958. Roth, L. M., and Willis, E. R.: The Medical and Veterinary Importance of Cockroaches, Miscellaneous-Smithsonian Collections 134: 16, 1957. Step, E.: Marvels of Insect Life, National Travel Club of New York, p. 298. Heiser, V. : An American Doctor’s Odyssey, 1936, W. W. Morton & Co., p. 312. Sheffield, G. J.: Prevalence of the German Cockroach-Blattela germanica (Linn.)-in the Habitations of Certain Ethnic Groups in Slums of the City of New York, Personal communication. Lopes, G., Masquelier, J., and Jensen, R.: Some Experimental Results and Personal Considerations on Dietary and Digestive Allergy. Livre des resumes des rapports et des communications, p. 25, 1963, presented at the 10 Congres International d’Allergie Alimentairc et Digestive, Vichy, France, June, 1963.
studies
Harry
M.D.,
S. Bernton,
M.D., and Halla
Brown,
Washington,
D. C.
F
or many years the role of insects as sources of both injectant and inhalant allergens has received much attention. l-4 A survey of these studies, however, reveals that the allergenic potential of the cockroach, a member of the family Bluttidae Orthoptera, has not been fully recognized. There are about 55 species of cockroaches in the United States, of which only seven or eight constitute a menace to indoor sanitation. Our studies were limited to the American cockroach, Periplaneta americana, and to the Oriental cockroach, Bktta orientalis.
MATERIALS Preparation
AND METHODS of extracts
Batches of live insects were stored in a deep freeze for 15 hours. The frozen bodies were crushed by mortar and pestle, extracted with ether, and dried at room temperature. Ten grams of the defatted residue was homogenized in a Waring Blendor in 100 ml. of Coca’s solution. The fine suspension was stored in the refrigerator for 1 week with frequent shaking. The extract was sterilized by Seitz filtration. Dialytic
fractions
Aliquots of the crude extracts of the two species of cockroaches were dialyzed in cellophane tubes against 3 volumes of distilled water, changed after 24, 48, and 72 hours, respectively. The combined dialysates from each species were lyophylized and redissolved in the original volume of 0.4 per cent phenol in From the Allergy Clinics of Freedmen’s Hospital, Providence Washington University Hospital. This study was supported by a grant from the John A. Hartford Presented at the Twentieth Annual Meeting of the American Feb. 13, 1964. Received for publication April 21, 1964.
506
Hospital,
and
Foundation. Academy
of
George Allergy,
Allergy
Volume Number
35 6
Table
I. Analyses of cockroach extracts Total nitrogen (w./ml.J
Preparation American Original Dialyzed Dialysate
roach 1: 10 extract residue
Oriental roach Original 1: 10 extract Dialyzed residue Dialysate
to cockroach
PNlJ
per
507
milliliter
1.08 0.10 0.84
52,000 7,700 28,000
1.27 0.059 1.03
52,000 4,400 29,000
order to restore the phenol lost from the Coca’s solution during the lyophylization procedure. The residue was dialyzed an additional 24 hours against running water and then dialyzed 24 hours against an equal volume of double-strength Coca’s solution. The volume change of the residue was insignificant. Table I records the total nitrogen and protein nitrogen units of the extracts of the two species of cockroach. EXPERIMENTAL Survey of skin
sensitivity
to cockroach
In our survey to determine the incidence of skin sensitivity to the cockroach, two groups were employed for testing. Group 1 consisted of 253 normal and apparently healthy persons, selected from the lowest social and economic strata of our population. Group 2 consisted of 114 patients, classified as allergic, who reported to our clinics for treatment. Thus, a total of 367 individuals was tested. For practical reasons, only the intracutaneous test was performed on sixty-four subjects in the younger age group (to age 16). A solution of the American roach was administered to fortyfour children and that of the Oriental to twenty others. As a result of many exploratory tests, a volume of 0.025 ml. of a dilution of 1 :lO,OOO was found adequate for this purpose. Readings were taken in 15 minutes. The remaining 303 subjects were tested cutaneously with extracts of both the American and Oriental cockroaches. Solutions of the extracts were adjusted in normal saline solution so that each milliliter contained 50,000 PNU. Two or three drops of these concentrations and of a negative control solution were applied to scratch marks on the anterior surface of the lower arm. Vigorous typical reactions with wheals, pseudopodia, and extensive areas of areola developed in the positive reactors within one-half hour of observation. Table II records the incidence of skin response of group 1 to cockroach extracts. The results show that 7.5 per cent of the members of this group give a positive skin response to cockroach extract. The results recorded in Table III indicate that thirty-two of the 114 allergic patients give a positive skin response to at least one of the cockroach extracts. This is an incidence of 28 per cent, as compared with an incidence of 7.5 per cent in the normal group. Group 1, our normal and control group, consisted of 253 subjects, 234 of
Age group (years) 5-10
11-19 20-30 31-40 41-50 51 and over Total
Table
111.Summary
of skin responses to cockroach extmct
in ~~roup S---nllergic
group gruup (wars)
Age
5-10 11-19
20-30 31-40 41-50 51 and over Total
Yegaticp
reactors
5 II 21 !I 15
23
Positive
reactors
3 2
6
11
6 4
whom reacted negatively, and nineteen, or 7.5 per cent, positively (Table II). One hundred and seventy-four of the nonreactors, or about three-quarters of the total, were individuals in t,he age groups 11 to 19 and 31 to 50. Most of the subjects were men. Practically every age, from 12 to 61 years, was represented in the nineteen positive reactors to the allergen of the cockroach. The members of group 2 were selected at random from patients who came to our clinics for treatment of allergic disorders. All of them had previously been subjected to the usua.1 routine tests. Of the 114 allergic patients, eighty-two reacted negatively and thirty-two, or 28 per cent, reacted positively to the allergen of the cockroach. Table III indicates the distribution of t,he reactors and nonreactors according to age. The age group 31 to 40 contained the majority of reactors (eleven). It is noteworthy that group 1, the normal group, registered 7.5 per cent of positive reactors as compared with 28 per cent of the allergic group 2. Of the fifty-one positive reactors found in groups 1 and 2, forty-three had been tested with extracts of both the American and Oriental cockroaches with the following results: thirty-two reacted to both species of cockroaches, eight reacted only to the American, and three only to the Orient,al. In the positive reactors among the children, three were tested intracutaneously with thtx extract of the Oriental cockroach and 5 with that of the American cockroach. A supplementary study was conducted with the original extracts ( 1 :lO), their dialyzed residues, and their dialysates. The total nitrogen and protein nitrogen content of these preparations is recorded in Table I.
Volume Number
Allergy
35 6
to cockroach
509
Fifteen of the positive reactors were tested cutaneously with each of the three designated solutions, derived from both the American and Oriental cockroaches. The former were placed on the right lower arm, and the latter on the left lower arm. Four other positive reactors were tested with the American cockroach reagents. A negative control solution was also placed on a fourth scratch site. It is noteworthy that the skin reactions resulting from the extract of the dialyzed residue have been of smaller dimension than those of the original extract, save in three test subjects. In only one instance, however, did the dialyzed residue fail to react; and in two cases the dialysate yielded positive reactions. Passive
transfer
studies
Blood sera were obtained from twenty-three positive reactors for passive transfer studies. The tests were performed in triplicate. Three recipients were inoculated intracutaneously with 0.05 ml. of serum from each of four pa.tients. As a negative control, 0.05 ml. of serum was injected intracutaneously in the other arm. This serum was obtained from an individual who reacted negatively to the cockroach allergen. The sensitized sites were challenged after an interval of 48 hours with 0.025 ml. of extract. Twenty-two of the twenty-three specimens examined showed the. presence of a. skin-sensitizing antibody. The challenging dose in four tests was 0.025 ml. of the extract of dialyzed residue containing 7,700 PNU per milliliter. In fourteen tests, a dilution containing 7’70 PNU per milliliter was used. A quantitative determination was made in four sera., using the extract in tenfold dilutions from 1:lOO to l:lOO,OOO. Two sites reacted positively when challenged with the extract 1 :lO,OOO (86 PNU per milliliter) and two other sites reacted with the extract 1 :lOO,OOO(8.6 PNU per milliliter). Sensitizing
capacity
of the allergen
It is noteworthy that two recipients developed marked redness and diffuse swelling of the arm which had been previously used for the passive transfer tests. The itching was intense. Swelling gradually subsided during the course of 7 days and was followed by a profuse scaling. No other part of the body was involved. Symptoms usually associated with a constitutional reaction were absent. The two sensitized subjects had each received seven intracutaneous injections of 0.025 ml. of the extract of the dialyzed residue, totaling 1,347 protein nitrogen units. To determine their suitability for passive transfer study, the entire group had been tested 12 days previously by the cutaneous method. Two minims of an extract of the American cockroach and one minim of the Oriental cockroach (each of 50,000 PNU per milliliter) were applied to scratch marks with negative results. Food
infestation
It must be emphasized that the cockroaches from which our extracts were prepared were laboratory born and bred. Their diet has been a constant and plentiful one, consisting of water and small bars of Purina Dog Chow.
Table IV. Tests indicating
the tkermostrrhility .Vormnl
Subjects
Unheated
1. Sensitiw to cockroach A. dialyzed residur of Amrwicnn zl
C: G: kHk. R.
Dialyzed L. R. C. G. w. M.
C. Dialyzed C. H.
2’. Nonwnsitive A. Dialyzed
R.
J. c. M. R. Dialyzed
residue
0
0
C.
Heated for
at 60” 1 how
C’.
4+ 4+ 4+
4+ 4+
a+ 4+
I:
3+ 3+ 3+
4+ 3+ 3+
0
2i
3+
0
0
:
:
Ei
0 0
a
0 0
0 :
0 0 0 0 0 0 0
0 0
0 0
0 0
PNr/ml.)
to cockroach residue of American 0
roach
PNl?/ml.)
(770
PNlr/ml.)
(77
thY+R. ’ J: c. s.
:0 0
residue
at 100” 1 how
4+ 4+ 4+
0
3+
residue
Heat& for
allergen
PNF/ml.)
(77
(7.7
cockroach
P.~~‘/ml.J
4+
0 0 0
J. E. E. D.
(770
i 0
L. c. R. M. G. D.
C. lklyzed
roach
control
4+ ‘I+ 4+ -I+
4+ 3+ 3+ residue
salt
solution,
of
(7.7
PiVO/ml.) 0 0
Extracts were prepared from a fresh supply of Purina Dog Chow bars and from bars which had been partially consumed by the insects. The latter were removed from the cages and presented the appearance of a honeycomb, in sharp contrast with the solid bars of the original material. The designation of chow I is applied to the fresh material and chow II to the partially consumed bars. The food materials were ground to powder form and washed in ether. Twenty grams of each dried lot was a,dd&l to 100 ml. of Coca’s solution and extraction continued for 6 days in the refrigerator. Sterilization was effected by Seitz filtration. As a preliminary step, nine cockroach-sensitive subjects were tested cutaneously with the powdered materials, moistened with N/20 NaOH. The results were negative. Then followed a series of exploratory tests with the extracts in various dilutions: 1:50, 1:500, 1:5,000, 1 :lO,OOO and 1:50,000. Ten cockroach sensitive and two nonsensitive individuals were available, and ninety-eight intracutaneous tests with the varying dilutions and negative controls were performed. It was noted that the dilutions of 1:5,000 and 1 :lO,OOO gave the more decisive results. Thus, with the dilution of 1~5,000, twenty tests with chow I extract of the original material yielded eight positive, three weakly positive, and nine negative reactions, whereas chow II extract yielded fifteen positive, three
Volume 35 Number 6
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weakly positive, and two negative reactions. With the dilution of 1 :lO,OOO,chow I extract gave three positive and seven negative reactions; chow II extra& gave seven positive and three negative reactions. The variations in the reactions may be explained by the irritant qualities of the extracts and especially by the different degrees of the allergen-sensitivity of the individual tested. The results indicate that the chow II extract, prepared from the partially consumed Purina Dog Chow bars, contains an allergenic ingredient. This initial study with its important implications will be extended to overcome the difficulties encountered. Effect of heat
on the allergen
The association of allergen with food supply raises the inevitable question concerning the effect of heat upon the allergen. Accordingly, one sample of the extract of dialyzed residue of the American roach was heated for 1 hour at 100’ C. and another sample was heated for 1. hour at 60° C. The unheated extracts were diluted 1 JO, 1 :lOO, and 1 :l,OOO. Five of our group of cockroach-sensitive subjects were each tested intracutaneously with 0.025 ml. of these dilutions. Table IV shows the results? which indicate that there has been no impairment of the allergenicity of the extracts as a result of heating. Interestingly enough, subject C. H. reacted positively with 0.025 ml. of the three dilutions, each containing 7.7 PNU per milliliter. Another subject, L. C., who presented ma#rkedly positive reactions, developed hives all over his body immediately after the test. Eleven nonsensitive subjects reacted negatively with the three preparations to which our sensitive group had responded positively. DISCUSSION
We are now confronted with the problem of determining the route or routes by which man is sensitized to cockroaches. The experience of the past with insect allergy affords some guidance. The role of the Hymenoptera as sources of injectant allergen has long been recognized. 5 Another concept, that of inhalant allergen, has been developed by the observation of Figley,G Parlato,? and Kern8 with the May fly, sand fly, and mushroom fly, respectively. The following conclusion of Feinberg and his co-workers1 is noteworthy: “Recent findings have indicated that dust from disintegrated insects may constitute a common type of antigen responsible for asthma and hay fever.” Equally challenging is the assertion of Perlman” : “Insects as inhalant allergens fulfill the postulates set down for pollinating plants causing hay fever and asthma.” We must now determine the applicability of all of these observations to cockroaches. A survey, therefore, of their habits and habitats is essential for formulating judgment. In the United States, there are thirty-five species, of which seven or eight can be regarded as domiciliary pests.s Their migratory tendencies in search of food and water present a constant problem to food handling establishments such as dairies, food and meat packing plants, mess halls, and restaurants. Even the kitchens of modern apartment houses do not escape infestation. Cockroaches are omniverous. They eat almost everything that man eats, wears, or otherwise uses. To allergists, the most significant phase of their activity
is tllc: contamination of food not adcclllatcly protc~ctc~cl.This c*ontaminal ion (*onsists of feces, vomitus, and dead insects.“’ Of equal interest are the man!- rc~fet~~es in the litcraturc? to bodil?- conta.ct with cockroaches.” A case in point is that of a 56-Scar-old woman (mployec~ of the Bureau of Entomology. After 2 years of such employmant, she obscr\-cd that, contact with only one species of cockroach, I,eucoljha8e~~,~)ZCI~~WC~C, causc~d itching of her hands. an. eruption of papules also appeared on the dorsal aspect of her hands and arms. The lesions, ho’wcvcr, did not extend aho\-c the elbows. This cast of contact dermatit,is denotes an allergic reaction of occupational origin. There are also records of cockroaches crawling OVPL the faces of sleeping persons and even biting some of tl1em.l” They arc attracted by the odor or taste of food or drink on the mouths and lips of their victims. Bodily contact is inevitable in the slum apartments of our metropolitan arclas. In New York (.‘ity,‘:’ for example, the living habits of certain economic groups have a decided influence upon the extent of cockroach infestation. Accordingly, contaminated food, dishes, and surfaces upon which food is prepared provide other routes for the transfer of allergen. In our limited experience to date, we have encountered four patients who have failed to react to the test allergens routinely used but hare reacted solcl:to the cockroach allergen. From the evidence at hand, we may conclude that in some instances symptoms a,ttributable to food allergy 1na.v be due to the food contaminant. In a recent paper Lopes, Masquelier, and eTensenld state : “They have studied allergies of various origins, in particular the insidious and frequent allergies of provoked by food-contaminating agents, whether chemical (derivatives salicylic acid, terpene, . . .) or biological (moths, shell fish, fungi). ” Because of its rarity, the incrimination of inscct,s in allergic reactions makes this paper noteworthy. Our work with the cockroach paves the way to determine the allergenic potentials of other insects associated with food infestation. The present era is, indeed, a counterpart of that of mold allergp of 35 years ago. SUMMARY
AND
CONCLUSION
1. Seven and five-tenths per cent of 253 normal persons show positive skin tests with extracts of cockroaches, as compared with 28 per cent of an unselected group of 114 allergic patients. 2. Skin-sensitizing antibodies are present in the blood sera of positive reactors. 3. Cockroach allergen has the capacity to sensitize. 4. Cockroach allergen has the capacity to provoke constitutional reactions in reactors. 5. Cockroaches contaminate food substa,nces with an allergen. 6. Cockroach allergen is thermostable. 7. The possibility that the cockroach allergen may bc of the ingestant or injectant variety must be given critical consideration. The advisability of adding cockroach allergen to the routine tests of allergic patients is clearly indicated.
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Desensitization with cockroach extract as the sole therapeutic measure, or as an adjunct, may reduce the number of refractory allergic patients, especially of those living in an insect-infested environment. This is the challenge. We express our thanks to Mr. Donald Clemmer, Director of the Department of Corrections of the District of Columbia Government, to Mr. John W. Beard, Superintendent of the Workhouse Division, and to their respective staffs for their cooperation in permitting inmates to volunteer for these studies. We are grateful to Mr. R. L. Walker and Mr. J. H. Fales, of the Entomology Research Division, Agricultural Research Service for the supply of insects essential for this investigation and to Mr. William V. Eisenberg, Chief, Microanalytical Branch of the Bureau of Biological and Physical Sciences of the Food and Drug Administration, for his guidance and counsel. The technical assistance of Miss Anita Louise (‘arter and of Mr. Peter Basa is also acknowledged. REFERENCES
1. Feinberg, Allergens, 2. Perlman,
2. 1961. 3. Wiseman, 4. 5. 6. 7. 8. Y lb:
11. 12. 13. 14.
A. J. F.:
R.,
Feinberg,
S. M.,
and
Pinto,
C. B.:
Asthma
and
Rhinitis
From
ALLERGY 27: 5,1956. Insect
Allergens:
Their
Interrelationship
and
Differences,
J.
Insect
ALLERGY 32:
R. D., Woodin, W. G ,.,. Miller, H. C., and Myers, M. A.: Insect Allergy as a Possible Cause of Inhalant Sensltlvitv. J. ALLERGY 30: 3. 1959. Perlman, F.: Insects as Inhalant Allergens, J. ALLERGY'~~: 4, 1958. Waddell, L. A.: Egyptian Civilization, London, 1930, Lazac Co. Figley, K. D.: Asthma Due to the Mayfly, Am. J. M. SC. 178: 338, 1929. Parlato, S. J.: The Sand Fly (Caddis Fly) as an Exciting Cause of Allergic Coryza and Asthma. II. Its Relative Frequency, J. ALLERGY 1: 307, 1930. Kern, R. A.: Asthma Due to Sensitization to a Mushroom Fly (Aphiochaeta agarici), J. ALLERGY 9: 6C4, 1938. United States Department of Agriculture: Cockroaches, Leaflet No. 430, 1958. Roth, L. M., and Willis, E. R.: The Medical and Veterinary Importance of Cockroaches, Miscellaneous-Smithsonian Collections 134: 16, 1957. Step, E.: Marvels of Insect Life, National Travel Club of New York, p. 298. Heiser, V. : An American Doctor’s Odyssey, 1936, W. W. Morton & Co., p. 312. Sheffield, G. J.: Prevalence of the German Cockroach-Blattela germanica (Linn.)-in the Habitations of Certain Ethnic Groups in Slums of the City of New York, Personal communication. Lopes, G., Masquelier, J., and Jensen, R.: Some Experimental Results and Personal Considerations on Dietary and Digestive Allergy. Livre des resumes des rapports et des communications, p. 25, 1963, presented at the 10 Congres International d’Allergie Alimentairc et Digestive, Vichy, France, June, 1963.