STATS E-480 ^,krrp. pLA»>*1 "" June 1939 United States Department of Agriculture Bureau of Entomology and Plant Quarantine RESULTS OF STUDIES ON DIPHENYLAMINE AS A WOUND PROTECTOR AGAINST THE SCREWWORM, COCHLIOMYIA AMERICANA C. AND P . By Roy Melvin, Henry E. Parish, Edward F, Knipling, and Raymond C. Bushland, Division of Insects Affecting Man and Animals Since the discovery by Cushing and Pattonl/ that Cochliomyia ameri- cana C. and P. is the primary screwworm fly, considerable study has been made on the biology and control of this important livestock pest. Laake, Cushing, and Parishg/ have published on the biology of the screwworm. The present paper deals with studies on improved methods on that phase of control which has to do with wound protection. Since most materials in general use as wound dressings depend on their repellent properties to prevent screwworm infestation, the discovery that phenothiazine,3/ while it would not prevent oviposition or the hatch- ing of eggs, would prevent, in the majority of cases, the young larvae from establishing themselves in the wound, paved the way for a new approach to the screwworm problem. During the past two years over 600 materials in powder or granular form were tested against screwworms in the hope of finding compounds with more effective larvicidal and wound-protective properties. All the compounds were tested in the laboratory on artificial screwworm- breeding media4/ as a means of selecting materials that would kill young 1/ Cushing, E. C, and Patton, W. S. Studies on the Higher Diptera of Medical and Veterinary Importance. Cochliomyia americana sp. nov., the Screw-Worm Fly of the New World. Ann. Trop. Med. and Parasitol. 27: 539-551, illus. 1933. 2/ Laake, E. W. , Cushing, E. C, and Parish, H. E. Biology of the Primary Screwworm Fly, Cochliomyia americana, and a Comparison of Its Stages with Those of C. macellaria. U. S. Dept. Agr. Tech. Bull. 500. 24 pp., illus. 1936. 3/ First noted by Melvin and Bushland while using artificially infested guinea pigs, and later confirmed by Parish and Bigham on natural screwworm cases in domestic animals. Unpublished data. 4/ Melvin, Roy, and Bushland, R. C. A Method of Rearing Cochliomyia americana C. and P. on Artificial Media. U. S. Dept. Agr., Bur Ent. and Plant Quar., Circular ET-88 (multigraphed) . - 2 - screwworm larvae. The more toxic compounds were tested on animals infested with screwworms. Of the many chemicals tested, several appear to be effective wound protectors. One of these promising materials, diphenylamine, has been studied extensively in the laboratory and on artificial and natural screw- worm cases. Although much remains to be determined regarding methods, time intervals of application, the degree of fineness for best performance, etc., the results seem worthy of presentation at this time, since there is a definite need for a more effective treatment than is now available to ranch- men. Artificially infested guinea pigs were used in the early labora- tory tests, while sheep and goats were used in the preliminary field tests. From the results of these studies the following determinations were made: 1. Finely ground diphenylamine adheres well to the tissues, es- pecially if the wound is moist; excessive bleeding, however, washes it away. 2. Flies oviposit rather freely on wounds treated with diphenyl- amine, and the majority of the egg masses hatch, but only a low percentage of the masses cause infestations, some of which consist of only a few larvae. 3. Diphenylamine will kill 3-day-old or younger larvae but will not kill all the 4-day-old larvae. When diphenylamine is applied to wounds containing nearly grown larvae, some of these larvae leave the wound but are not killed. 4. Diphenylamine was approximately twice as effective as pine-tar oil in protecting wounds from reinfestations. With the above information, field studies were carried out on natural screwworm infestations on several classes of range livestock. In these studies a comparison of the efficiency of diphenylamine and pine-tar oil as wound protectors was made by treating 444 natural screwworm cases with diphenylamine and 250 with pine-tar oil. The general procedure is described below. 1. Benzol was used to kill the larvae in the wound and was applied as follows: The wound was first swabbed with cotton to remove blood and exudate, a little benzol was applied, and then the wound was plugged with dry cotton to which more benzol was applied. The cotton plug was allowed to remain in the wound at least 3 minutes to kill the larvae present. 2. Soiled wool or hair was removed from the region of the wound with ordinary hand shears. 3. Most of the larger dead worms were removed from the wound with blunt-pointed forceps. r - 3 - 4. The test material was applied (pine-tar oil by means of a brush, and diphenylamine by hand), enough being used to fill all pockets and to cover the entire wound. For the most part the animals were examined daily to obtain records on the number of egg masses deposited on the wounds and to record reinfesta- tions. The condition of the wounds and general condition of the animal were also noted. Wounds were not re-treated unless reinfestations occurred, in which case larvae present were killed with benzol and the wounds re- treated with diphenylamine or pine-tar oil. Table 1 presents the results of the tests with diphenylamine and pine-tar oil, showing (1) the average number of reinfestations per animal, (2) the average number of days from the time of original treatment until the first reinfestation occurred, (3) the average number of days ensuing before subsequent reinfestations occurred, (4) the average number of days required for wounds to heal, and (5) the percentage of mortality in treated animals . Table 1 Test Average number Average number of days Average number Percent material of reinfes- of protection of days mortality tations per Initial Subsequent required to animal treatment treatment heal Diphenylamine 1.3 14.7 13.1 32.0 16.8 Pine-tar oil 3.2 9.2 7.9 35.8 21.0 Table 2 shows the percentage of cases reinfested at various intervals up to 5 days, after treatment. Table 2 Diphenylamine Pine-tar oil (Percent) (Percent) 0.5 10.8 4.2 32.1 12.4 48.9 21.7 60.4 30.0 66.5 Cases reinfested within 1 day after treatment Cases reinfested within 2 days after treatment Cases reinfested within 3 days after treatment Cases reinfested within 4 days after treatment Cases reinfested within 5 days after treatment The results of these tests show clearly the superiority of the di- phenylamine treatment over that of pine-tar oil. Diphenylamine proved over twice as effective as pine-tar oil in preventing reinfestation, as indicated by the difference between the average number of reinfestations per animal in one group as compared with the other. In screwworm-infested animals treated with diphenylamine only 12.4 percent were reinfested within 3 days following the treatment, whereas 48.9 percent of those treated with pine-tar oil were reinfested during the same period; and 2 days after treatment the percentages of reinfestation were 4.2 and 32.1 respectively. UNIVERSITY OF FLORIDA ■•■IJIHII In view of the evidence presented herein, it seems reasonable to conclude that applications of this material to wounds every third day will give satisfactory protection against injury by screwworms. 5/ Further- more, following this schedule of treatment, it would not be necessary to use benzol for killing the larvae in reinfested wounds since only 0.5 percent of the wounds will contain larvae approaching 3 days old, and diphenylamine is effective in killing screwworms of this age. It is realized that in seasons of extremely great abundance of screwworm flies, 2-day intervals between applications with diphenylamine may become necessary for satisfactory protection against reinfestations. The data indicate, however, that, even during severe screwworm outbreaks, several times greater wound protection may be expected when treating with diphenylamine at regular intervals of from 1 to 3 days than may be expected under similar conditions when treatments are made with pine-tar oil. 5/ Directions for using diphenylamine for the control of screw- worms are given in Bur. of Ent. and Plant Quar. Circular E-481, entitled "Diphenylamine as a Wound Protector against the Screwworm, Cochliomyia americana C. and P."