PARARAMA, A DISEASE CAUSED BY LARVAE: 72 EXPERIMENTAL FINDINGS’

Leonidas Braga Dias, M.D., and Miguel Cordeiro de Azevedo, M.P.H.3

Contact with hairs (setae) from larvae of the moth Premolis semirufa is known to have painful or crippling effects on the fingers of Brazilian rubber workers. Research on mice exposed to these setae, reported here, provides new information about how this occurs.

A caterpiller called “pararama,” larva of the patient of the use of one or more fingers, moth Premolis semirufa, has previously been presenting a clinical picture corresponding to reported by Vianna and Azevedo (I) to have ankylosis. affected at least 24 rubber workers at a Martins (4) and Dias (5) have confirmed plantation named Granja Marathon (Marathon observations in Belterra, where Machado (6) Farm) in the municipality of Sa’o Francisco, says that the proportion of rubber plantation which is located in Brazil’s north-central state workers affected by this diseasehas been as _ of Para. These authors have termed the delayed high as 40 per cent. Ma&do (T), in a radio- clinical manifestations of the condition “para- logical study, found no alterations on the rama,” using the caterpiller’s local name. (The surface of finger joints, but notedperiarticular, larvae are also frequently referred to simply as edematousand fibrous alterations of underlying Zagar&s-caterpillers.) tissues. The presenceof these larvae has been noted Lacking further data of this type, we under- for many years at another location as well, the took experimental research on mice, using the rubber plantations of Belterra in the Para larvae indicated by rubber workers asthe cause municipality of Santarem. of their lesions, to investigate development of The lesions frequently observed after ac- the articular injuries cited in these clinical and cidental contact with the larvae of certain radiological reports. , their cocoons, or their adult forms are itching, blistered, pruriginous processesin- Materials and Methods volving allergic and toxic manifestations. Both isolated casesand epidemicshave been reported Well-developedlarvae (Picture 2) collected (Gusmao,et aZ.,-2); more recently, such lesions from the trunks of rubber trees (Heveabrasil- have also been considered an occupational iensis) on Granja Marathon were brought to the disease(Katzenellenbogen-3). laboratory in screenedboxes, where they were Contact with a pararama causesimmediate kept, and were fed with leavesfrom the same intense itching (pruritus), hyperemia, and local trees. Several of them formed cocoons during edema. It also gives rise to chronic manifesta- the first 24 to 48 hours and molted a few days tions later on, and these sometimesdeprive the later. Some adult forms were mounted and sent to Professor Lauro Travassos Filho at the Sa’o ‘Work carried out at the Evandro Chagas Insti- Paulo State Agriculture Ministry’s Zoology tute-Foundation of the Oswald0 Cruz Institute, Beldm, Pari. Department; there the specieswas identified as 2Chief of the Pathology Section of the Evandro Premolis semirufa (Walker, 1856; Hampson, Chagas Institute; Assistant Professor of the Center f?r Biological Sciences of the Federal University of Para. 1901) a moth of the superfamily , SDirector, Evandro’Chagas Institute. Family Arctiidae (seephotograph). In addition, 9 10 PAHO BULLETIN . voz. VII, No. 3, I973

some mature larvae were subjected to macro- setae had inflicted injury, which occurred scopic and histologic examination. Three types regardless of whether they came from living of surface hairs (setae) were macroscopically larvae, dead and dried larvae, or cocoons. isolated; these have been classified as large, Therefore, subsequent experiments used only medium, and small (Picture 2). The large ones, dried larvae from which the large and medium measuring up to 3 cm in length, are located : setaehad been removed. chiefly on the lateral surfaces of the thoracic and abdominal segments,emerging from small protuberances. The medium-length ones are Experiment A: about 1 cm long and are located on the dorsal Five three-day-old mice were put in a glass surfaces of the first and seventh abdominal segments,there being two tufts of setaeon each container with somedried larvae and kept there segment. The small ones, measuring 1.5 to 2 for five minutes. The container wasshaken four mm in length, are also dorsally situated, and are times, using circular and lateral movementsto found on abdominal segments two through increase the ’ contact with the larvae. eight; they occur in two pairs of tufts on each One mouse was sacrificed two hours later, two segment, were sacrificed 24 hours after exposure,and the The various types of setae are also woven last two were sacrificed 48 hours after expo- into the pararama’sdun-colored cocoon, which sure. is shapedlike a spindle (except that one of the sides is flattened where the cocoon is attached Experiment B: to a surface). The small setae are erect and disposed perpendicularly about the outer sur- Eight seven-day-old mice were exposed to face, giving the cocoon a velvety appearance larvae as in Experiment A; in addition, before (Picture 3). adding the mice the inside of the container was sprayed with small setae extracted from another larva. The animals were exposedfor 10 minutes and the receptacle was shaken six To prepare slides for histologic examination times. Four animals were sacrificed 24 hours (Pictures 4 and 5) larval segmentswere sliced later and the others were sacrificed on the transversely,fixed in formaldehyde, and treated fourth day after exposure. with paraffin. Dead larvae were dried in an oven at 37’C Experiment C: and were stored for later use in the experiments reported here. Five full-grown mice were exposed as in Experiment B. Three were sacrificed after 24 hours and the other two on the fourth day after Experiments exposure. Preliminary Test Experiment D: Setae from living larvae, dead and dried larvae, and cocoons were placed on the shaved Four full-grown mice were exposed as in abdomens of adult mice and affixed with Experiment B on four separate occasions, at adhesivetape. Each kind of setafrom each type intervals of four days. They were then sacri- of source was tested on two animals,so that 18 ficed 18, 25, 32, and 39 days after the last mice were usedin all. exposure. When these mice were sacrificed 24 and 48 8 8 m hours later, it was observedthat only the small Braga Dias and Cordeiro de Azevedo . PARARAMA 11

(1) Adult form of the moth Premolis semirufi. the small setae and their sharp tips. (Carbol-fuchsin (2) The larva pararama, after being fixed in for- light green solution, approximately 120X.) maldehyde. Arrows indicate the large (G), medium (6) Section of a mouse foot 18 days after expo- (M), and small (P) setae. sure, showing part of a seta in a synovial bursa (3) Pararama’s spindle-shaped cocoon. The surface evidencing a fibrous and slightly granulomatous re- is covered with small setae. sponse. (Hematoxylin-eosin, approximately 250X.) (4) Tissue slide made from a tuft of small setae; (7) Section of a mouse foot 25 days after expo- secretory cellular elements may be seen under the sure, showing a seta under the periosteum accom- cuticle. (Carbol-fuchsin light green solution, approxi- panied by a fibrous, histiocyte-producing reaction. mately 50X.) (Carbol-fuchsin light green solution, approximately (5) Detail of a tuft, showing the tubular nature of 250X.) 12 PAHO BULLETIN . Vol. VII, No. 3, 1973

All the animals were anesthetizedwith ether cytic infiltration and edema.Occasional hemor- prior to being sacrificed. The newborn animals rhagic foci were also observed in the lungs, were then fixed whole, after their cranial, although no setae were observed in the lung thoracic, and abdominal cavities had been tissuesamples examined. opened. To examine the adult animals, slides Similar results were observedin the Experi- were made from portions of the abdominal ment B mice sacrificed24 hours after exposure. wall, snout, perianal tissue,hind feet and lungs, In addition, setae were found in the deep A 10 per cent formaldehyde solution was used tissuesof the feet, in the corium of the mucosa to fix the samples,After fixation, the feet were of the mouth and tongue, and frequently in the decalcified in an aqueoussolution of S per cent corium of the snout. The mice sacrificed after formic acid. Standard techniques for embed- four days also showedsetae in theseareas, there ding in paraffin and sectioning were used; the having meanwhile been changes in the local resulting 6-micron sections were stained with reaction, with partial replacement of neutro- hematoxylin-eosin and carbol-fuchsin light philic elements by histiocytes, giving the ap- green solution. pearanceof a granulomatousreaction to foreign bodies. Experiment C animals sacrificed 24 hours Results after exposure showed some setae within the abdominal wall, in the perianal tissue, in the There was virtually no evidence of large or dermis of the snout, and in the feet. The local medium setaein the tissuesof mice exposedto changespreviously mentioned (neutrophilic in- these types in the preliminary tests. There was filtration and edema) were observed. The two just one case-that of a mouse that had been animals sacrificed after four days showed sacrificed 24 hours after exposure to medium results similar to those obtained in the Experi- setae-in which we found two reactive dermal- ment B mice sacrificed after four days. Setae epidermal foci with very superficial leukocytic were found in the sametissues, accompanied by infiltration. However, the small setaedid pene- a similar granulomatousresponse. In one trate the tissues of the exposed animals, being a granuloma in the pulmonary parenchymawas found later at the opening of sebaceousglands, found to have formed around a seta. throughout the epidermis, at various levels of In Experiment D, only the hind feet of the the dermis, sometimesin the abdominal mus- animals were examined. Lesions were found in cles, and less frequently in the subperitoneal the dermis, tendon sheaths, periostea (Picture tissue. The presenceof setaein the connective 7), bone marrow, and less frequently in tissue was accompaniedby neutrophilic leuko- synovial bursae (Picture 6); setaewere found at cyte inflitrations and by edema,the latter being the center of foreign body granulomas. Little less intense in the animals sacrificed after 48 variation in cellular composition or fibrous hours. tendency of the lesions was observed in the In Experiment A, the animal sacrificed two Experiment D animals kept alive a relatively hours after exposure had small setaeat various longer time. levels of its epidermis and dermis, and occa- sionally in its cutaneous muscles;some edema Discussion was observed, but there was no evidence of leukocytic infiltration. In the animals killed During the preliminary tests,as well as in the after 24 and 48 hours, severalsetae were found other experiments, exposure to small setae in the dermis of various parts of the body- produced extreme restlessnessin the exposed including the snout-and in the periostea of animals almost at once, indicating the setaein some ribs, the jaws, and other bones. The their tissues caused itching similar to that presenceof these setae was marked by leuko- experienced by accidentally exposed humans. Braga Dias and Cordeiro de Azevedo - PARARAMA 13

In man, itching and edema are the initial experimentally lead us to suppose that more acute manifestations normally encountered than one factor is responsiblefor the lesions. As (along with hyperemia); thesetend to disappear is true of other lepidoptera setae, the para- after a few hours, or after a week at the most. rama’s hollow setae intimately linked to glan- In some patients, however, the edema or dular elements presumably carry chemical sub- swelling of the joints persists, along with stances-which in this caseare assumedto cause immobility of the affected fingers, these the immediate itching, edematousreaction, and symptoms characterizing chronic casesof para- neutrophilic infiltration that occur in the first rama (7). phaseof the disease.The physical persistenceof Histopathologic examination of mice sacri- these setae-in probable conjunction with their ficed two hours after exposure to small setae chemical make-up-provokes the chronic granu- showed only edema, but this was accompanied lomatous and fibrous response.The number of by neutrophilic infiltration in animals sacrificed setaeinvolved and the particular circumstances after 24 and 48 hours. The mice sacrificed four prevailing in each casemay be cited to explain or more days after exposure showed steady why the chronic form of the condition has been replacement of their acute reaction processes experienced by only a small portion of the by a chronic granulomatous response to the affected rubber workers. presence of setae in the tissues.‘Thirty-nine There is little published literature on occu- days after exposure the setae still persisted in pational hazards, such as those referred to here, the tissues, and were accompanied by well- involving lepidoptera larvae. Katzenellenbogen defined foreign body granulomas with fibrous (3) refers to a dermatitis causedby caterpillers proliferation of the peripheral connective of Thaumetopoea pinivora (wilkinsoni), among tissue. forest workers in Jerusalem.The setaeof dead These results show that the small setae’s larvae were shown by experiment to cause marked penetration ability permitted them to dermatitis, and histologic examination revealed reach the tendon sheaths, periostea, and acute inflammation; but setaefrom theselarvae synovial bursae of the exposed mice. This in were not found to have penetrated tissue to turn leads to the supposition that the setaecan which they were exposed. reach the same sites in the hands of exposed In the last analysis, the presence of setae rubber workers, though up to now there has from Premolis semirufa larvae in the soft tissues been no direct proof of such natural pathologic of mouse paws, and in perichondria, periostea, behavior. At the sametime, the ability of setae tendon sheaths, and synovial bursae of the to persist in tissues for over 30 days without experimental animals-accompanied by forma- showing any signs of absorption supports the tion of foreign body granulomas and a tend- theory of chronic pararama evolution in man. ency toward fibrosis-provides a parallel to the The clinical progress of affected patients clinical manifestations and radiological findings and the histopathologic changes observed in chronic human casesof pararama.

SUMMARY Pararama is an occupational disease of Until now, however, little was actually Brazilian rubber workers apparently caused by known about the disease process itself. The contact with caterpillers of the moth species experimental research on mice, reported here, Premolis semirufa. The chronic form of the sheds new light on this matter. condition involves long-term disability in the On penetrating the skin of laboratory mice, fingers of affected persons. the larva’s small dorsal hair3 (setae) almost 4 14 PAHO BULLETIN . Vol. vrr, No. 3, 1973 4 immediately cause acute edemous inflamma- The presence of lesions in the peripheral tion. Infiltration by neutrophilic leukocytes connective tissues of the joints parallels the follows, and this stage in turn is superceded by clinical and radiological picture associated with fibrosis and formation of foreign body granu- pararama cases affecting the fingers of workers lomas. Lesions caused by the setae have been who tap latgx from the rubber tree Hevea found in the animals’ skin, tendon sheaths, brasiliensis. periostea, bone marrow, and other tissues,

‘? REFERENCES I

(I) Vianna, C. M., and M. C. Azevedo. “Pararama, (3) Katzenellenbogen, 1. “Caterpiller Dermatitis as an doeqa dos seringais.” Work presented at the Occupational Disease ” Tropical Diseases Bul- , First Congress of the Medical-Surgical Society Zetin 51: 1292-1293, 1954 (summary). of Para, held at Belem, Brazil, in October 1967. (4) Martins, J. C. Personal communication. (2) GusmHo, H. H., P. Forantini, and A. Rotberg. (5) Dias, R. M. Personal communication. “Dermatite provocada por lepidbptero do (6) Machado, A. Correio da Manhd (newspaper), Rio Y genera Hylesia.” Rev Inst Med Trop Sao Paul0 de Janeiro, 31 December 1961, section 1,-p. 4. b 3: 114-120. 1961. (7) Macedo, R. Personal communication.

WORLD SMALLPOX SURVEILLANCE

A total of 96,951 smallpox cases had been reported to the World Health Organization by 19 September 1973, approximately twice the number reported by that date last year. The sharp increase was attributable to major smallpox epidemics in the three remaining endemic countries of Asia: Bangladesh, India, and Pakistan. This year all three countries will record their highest total number of cases since 1967, the first year of the global eradication program. While smallpox incidence did show its expected seasonal decline, the number of cases recorded during August in each of these countries remained well above the levels recorded in August 197 1 and August 1972. Ethiopia is still the only African country where smallpox is known to be endemic, Smallpox incidence in Ethiopia is now 73 per cent below what it was at this time last year and is continuing to decline; less than 100 casesper week were reported over the past 12 weeks. However, the eastern province of Harar was the source of two cases imported last July into the French Territory of the Afars and the Issas. [Weekly Epidemiological Record of the World Health Organization, Vol. 48, Nos. 32 and 38, pp. 316 and 370.1