isolation of leishmania braziliensis from lutzomyia ...

Am. I. Trop. Med. Hyg.. 46(4), 1992, pp. 465—468

Copyright ©1992 by The American Society of Tropical Medicine and Hygiene

ISOLATION OF LEISHMANIA BRAZILIENSIS FROM LUTZOMYIA OVALLESI (DIPTERA:PSYCHODIDAE) IN GUATEMALA EDGAR D. ROWTON, MARGARITA DE MATA, NIDIA RIZZO,

CHARLES H. PORTER, ANDTHOMAS R. NAVIN Department of Entomology. Walter Reed Army Institute of Research. Washington. DC; Medical EntomologyResearchand Training Unit,Universidad delValle, GuatemalaCity. Guatemala; Division of Parasitic Diseases. National Center for Infectious Diseases. Centers for Disease Control, Atlanta, Georgia Abstract.

Leishmania

braziliensis

is endemic

in Guatemala

and Belize in Central Amer

ica. To help identify the vector(s) of this parasite in Guatemala, phlebotomine sand flies that were aspirated from the clothing of collectors at Tikal National Park in the Department of the Peten were examined for flagellates. Lutzomyia ovallesi was found infected with flagellates that were identified as L. braziliensis by isoenzyme electrophoresis. The isoen zyme profile of this isolate matched those from humans from the same area. Leishmania

braziliensis was first reported north

of Costa Rica in 1984 in Belize.' It was subse quently found in neighboring Guatemala, in the department of the Peten.2 In both studies, L. braziliensis

was the most common

ken canopy coverage over the site. Site 2 was at the edge ofthe cleared area, next to the secondary forest. Site 3 was located approximately 100 me ters into the forest from site 2.

Leishmania

species isolated frompatients. Althoughdetailed Collection methods studies of Leishmania vectors have been con ducted in Belize,3 only L. mexicana has been Collections were made at ground level for two isolated from sand flies. This was also true in evenings during the last week of September 1989. Guatemala, whereL.mexicanawasisolated from Sand flies attracted to collectors were aspirated Lutzomyia ylephiletor.4 During initial efforts to from their clothing from 7:00 PM to 10:00 PM. establish and test procedures for a vector study, To reduce the risk of being bitten, an N,N-die we were able to isolate and identify L. braziliensis thyl-3-methylbenzamide (DEET)—based repel from Lu. ovallesi. This is the first identification lent was applied to the exposed skin of each col of L. braziliensis from a human-biting sand fly lector. Each wore a beige, cotton twill uniform in Central America. consisting of a long-sleeved shirt and full-length trousers. Two collectors worked at each site, with one aspirating sand flies into a carton as they MATERIALS AND METHODS landed on the uniform of the other. At the end of the collection period, the sand Study area flies were taken to a field laboratory in Tikal, where collection data were recorded and the flies The study area was Tikal National Park, Gua prepared for freezing in liquid nitrogen. The temala (17°38'N, 89°38'W). It was chosen be freezing technique was similar to that of Young cause several cases of L. braziliensis among hu mans have been identified there (T. R. Navin, and others5; however, a different cryoprotectant (10% glycerol in saline) was used, and the prep unpublished data), and because the road access and facilities provided by the park made the pro aration of flies was modified to minimize con ject logistically feasible. tamination in subsequent culture attempts. Live Three sites were used in this study. Site 1 was flies were inactivated by cooling on ice or in a located in a closely mowed, mostly open area refrigerator, and then blown with a mouth as surrounded by buildings that were used for main pirator into a 50-ml tube containing approxi tenance and storage of equipment. Although the mately 30 ml of saline and 3—4drops of an io dine-containing soap solution (Pharmadine; secondary forest was 30 meters away, several large trees within the complex provided unbro Sherwood Pharmaceutical Co., Mahwah, NJ). 465

466

ROWTON AND OTHERS LP

OPt

MPI

Dissection

and

Dissection,

;1ttt—ftH;i--H1+@ 12345

2345

12345

6PGDH

12345

PGM

12345

Fiouar 1. Diagrammatic representations of iso enzyme patterns for the five enzymes used to determine the identity ofthe Leishmania isolate from Lu:zomyia ovallesi. 0 indicates the origin. The anode is at the top ofeach diagram. Lane 1, L. mexicana; lane 2, L. pan amensis; lane 3, L. braziliensis(WHO reference strain); lane 4, isolate from Lu. ovallesi; lane 5 L. braziliensis human isolate (G648). LP = peptidase; GPI = glucose phosphate isomerase; MPI = mannose phosphate isomerase; 6PGDH = 6-phosphogluconate dehydro

genase; PGM = phosphoglucomutase.

The tube was capped and shaken vigorously for 5—10sec. and the contents were emptied into a sieve made of a white plastic cylinder approxi mately 3 cm in diameter with a fine mesh screen glued to one end. The soap solution was dis carded, and the sieve with the retained flies was immediately placed for one minute in a 1% bleach solution followed by a soaking for 3—5mm in sterile saline. The cleansed flies (up to 25/tube) were transferred with forceps into cryotubes con taining cryoprotectant. Liquid nitrogen dry ship pers were used in the field for stage freezing and storage of the flies. After transport, the flies were transferred into a liquid nitrogen refrigerator. In conducting this research, informed consent was obtained from each volunteer and the in vestigators adhered to the policies regarding the protection of human subjects as prescribed by 45 CFR 46 (Protection of Human Subjects). An imal research was conducted in compliance with the Animal Welfare Act and other Federal stat utes and regulations relating to animals and ex periments involving animals and adheres to principles stated in the Guide for the Care and Use of LaboratoryAnimals, 23.

NIH publication

85-

isolation

microscopic

examination,

and

identification of sand flies, and culturing and identification of flagellates were done at the Universidad del Valle de Guatemala in Guate mala City. Vials containing frozen flies were thawed in warm water, and the contents were emptied into a petri dish containing cold saline. Individual sand flies were placed on an alcohol sterilized glass slide in a drop ofSchneider's Dro sophila medium (SDM), where the gut was re moved from the abdomen and thorax with dis secting needles and overlaid with a cover slip. The preparation was examined by phase-con trast microscopy for the presence of flagellates, and to identitify the sand fly by spermathecal morphology. When flagellates were found, the cover slip was removed and a hole was made in the gut wall with a dissecting needle. The gut was then picked up with the needle and transferred to a blood

agarslantoverlaidwithSDMsupplementedwith 20% fetal bovineserum,penicillin (100gsg/ml), and amikacin(100 @ig/ml).2 The medium re mainingon theslidewas collected in a 1-cm3 syringe fitted witha 26.5gaugeneedleand in jected subcutaneously intothedorsumoftheright hindfoot ofa Syrianhamster. Cellulose acetate electrophoresis

(CAE)

Positive cultures wereclonedby streaking on blood agar plates. Test lysates were prepared from

pre-andpost-clone cultures. Control lysates were preparedfrom a L. braziliensis isolate (G648) froma patient intheendemicareaand fromthe World HealthOrganization (WHO) reference strains ofL.braziliensis (MHOM/BZJ75/M2903; WR 675),L.mexicana(MHOM/BZ/82/BEL21; WR 667),and L.panamensis(MHOM/PA/71/ LS94; WR 676), which were obtained

from the

WalterReed Army Institute of Researchcryo bank.Lysates werestored inliquid nitrogen until identified by CAE and enzyme staining, accord ingtothetechniques ofKreutzer andChristensen6 and Kreutzerand others.7 At least one sample ofeachcontrol lysate was runconcurrently with thesandflyisolate. Fiveisoenzymes wereexaminedby CAE: pep tidase (LP,E.C.3.3.l 1), glucose phosphateisom erase(GPI, E.C.5.3.1.9), mannose phosphate isomerase (MPI,E.C.5.3.1.8), 6-phosphoglucon

L. BRAZJLJENSIS

467

IN LU. OVALLESI

TAmI 1 Relative abundance of Lutzomyia species collected at each site from the clothing of collectors collectedSite 3TotalLu. Lutzomyia speciesNumber(%)

1Site

2Site

(0.3)Lu. (0.3)1 (0.4)2 cruciata01 (82)218(86)537(85)Lu.panamensis1(3)42(12)18(7.6)61(9.6)Lu. ovallesi28(88)291 (0.5)Lu. shannoni02 (0.2)Lu. undulata1 (3)001 ylephiletor2(6)12(3)15(6)29(4.6)Total32(5)348

(0.6)1

(0.4)3

(55)253

(40)633(100)

ate dehydrogenase (6PGDH, E.C. 1. 1. 1.44), and phosphoglucomutase, (PGM, E.C.5.4.2.2). At least six electrophoretic runs were made with each isoenzyme. The migration distance (MD) was measured to the nearest millimeter from the origin to the middle ofband. The MD values for each isozyme are shown diagramatically in Fig ure 1. A representative fraction (Ri) value was calculated by dividing the MD of the sand fly isolate by the MD of L. braziliensis (MHOM/ BZ/75/M2903).

parasite in nature, and implicates Lu. ovallesi as a vector of L. braziliensis in Guatemala. Lu:zomyia ovallesi has been implicated as a vector in other areas of Central and South America. In Panama, Lu. ovallesi is a suspected vector of L. panamensis because it feeds both on human and reservoir hosts.8 Similarly, in Carabobo State, Venezuela, Lu. ovallesi is a suspected vector of L. braziliensis because it is abundant in the en demic area, it will bite humans, and flagellates have been observed in the hind triangle of this fly.9 Flagellates isolated from Lu. ovallesi col lected in Lara State, Venezuela caused lesions RESULTS when injected into hamsters. Amastigotes iso lated from these lesions were morphologically During 18 person-hours, 658 sand flies rep similar to L. braziliensis; however, all attempts resenting sixLutzomyiaspecies were aspirated at culturing these isolates failed because of con from the clothing of the collectors (Table1). tamination, which prevented confirmation by Twenty-five ofthesandflies weremales.Of the isoenzyme ‘° 633 femalesandflies dissected, a single Lu.oval Unidentified flagellates have also been found lesi, containing a partial bloodmeal,was found in Lu. ovallesi collected in Belize,3 Colombia,5 with flagellates in both the hindgut and midgut. and Panama.3 Lutzomyia ovallesi has been cap Attemptsto culture theflagellates in biphasic tured at both canopy and ground levels,3 which

culture mediaandby transient growthinthefoot may indicate thatthisspecies hastheability to ofa hamsterweresuccessful. traverse smallclearings by traveling throughthe For theisolate from Lu. ovallesi, theaverage canopy,and thendown treetrunks. InthePeten, Rf values(range) of theisoenzymesexamined thisability would permitLu. ovallesi to have were as follows: LP, 0.99 (0.95—1.00); GPI, 0.99 increased contact withhumans,sincemany local (0.97—1.00); MPI, 1.01(1.00—1.03); 6PGDH, residences arebuiltbeneaththeforest canopy 0.99(0.98—1.00); and PGM, 1.00(1.00). This and onlyunderbrushiscleared. isoenzymeprofile matchesthoseofL.brazilien sis (MHOM/BZ175/M2903 and G648) and dif fers from profiles of the other WHO reference

Acknowledgments: We are grateful to Lic. Flora E. Arana and Dr. Byron A. Arana for help and assistance,

strains (L.mexicanaand L.panamensis).

toa groupofbiology students attheUniversidad del Valle deGuatemala forhelpwiththedissection ofsand flies, andtoDrs.RonaldWardandPhilip Lawyerfor critical review of the manuscript.

DISCUSSION

The biochemical identification ofL.brazilien Disclaimer: The views of the authors do not purport sis isolated from feral collections

of Lu. ovallesi

verifies thecapability ofthissandflytocarrythe

to reflect the position of the Department of the Army or the Department of the Defense (pars. 4-3), AR 360-5.

468

ROWTON AND OTHERS

Authors' addresses: Edgar D. Rowton, Department of

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Entomology, Walter ReedArmy Institute ofResearch,