Natural infection of Phlebotomus perfiliewi with Leishmania infantum in a cutaneous leishmaniasis focus of the Abruzzi region, Italy

5% TR~~NSACTIONS OF THE ROYALSocr~n OF TROPICAL. MEDIC~.JE AND HYGIENE(1987)81, 596-598

Natural Leishmania

infection of Phlebotomus infantum in a cutaneous of the Abruzzi region,

perfiliewi with leishmaniasis focus Italy

M. MAROLI, M. GRAMICCIAAND L. GRADONI Istituto Superiore di Sanith,

Laboratorio

di Parassitologia,

Viale Regina Elena,

299, 00161 Rome, Italy

Abstract

One out of 213 Phlebotomus perfiliewi caught from an endemic cutaneous leishmaniasis focus in Abruzzi region (Italy) was naturally infected with promastigotes. The parasiteswere grown in culture medium and in a hamster, typed by the examination of 11 isoenzymes, and found to be indistinguishable from Leishmania infantum s.st. (Montpellier zymodeme 1). The probable role of P. perjilhi in the transmission of cutaneous leishmaniasis due to L. infantum s.1. in the focus is discussed. Introduction

There is much evidence that Phlebotomus perfilewi Parrot, 1930 (Diptera:Psychodidae) is responsible for the transmission of cutaneous leishmaniasis (CL) in Italy. VANNI (1939) found promastigotes in one of 1600wild P. perfiliewi examined from an endemic CL focus in Abruzzi (Italy). CORRADETTI (1962) reported that all CL foci in Italy are in the area of distribution of P. perjiliewi, and that there is no place where oriental sore occurs in the absence P. petjiliewi. However, the possibility that P. perjiliewi transmit-

ted visceral leishmaniasis (VL) in Emilia Romagna during the epidemic of 1971 cannot be excluded (KILLICK-KENDRICK,

1977). MAROLI & BETTINI

(1977) suggest that, in some VL foci in Tuscany, the speciescould also be suspectedof playing a role in the transmission of VL. P. perfiliewi is a suspected vector of canine leishmaniasis in Tunisia (DANCESCO et al.. 1970:, DEDET. -----I 1971; LEGER et al.,‘l979) and VL d Greece (PERFIL’EV, 1968), Rumania (ADLER, 1964) and Yugoslavia (ZIVKOVIC’, 197%. P. Wrr%ewi is aDDarentlv invoived in the~trans&ssion~of~either

CL b; VL i;

different foci of the disease. Recently, isolates from CL casesin endemic foci of Abruzzi h&e been typed as Leishmania infantum s.1. (GRAMICCIA et al., 19871. In the Dresent note we

deport the disco&y

&d isoenzyie

typing of a

in P. petfiliewi from an area where there are CL cases due to L. infantum s.1.

Leishmania infection

Materials and Methods Locality

The area under study lies along the Pescarariver between the towns of Pescaraand Chieti. The locality “Cavaticchi”, where the sandflies were collected, is in the commune of Spoltore (Pescaraprovince), SO-100m above sealevel and 10 km from the Adriatic coast (see Map); it is an agricultural area with many human settlements. The collecting places were farmhouses in which 4 to 6 people were living. On all farms there were cows, sheep, fowl, rabbits and dogs.

Sa@iy collectionsand Leishmania isolation Sandties were collected from 9-13 September 1985, at the end of the sandfly seasonin Italy, mainly with C.D.C. light traps but also with hand aspirators in stables. Female ilies were identified by the morphology of pharyngeal armatures

and spermathecae.Male flies were identified by the morphology of the genitalia. Before dissection the flies were anaesthetized with CO2 and stored for 15 min in phosphate-buffered saline containing 500 @ml of gentamicin and 500 @ml of S-fluorocytosine. Freshly engorged sandflies caught in the stables were dissected after oviposition (6-7 days after their blood meal). When flies were found to be infected, a few drops of EMTM liquid phase (Evans, 1978) containing 500 @ml of gentamicin and 500 @nl of S-fluorocytosine were added to the dissected material. The gut was then aspirated and inoculated into screw-top vials containing EMTM solid phase, and intraperitoneally into 2 hamsters. Characterization of Leishmania The methods and techniques employed in starch-gel electrophoresis and enzyme staining were based on those of MAAZOUNet al. (1981) and MILES et al. (1980) with slight modifications. Eleven enzymes were tested: ASAT (E.C.2.6.1.1), PGM (E.C.2.7.5.1), GPI (E.C.5.3.1.9), 6PGD (E.C.1.1.1.44), G6PD (E.C.1.1.1.49), ME (E.C.1.1.1.40), MDH (E.C.1.1.1.37), IDH (E.C.1.1.1.42), MPI (E.C.5.3.1.8), NH, and NH2 (E.C.3.2.2.1). Four reference strains were used: MHOM/IR/65I~for. L. tropica (Montpellier zymodeme 6); MRHO/SU/59NealP for L. mujor (Montpellier zymoderne 4); MHOM/FR/78/ LEM75 for L. infanturn (Montpellier zymodeme 1) and MHOM/IT/84/ISSlOO-IDA for L. infanturn NH variant (zymodeme to be delined). Results and Discussion

213 sandflies were caught and dissected (Table). These and 66 male specimens collected at the same time were all P. pefjiliewi. One of 6 freshly engorged females, caught in a stable on “Di Muzio” farm and dissected after oviposition (6 days after the blood-meal), was infected. Promastigotes were seen in the anterior midgut (attached to the stomodaealvalve), but not in the head. Promastigotes developed in cultures and, after 2 months. the inoculated hamsters disolaved tvoical visceral-distribution of amastigotes. The koek$mes and the new isolate (code: IPRF/IT/8MISS174-Prfl1 were indistinguishable from the reference strain for L: infantum Nicolle, 1908, s.st. (Montpellier zymodeme 1). P. perfiriewi can now be added to the list of sandfly speciesfound naturally infected with typed isolates of

M.

MAROLI

et al.

Mapshowing the distribution of CL cases typed as L. infrmhnn NH variant (A), L. infanturn s.sf.m (GRAMICCIA et al., in press),and the collecting stations for P. pqrX6wi (0). Table-Collecting

stations

aad numbers of P. perfiliewi

females dissected

Locality (Spoltore)

dates (nights) (September)

Number of specimens dissected

NUIIlb-3 infected (%)

: 3

“Cavaticchi” farm no no: 31* 32 “Di Muzio” farm

9-1012-13 and 12-13 11-12 and 12-13

58 13 115

i l(O.80)

4

300 m from “Di Muzio” farm

collecting

Station number

9-10

27

0

Collecting methods light traps light traps light traps (6 engorged females collected in stable) hand aspirators in car

Total 213 l(O.46) *A case of CL was diagnosed in 1984 and the parasite typed as L. infanturn s.1. L. infanturn s.st., along with P. ariasi (see RIOUX et al., 1984)and P. perniciosus (seeBETTINI et al., 1986). The identity of the parasite isolated from P. petjiliewi suggests that this species of sandfly might have been responsible for the transmission of VL in

Emilia Romagna and in Tuscany as suspected by

KILLICK-KENDRICK et al. (1977) BETTINI (1977), respectively.

and

MAROLI

&

However, in the area under study, there are CL casesdue to L. infanturn s.1. (see GRAMICCIA et al., 1987), but no VL casehas been reported in the last 10 years, and canine leishmaniasis appears to be very

rare. It is notable that in the Abruzzi P. per/&k has a distribution in accord with that of CL, it is abundant and there is an apparent absence of other species of Phlebotomus.

These epidemiological data, the anthropophilic behaviour of P. petjliewi in the Abruzzi (CORRADETTI, 1936), and our isolation from it of L. infanturn s . st . , meet 3 of the essential conditions required to incriminate this sandAy speciesas a vector of CL. Nevertheless the complete life cycle of the parasite in the fly should be elucidated, and experimental transmission by the bite of P. perjliewi

P. perjiliewi

598

TRANSMUTING

should be carried out, to satisfy all the criteria for its incrimination (KILLICK-KENDRICK & WARD, 1981). The finding of P. pe@iewi infected with L. infanturn s.st., the typing of CL isolates as L. infanfum s.1. in the same focus, and the absence from the focus of P. sergetui, the only proven vector of L. tropica (see ADLER & THEODOR. 1957; LE BLANCQ & PETERS, 1986), suggest the a&ence bf L. tropica from the Ci focus of the Abruzzi region. Acknowledgements The authors express their appreciation to Dr R. KillickKendrick for reviewing the manuscript and thank Mr E. Guandalini for technical assistancein sandfly collection and dissection. This work received financial support from the Technical Services Agreement (B), WHO, Geneva and from CCE, project No. TSD. 386.1 (TT). Adl;;,9.

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Accepted for publication

6, 37-43. 8 July

1986