315 TRANSACTIONSOF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, VOL. 71, No. 4,1977.

(I) An investigation

Leishmaniasis in Tuscany (Italy) : on phlebotomine sandflies in Grosset0

Province

M. MAROLI AND S. BETTINI Laboratorio

di Parassitologia,

Istituto

Superiore

(Technical assistance by A. M. Lopomo of the Laboratorio Grosseto, Italy)

Summary A survey of phlebotomines has been made in the area of the Province of Grosset0 (Tuscany, Italy) where human cases of both visceral and cutaneous leishmaniasis have recently occurred. 12 collecting stations, placed around the leishmaniasis foci, were divided into three groups of localities, the Argentario Promontory, coastal plains territory and hilly inland territory. Sandflies were collected with lit and unlit oil traps, and by hand. A total of 11,032 specimens (57 % males) were caught and identified. Of these. 86.6 % were Phlebotomus perfiliewi, 10.8 % P. perniciosus and 2.3 % Sergentomyia minuta; only a few specimens were P. papatasi and P. mascittii. In the first group of localities, P. perniciosus was very abundant (87.8 %) and S. minuta scarce (12.0 %); in the second group, P. per$liewi was the only species collected; in the third group, P. perfiliewi was very abundant (98.0%), S. minuta was very scarce (1.1%) and P. perniciosus even more so (0.7 %). P. perniciosus is thought to be the probable vector of visceral leishmaniasis in the area of Monte Argentario and P. perfliewi is strongly suspected of playing a role in transmission of the cutaneous form in the third group of localities. The species responsible for transmitting visceral leishmaniasis in some of the foci in this area is yet to be found. l”

Introduction 70 cases of visceral leishmaniasis (VL) and 53 of cutaneous leishmaniasis (CL) have been reported in Tuscany between 1933 and 1975, the first record in the Province of Grosset0 being in 1936 (BETTINI et al., 1977a). The leishmanin skin test (LST) has been recently performed on subjects of different age groups from the Provinces of Grosset0 and Siena where relatively high positivity frequencies (up to about 30 “/,) have been found in different localities @ETTINI et al., 1977b), thus proving that leishmaniasis is endemic in certain areas of Grosset0 and Siena Provinces. These findings led us to carry out an investigation on phlebotomine sandflies, limited to the Province of Grosseto, with the aim of acquiring information on the local vectors of leishmaniasis. The only previous investigation on sandflies in Tuscany was carried out by COWETTI & NERI (1955) in the area of Monte Argentario (Grosseto) where the prevalent specieswas found to beP. perniciosus, only a few specimens of P. papatasi and S. minuta being collected. In the present note, the geographical distribution and densities of the phlebotomine species collected from June to November 1975 in 12 localities of the Province of Grosset0 are reported. Repeated attempts to collect

di Sanitd,

Rome, Italy

Provinciale

di Zoologia

Medica,

sandflies from January to May in the same localities gave negative results. Materials and methods The methods used for collecting adult phlebotomines were those described by RIOUX et al. (1967). Light traps having a castor oil-treated collecting surface of 0.3 m2, similar to that designed by RIOUX but slightly modified (Fig. l), were employed. The traps’ lights were turned on at sunset and turned off at 9 a.m. Fresh oil-treated paper sheets were left in place for the whole period the trap was kept unlit (=dark trap), i.e. usually about one week. For each species and in different stations data on phototropism were obtained by placing a control dark trap in a place where other lamps could not be seen. Each time the traps were lit, sandflies were also caught with mouth aspirators for one hour. After exposure, the oil-treated sheets were brought to the laboratory and the samlilies were collected with a fine paint-brush soaked with 90% ethyl alcohol, kept in 90% alcohol for a few hours, and then separated according to sex. The material was thence transferred into Bless liquid (CORRADET~ et al., 1961). Before mounting the flies in Faure’s liquid, they were cleared in chlor-lactophenol at 40°C for five days. Classification was based on the female pharyngeal armature and spermatheca, and on male genitalia, according to THEODOR’S keys (1958). All collected sandflies were identified.

Fig. 1. Light trap with castor oil-treated papers, Collecting Stations The 12 collecting stations were chosen according to the recent occurrence of cases of VL or CL, i.e. from 1965-75

316

LEISHMANIASIS

IN TUSCANY:

(i) INVESTIGATION

ON PHLEBOTOMINE

SANDFLIES

(BETTINIet al., 1977b). The distribution of these localities with indication of the recent cases of leishmaniasis is shown in the map (Fig. 2). The 12 collecting stations were

H 10 km

Fig. 3. A: Collecting Station No. 1

Fig. 2. Map of the Province of Grosseto. Distribution of collecting stations (numbers, in circles, correspond to localities listed in Table I) and of VL and CL cases reported from 1965 to 1975 (number of cases in triangles). divided into three groups according to their geographical distribution (Table I). The tirst group (Nos. l-4) were in localities of the Commune of Monte Argentario, which consists of a mountainous promontory (maximum altitude 635 m above sea level) connected with the coast by two sand dunes. This territory, mainly covered by Mediterranean-type maquis, includes small cultivated areas. Cala Galera (Fig. 3A) is situated a few metres above sea level and the other localities of this group are at about 100 m above sea level. The second group consists of only two collecting stations (Nos. 5-6), situated on the coastal plains north-west of the town of Orbetello, a few metres above sea level. This territory is in part covered by the lagoon and swamps near Orbetello and the rest is intensively cultivated. Typical of this group is Station No. 6 (Fig. 3B). The third group comprises six collecting stations (Nos. 7-12), situated in a hilly, inland, territory extending from the plains to the province’s north-eastern boundary, partly cultivated and partly covered by maquis and woods, and by gullies with clay soil. Station no. 12 is shown in Fig. 3C. The different species of farm animals, their number in each station, and the results of LSTs previously carried out (BETTINI et al., 1977b) on the inhabitants of these localities are also listed in Table I.

B: Collecting Station No. 6

C: Collecting Station No. 12. specimens collected, followed by P. perniciosus, S. minuta, and P. mascittii. The percentage of males collected was highest in P. perniciosus (81 .I %), followed by P. perfiliewi (53.8 %) and S. minuta (44.6%).

P. papatasi

Results All stations were positive for sandflies. 11,032, of which 57% were male specimens, were collected. Table II shows the distribution of the five species found and the density per station. The highest number was collected at Poggio Abechi and the lowest at Convent0 Frati. The following species were identified: Phlebotomus perfiliewi, P. perniciosas, Sergentomyia minuta, P. papatasi and P. mascittii. P. perfiliewi ranked first for number of

Distribution stations

and densities of species according to group of

As shown in Table III, in the first group of stations (Nos. l-4), situated around Monte Argentario, P. perniciosus was found to be very abundant, its highest number being collected at Cala Galera. S. minuta ranked second

M.

MAROLI

AND

317

S. BETTINI

Table I - Positivity of inhabitants to LST and presence of farm animals in the collecting stations LST pos. subjects Group

Station No.

I

II

Description

Cala Galera (Monte Argentaria)

2

Convent0 Frati (Monte Argentario)

3

Campone (Monte Argentaria)

4

Lividonia (Monte Argentario)

5

Parrina (Orbetello) Polverosa (Orbetello)

Farm house with 4 people Farm house with 11 people

Campana (Scansano) Poggio abechi (Campagnatico) Pianetto (Campagnatico)

Farm house with 9 people Farm house with 9 people Farm house in rural district, with 10 people Farm housewith 3 people Farm housewith 2 people Farm house with 8 people

6

8 9 10 11 12

S. Antonio (Campagnatico) Mille Miglia (Campagnatico) Cinressino (Caste1de1Piano)

__-

Subjects

1

7

III

Locality (Commune)

Farm animals

tested

Cattle

-

-

Uninhabited; about 500 m from summer resort and tourist port Uninhabited; about 50 m from monastery with about 20 people Uninhabited during night; surrounded by similar rural houses Dog housenext to old building with about 50 people: (Lividonia is a peripheral district of Port0 S. Stefano)

in frequency, while P. mascittii was very scarce. No or P. papatasi were collected. In the second group of stations (Nos. 5-6) north-west of Orbetello, P. perfiliewi was the only species collected and although not abundant, its highest number was collected at Polverosa. Jn the third group of stations (Nos. 7-12), scattered in a hilly, inland area, P. perfliewi was found to be very abundant, its highest number being collected at Poggio Abechi; S. minuta showed a much lower density and P. perniciosus even less, their highest numbers being also collected at Poggio Abechi. A few specimens of P. papatasi were collected at Cipressino and only one of P. mascittii at Campana. P. perfliewi

Frequency of species, or sex, according to different collecting methods P. perfiliewi. The first specimen of this species was

Sheep Pigs Fowl

Rabbits

Dogs

Cats

-

-

-

-

-

-

-

-

--

--

-

-

-

-

-

-

-

-

-

-

-

2

-

l/4

-

-

-

15

15

2

-

2/11

-

-

2

40

15

2

-

319

-

92

7

35

-

3

4

4/g

9

49

7

100

2

5

-

2/10

-

-

3

150

1

2

4

-

-

-

7

30

2

1

-

-

-

-

116

10

-

-

3 3

50

3 -

1 1

4

collected on June 16, the last on October 20. Of the 9,571 collected, 86.2% were caught with light traps, 5.9% with dark traps and 7.9% were hand collected. It was also found that the percentage of sexes varied according to the collecting method; male flies collected with light traps amounted to 55.9%, with dark traps to 61 .l “/, and by hand collecting to 20.8 % (Table IV). The graphs of Fig. 4 were plotted by using the data from the four collecting stations with the highest number of flies caught with light traps. The sandily frequency from June to October follows a bell-shaped trend in all four stations. Isolated peaks of male flies appear in all stations. P. perniciosus. The first specimen was collected on June 9, the last on October 22. Of the 1,193 specimens collected, 65.2% were caught with light traps, 27.5% with dark traps and 6.5% were hand collected. Male flies caught with dark traps were 93.6 %, with light traps

318

(i)

LEISHM.~~SIS~TUSC.~~:

INVESTIGATIONONPHLEB~T~MINJ~S.~~FL~~

Table II - Number of specimensof different phlebotomine species collected in the 12 stations from June to October 1975

Locality

Total of collected specimens

1 2 3 4

Cala Galera Convent0 Frati Campone Lividonia

1,004 15 34 221

5 6

Parrina Polverosa

Station No.

P. perfiliewi

16 107

16 107

P. pevniciosus

S. minuta

910 12 19 178

94 1 15 43

7 8 9 10

Campana Poggio Abechi Pianetto S. Antonio

2,780 3,334 103 572

2,753 3,218 96 566

10 29 5 6

16 87 2

11 12

Cipressino Mille Miglia

2,821 25

2,799 16

1:

1

Totals (% of total)

11,032

‘A of males

9,571 (86.7)

57.0

A

1,193 (10.8)

53.8

P. papatasi

2

1

6

259 (2.3)

81.1

P. mascittii

44.6

6

3

83.3

0.0

Table III - Frequency distribution of different species collected in the three groups of localities Groups of Localities

Total of collected specimens

1

1,274 123 9,635

II III

600

P. pevfiliewi

P. perniciosus

100~0% 98.0 %

q m

S. minuta

P. papatasi

P. mascittii

87.8 %

12.0%

-

0.15%

0;7%

l-1%

0.06%

O.G%

A

w

600

F’ M 500

500

400 i

400 300 200 100 0

JUN

’

JUL

’

AU8

’

SEP

’

OCT

JUM

’

JUL

’

AUO

’

OCT

Fig. 4. Trend of P. perJiliewi frequency in four collecting stations (light trap only), from June to October 1975. A: Station No. 8; B: StationNo. 7; C: StationNo. 10; D: StationNo. 12.

M.

MAROLI

AND

319

S. BETTINI

76.8 % and hand collected 71.7 % (Table IV). The graphs of Fig. 5 were plotted using data from two collecting stations (Cala Galera and Lividonia) with the highest number of flies (males and females) caught with light traps. The graphs show a gradual increase of fly density reaching a maximum in August to September after which the values drop abruptly. Two peaks due

mainly to males are observed in July and August to September. 5’. minuta. The first specimen was collected on June 25, the last on September 22. Of the 259 collected specimens, 52.1% were caught with light traps, 45.4% with dark traps and 2.3 % were hand collected. Male flies collected manually amounted to 50.0 %, with light traps to 47.4 %

Table IV - Frequency of samples, and male (M) percentage (in parenthesis), of different species collected with various methods. LT=ligbt traps, DT=dark trap, MC=manual collecting.

I

I

Species P. perfiliewi

(Ml P. perniciosus

CM) S. minuta

04) P. papatasi

Of) P. mascittii

(M)

86.2 (55.9)

(6::;)

65.2 (76.8)

27.5 (93.6)

(7%

52.1 (47.4)

43.5 (36.4)

2.3 (50.0)

16.6 (20.0)

66.6 (60.0)

16.6 (20.0)

66.6 -

33.6 -

“0 t 2

20

: 10

-

0 JUN

’

JUL

AUG

SEP

Fig. 6. Trend of S. minuta frequency in two collecting stations (light and dark traps, and manual collecting) from June to September 1975. A: Station No. 8; B: Station No. 1.

CID F M

JUN

JUL

BUG

SEP

OCT

Fig. 5. Trend of P. perniciosus frequency in two collecting stations (light trap only) from June to October 1975. A: Station No. 1; B: Station No. 4.

320

LELSHM~L~SISIN

TUSCANY: (i) INVESTIGATIONONPHLEBOTOMINESANDFLIES

and with dark traps to 36.4% (Table IV). The graphs of Fig. 6 were plotted on the data obtained from the two stations with the highest number of flies (males and females) collected by all three methods. Higher densities were registered in July to August. The small number of specimens collected does not allow an evaluation of the density trend. Little can be said about P. papatasi and P. mascittii of which very few were collected, only six and three. respectively. Discussion The investigation carried out in the Province of Grosset0 shows that high densities of sandflies occur in the areas of endemic leishmaniasis, and that a wide difference exists between the two dominant species P. perfiliewi and P. perniciosus in regard to the frequency of individuals, collected with the same techniques and under the same conditions, in various stations. This, however, does not prove, but only suggests, that actual differences in density exist between the two species in the localities under study. From the comparative findings with different collecting methods it appears that P. perfiiiewi seems to be much more attracted to light than does P. perniciosus and that S. minuta appears to be indifferent, the frequencies of light trap collected specimens being respectively 86.2, 65.2 and 52.1%. The above observations on P. perfiliewi, P. perniciosus and on S. minuta are in accordance with those of other authors [LIL’P (1952) and KOZHEVNIKOV et al. (1947), as reported by PERFILE'V (1968), and RIOUX & GOLVAN (1969)], though the attraction to light of different species probably varies according to geographical regions. From our results it appears also that light was more attractive to males of P. perniciosus than to males of P. perfliewi. The occurrence of “peaks” of male P. perfiliewi in the frequency distribution of flies from the localities where this speciesis abundant could be due to massive emergence of individuals. These peaks, however, did not appear at the same time in the different localities, their occurrence varying from July to the end of August. The second peak, observed at the end of September in S. Antonio, could be due to a second generation of the local sandfly population. Though this event is questionable, not having been reported elsewhere for P. perfiiewi, it is however possible considering that the pre-imaginal stages of this species last 46 to 56 days (PERFIL’EV,1968). If this is the case, it remains to be explained why the sudden increase of males was not immediately followed by that of females [phlebotomines sex ratio is 1.2 :l .O (KILLICK-KENDRICK et al., 1975)]. No increases in temperature preceding the male density peaks were recorded. A “diphasic evolution” of P. perniciosus, similar to the one shown by our graph, was also reported by ADLER & THEODOR (1931) for fed females caught during the morning hours in Catania, Sicily, by PARROTet al. (1933) in Algeria and by PETRISHCHEVA (1962) in Russia, but not by RIOUX & GOLVAN(1969) in southern France. The distribution of species in the area under study varies according to the groups of localities. In the territory of Monte Argentario P. perniciosus is very abundant (87.8%) and S. minuta less so (12.0%). In this area VL cases with prevalence in children have been reported throughout the years, but not a single case of CL has ever been recorded (BETTINI et al., 1977a; BETTINI et al., 1977b). Since S. minuta does not feed on man (ADLER & THEODOR,1957), P. perniciosus is suspected of being the only species responsible for VL

transmission there. This is in accordance with the findings of CORRADETTI(1962). In the second group of localities covering the coastal territory, P. perfiiewi was found to be the only species present. Cases of VL were recorded from the same localities where the collecting stations had been installed (BETTINI et al., 1977a). No cases of CL have recently been recorded from this territory. A case from Albinia (I 2 km from Orbetello), in a 60-year-old shepherd who had probably spent the summer with his flock on the inland hills, was reported by ZANCHI (1953). P. perfiliewi was also very abundant (98.0%) in the third group of localities (inland hills), while P. perniciosus was very scarce (0.7%). In the southern part of this area, cases of both VL and CL occurred in recent years (parasites were also cultured from an infected dog from Baccinello), while cases of CL only were reported from the northern part (Cipressino). Further north, in the Province of Siena, a focus of both VL and CL is present in the town of Asciano and environs (BETTINI ei al., 1977a) where, in a preliminary survey, P. perfilewi and P. perniciosus were found in about equal numbers. In the second and third group of localities, therefore, P. perfiliewi is strongly suspected of being the vector of CL, as it has been already indicated for the Abruzzi and Emilia-Romagna (VANNI, 1938, 1939; CORRADETTI, 1952, 1962; POGGI,1970). In the same area P. perfiliewi could also be suspected of playing a role in the transmission of VL. In northern Tunisia this species appears to be a secondary vector of canine Kala-azar (DANCESCOet al., 1970; DEDET, 1971) and in Serbia is suspected to transmit VL in man (ZNKOVIC, 1975) On the other hand P. perniciosus was either not found, or only occasionally collected by us in the stations of the second and third groups of localities respectively. This species was proved, by ADLER & THEODORduring their work on the Kala-azar Commission of the Royal Society, London, 1931-35: to be the vector of VL in Malta, Sicily and southern Italy (THEODOR,1976). Even although it is difficult at this stage to implicate any species other than P. perJiliewi, the only species present, as the vector of VL in the localities of Parrina and Polverosa where at least one patent case of the disease has occurred, the low number of collecting stations placed in our area cannot give a satisfactory picture of the local phlebotomine population. ADLER & THEODOR(1931) in fact reported that the distribution of P. perniciosus was “irregular” in the sandfly area of Catania. The need for a more accurate study of the sandfly distribution, to determine the role played by each species in leishmaniasis transmission, is supported by the observations of PAMPIGLIONEet al. (1975, 1976) on the “focal endemicity” of both VL and CL. On the other hand, the fact that a species of Phlebotomus is common in a “Kala-azar house” (or its density in different traps is high, as in our case) is no proof that it is the vector of the disease (THEODOR, 1976). Studies of arthropod-host relationships are, therefore, much needed. A similar problem was present in the Emilia-Romagna region where in 1971-72 an outbreak of VL occurred (PAMPIGLIONEet al., 1974). The survey carried out by KILLICK-KENDRICK (1973) showed that P. perfliewi was by far the most abundant species present in some localities where autochtonous cases had been reported. In the areas where P. perniciosus was found in low numbers, its role in transmission could be justified only if its transmission capacities were very high as compared with those of other more abundant species.

M.

MAROLI

Acknowledgements We thank Dr. D. J. Lewis for his critical review of the manuscript. Thanks are also due to Mr. R. Fei and Mr. E. Vichi for their technical help. The present work was made possible by the facilities placed at our disposal by the Laboratorio provinciale di Zoologia Medica, Grosseto. Bibliography Adler, S. & Theodor, 0. (1931). Investigation on Mediterranean Kala-azar. III. The sandflies of the Mediterranean basin. Distribution and bionomics of sandflies in Catania and District. Proceedings of the Royal Society London, B, 108,464480. Adler, S. & Theodor, 0. (1957). Transmission of disease agents by Phlebotomus sandflies. Annual Review of Entomology,

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Dedet, J. P. (1971). EpidCmiologie de la leishmaniose viscerale en Tunisie. Etude des r&,ervoirs de virus. Incidence et r&partition de la leishmaniose canine. Dr. de Sp&ialitB, thesis. Universite Languedoc, France, 163 pp. Killick-Kendrick, R., Leaney, A. J. & Ready, P. D. (1975). Methods for colonization of the phlebotomid sandfly Lutzomyia longipalpis. WHO/Leish.75.12. [Unpublished document.] Killick-Kendrick, R., Ready, P. D. & Pampiglione, S. (1973). Notes on the prevalence and host preferences of Phlebotomus perfiliewi Parrot, 1930 in EmiliaRomagna Italy. (Unpublished.) Pampiglione, S., La Placa, M. & Schlick, G. (1974). Studies on Mediterranean leishmaniasis. I. An outbreak of visceral leishmaniasis in northern Italy.

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Rioux, J. A. & Golvan, Y. J. (1969). Epid&miologie des leishmanioses dam le sud de la France. Paris: Institut National de la SantB et de la Recherche M6dicale. Monographie No. 37, p. 223. Rioux, J. A., Golvan, Y. J., Croset, H., Houin, R., Juminer, B., Bain, 0. & Tour, S. (1967). Ecologic des leishmanioses dans le sud de la France. I. Les Phlhbotomes. Echantillonnage-Ethologie. Annales de Parasitologie Humaine et CornpaGe, 42, 561-603.

Theodor, 0. (1958). Die Fliegen der Palaearktischen Region. Lief. 201. 9c. Psvchodidae. Phlebotominae. E. >chwrizerbart’sche ~erla&buchhan~lung, pp. l-55. ’ Theodor, 0. (1976). Transmission of kala-azar in Malta. Transactions of the Royal Society of Tropical Medicine and Hygiene, 70,259-260.

Vanni, V. (1938). Ricerche sulla leishmaniosi cutanea endemica degli Abruzzi. Annali d’lgiene, Roma, 48, 520-528. Vanni, V. (1939). Osservazioni e ricerche sperimentali in una endemia di leishmaniosi cutanea. Memorie della Reale Accademia d’ltalia, 10, 87-104. Zanchi, M. (1953). Sulla diffusione della leishmaniosi cutanea nelle provincie di Siena e Grosseto. Medicina Internazionale, 61,208-212. Zivkovic, V. (1975). Recherches rkcentes sur les phl& botomes (Diptera, Psychodidae) dans un foyer endemique de leishmaniose viscerale en Serbie (Yougoslavie). Acta Parasitologica Jugoslavica, 6, 37-43. Accepted for publication

21st April, 1977.