phlebotomine sand flies (diptera: psychodidae) associated
TRANSCRIPT
3535353535Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 102(1): 35-40, February 2007
Phlebotomine sand flies (Diptera: Psychodidae) associated withchanging patterns in the transmission of the human cutaneous
leishmaniasis in French GuianaFlorence Fouque/+, Pascal Gaborit*, Jean Issaly*, Romuald Carinci*,Jean-Charles Gantier**, Christophe Ravel***, Jean-Pierre Dedet***
Cellule d�Intervention Biologique d�Urgence, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris cedex 15, France*Laboratoire d�Entomologie médicale, Institut Pasteur de la Guyane, Cayenne cedex, French Guiana **Laboratoire de ParasitologieMycologie, Faculté de Pharmacie, Châtenay-Malabry, France ***Laboratoire de Parasitologie, Centre National de Référence des
Leishmania, Montpellier, France
Between March 2000 and December 2001 a survey of the sand flies (Diptera: Phlebotominae) of FrenchGuiana was carried out during 14 nights of captures with CDC light-traps and Malaise traps, and resulted inthe collection of 2245 individuals of 38 species. The most abundant species were Lutzomyia (Trichophoromyia)ininii Floch & Abonnenc, Lu. (Psychodopygus) squamiventris maripaensis Floch & Abonnenc, and Lu. (Nyssomyia)flaviscutellata Mangabeira. Half of the collected sand flies females were dissected under field conditions andfive species were found harboring Leishmania-like parasites. The Leishmania (Kinetoplastidae: Trypa-nosomatidae) species were identified by molecular typing, and for the first time Lu. (Nys.) flaviscutellata wasfound harboring Leishmania (Viannia) guyanensis and Lu. (Tri.) ininii harboring unknown Leishmania. The firstrecord for French Guiana of Lu. (Psy.) squamiventris maripaensis harboring L. (V.) naiffi, was also reported. Thepatterns of diversification of the human cutaneous leishmaniasis transmission in French Guiana are discussed.
Key words: sand flies - Lutzomyia - Leishmania - human cutaneous leishmaniasis - French Guiana
With about 300 cases of human cutaneous leishma-niasis (HCL) reported annually (Carme 2001, Lightburnet al. 2001) and some extreme situations (Couppié et al.2004), this disease still represents a major public healthproblem in French Guiana. The first HCL cases wereconfirmed in French Guiana in 1943 (Floch 1943) andpast studies showed that Leishmania (Viannia) guy-anensis is the predominant species of Leishmania(Floch 1957, Dedet et al. 1989, Raccurt 1996) but L.(L.) amazonensis, L. (V.) braziliensis, L. (V.) lainsoni,and L. (V.) naiffi have also been diagnosed (Dedet et al.1985, Raccurt et al. 1995, Basset et al. 2001, Pratlonget al. 2002). The sand flies of French Guiana have beenextensively studied until the 1990s and 77 sand fliesspecies were reported (Floch & Abonnenc 1952, Légeret al. 1977). The entomological studies have shown thatthe sand fly Lutzomyia (Nyssomyia) umbratilis Ward &Fraiha was the major vector of L. (V.) guyanensis (LePont & Pajot 1980, Pajot et al. 1982). The other sandflies species found infected with Leishmania parasiteswere Lu. (Nys.) flaviscutellata Mangabeira, harboringL. (L.) amazonensis (Dedet et al. 1989) and Lu. (Lut.)gomezi Nitzulescu and Lu. (Psy.) yullii pajoti Abonnenc,
Financial support: The French Ministry of Health+Corresponding author: [email protected] 1 June 2006Accepted 21 November 2006
Léger & Fauran harboring unidentified Leishmania spe-cies (Killick-Kendrick 1990, Young & Duncan 1994).The sand flies species transmitting L. (V.) braziliensis,L. (V.) lainsoni, and L. (V.) naiffi are partly known in otherSouth-American countries (Naiff et al. 1991, Young &Duncan 1994, Feliciangeli 2004), but have not been sofar identified in French Guiana. Furthermore, in the lastten years, the geographical distribution of the HCL trans-mission in French Guiana has changed, previous foci havedisappeared and new foci are reported. The people at riskfor HCL contamination have also changed and now themilitary personnel represent 25 to 85% of the annualnumber of cases (Lightburn et al. 2001). Finally, the en-vironment of French Guiana is continuously changingdue to human pressure on the forested area for road con-struction, housing development, and gold mining.
For these reasons, a research program to update ourknowledge on the HCL transmission in French Guianawas started in 2000. The objectives of the program wereto identify the species of sand flies acting as vector ofHCL in the probable contamination places, to identifythe Leishmania parasites when present in the collectedsand flies, to characterize the foci and finally to collectinformation on the biology and ecology of the species.
MATERIALS AND METHODS
The study sites - From March 2000 to December2001, 14 collections of sand flies were carried out indifferent areas of French Guiana. The prospected loca-tions were chosen in relation with the report of HCLcases during the past weeks or the month before the cap-
3636363636 Leishmaniasis vectors in French Guiana � Florence Fouque et al.
tures. The report of the HCL cases remained anonymousand only geographical locations were given by the Di-rection Départementale des Affaires Sanitaires et So-ciales (DDASS) reports and the military headquartersfor the study. The procedures of the report of humancases were in accordance with the ethical standards andthe research program was approved by the DDASS ofFrench Guiana representing the French Ministry ofHealth.
Different ecological conditions of French Guianawere prospected for sand flies collection at differentperiods of the year. The forested areas along the littoralat Regina were prospected in March, April, October, andNovember 2000, and at Nancibo in December 2001 (Fig.1). The forested areas in the interior of the country atMaripasoula and the Upper Maroni River were pros-pected in October 2000 and at Saül in December 2000.The savannas areas at Montsinery were prospected inFebruary and March 2001 and at Sinnamary in Septem-ber 2001. Finally, some agricultural areas were also pros-pected at Cacao in June 2001, Montsinery in July 2001and Saül in October 2001 (Fig.1, Table).
The sand flies collections and species identifica-tion - Each collection of sand flies included two con-secutive nights of capture with three types of traps, oneCDC light-trap (Bioquip model), one modified CDClight-trap (with a green plastic hat and a more powerfullight-bulb, Gantier model) and one Malaise trap. One ortwo traps of each type were left between 6 p.m. and 7a.m. in different places (close to trees, bushes, armadil-los� hole, etc.) and at different heights, varying from 0.30to 10 m. The sand flies were collected in the traps atregular intervals during the nights. The intervals were inmost cases, each 3 h, which is at 9 p.m., 12 p.m., 3 a.m.,and 6 a.m.
The collected sand fly females were dissected underfield conditions just after the empting of the trap, tosearch for natural infection to Leishmania spp. The in-fected sand flies were kept in Dulbecco�s ModifiedEagle�s Medium (ref D5523, Sigma Chemicals Co.)supplemented with fetal bovine serum (ref F2442, Sigma)and brought to the Laboratory of Immunology of the Pas-teur Institute of French Guiana for a tentative culture ofthe Leishmania parasite. Some of the positive samples
Fig. 1: map of French Guiana with the areas prospected (underlined) during the sand flies survey from March 2000 to December 2001.
3737373737Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 102(1), February 2007
TAB
LELi
st of
the s
and
flies
spec
ies c
olle
cted
bet
wee
n M
arch
200
0 an
d D
ecem
ber 2
001
in F
renc
h G
uian
a. A
mon
g th
e 14
colle
ctio
ns, i
n on
ly o
ne co
llect
ion
the i
ndiv
idua
ls co
uld
not b
e ide
ntifi
ed.
The n
umbe
rs in
par
enth
esis
are t
he n
umbe
r of d
isse
cted
fem
ales
for t
he sp
ecie
s fou
nd p
ositi
ve fo
r Lei
shm
ania
par
asite
s. Th
e rep
ort o
f vec
tor s
peci
es is
bas
ed o
n Yo
ung
and
Dun
can
(199
4)
Plac
e an
d da
te o
f col
lect
ion
Tota
l
Régi
naM
aron
i Riv
erSa
ülM
onts
iner
yC
acao
Sinn
amar
yN
anci
bo
Mar
chA
pril
Oct
ober
Nov
embe
rO
ctob
erD
ecem
ber
Oct
ober
Febr
uary
Mar
chJu
lyJu
neSe
ptem
ber
Dec
embe
r20
0020
0020
0020
0020
0020
0020
0120
0120
0120
0120
0120
0120
01
List
of s
peci
esm
fm
fm
fm
fm
fm
fm
fm
fm
fm
fm
fm
fm
fm
f
1 B
rum
ptom
yia (B
rum
ptom
yia) p
into
i (Co
sta L
ima 1
932)
11
0 2
Bru
mpt
omyia
(Bru
mpt
omyia
) tra
vass
osi (
Man
gabe
ira 1
942)
11
11
3 L
utzo
myia
(Ara
.) ar
agao
i (Co
sta L
ima 1
932)
22
12
33
7 4
Lut
zom
yia (A
ra.)
bras
ilien
sis (C
osta
Lim
a 193
2)1
01
5 L
utzo
myia
(Dre
.) dr
eisba
chi (
Caus
ey &
Dam
asce
no 1
945)
31
121
193
28
42
3529
6 L
utzo
myia
(Eva
.) br
achy
phal
la (M
anga
beira
1941
)1
43
11
41
17
9 7
Lut
zom
yia (E
va.)
infra
spin
osa (
Man
gabe
ira 1
941)
23
43
413
510
34
32
58
181
231
59 8
Lut
zom
yia (E
va.)
mon
struo
sa (F
loch
& A
bonn
enc 1
944)
11
12
14
9 L
utzo
myia
(Mic.
) micr
opyg
a (M
anga
beira
1942
)4
04
10 L
utzo
myia
(Mig
.) pa
cae (
Floc
h & A
bonn
enc 1
943)
11
11
11 L
utzo
myia
(Nys
.) an
duze
i (Ro
zebo
om 1
942)
a1
10
12 L
utzo
myia
(Nys
.) fla
viscu
tella
ta (M
anga
beira
1942
) b1
11
22 (7
)3
153
25
56
16
725
104
3816
9 (4
3)13
Lut
zom
yia (N
ys.)
umbr
atili
s (W
ard
& F
raih
a 197
7) a
81
11
117
274
12
97
181
22
3864
14 L
utzo
myia
(Nys
.) yu
illi p
ajot
i (A
bonn
enc,
Lége
r & F
aura
n 197
9)7
2435
2442
15 L
utzo
myia
(Osw
.) ro
rota
ensis
(Flo
ch &
Abo
nnen
c 194
4)1
68
11
98
16 L
utzo
myia
Pre
ssat
ia sp
.1
01
17 L
utzo
myia
(Pre
.) ch
oti (
Floc
h &
Abo
nnen
c 194
1)2
11
11
418
Lut
zom
yia P
sath
yrom
yia sp
.1
01
19 L
utzo
myia
(Psa
.) pu
nctig
enicu
lata
(Flo
ch &
Abo
nnen
c 194
4)1
01
20 L
utzo
myia
(Psa
.) sh
anno
ni (D
yar 1
929)
11
20
21 L
utzo
myia
(Psy
.) am
azon
ensis
(Roo
t 193
4)1
01
22 L
utzo
myia
(Psy
.) ay
roza
i (Ba
retto
& C
outin
ho 1
940)
21
11
1010
523
Lut
zom
yia (P
sy.)
claus
trei (
Abo
nnen
c, Lé
ger &
Fau
ran 1
979)
64
103
29
53
21
2144
3872
24 L
utzo
myia
(Psy
.) co
rros
soni
ensis
(Le P
ont &
Pajo
t 197
8)1
01
25 L
utzo
myia
(Psy
.) da
visi (
Root
193
4)1
11
126
Lut
zom
yia (P
sy.)
guya
nens
is (F
loch
& A
bonn
enc 1
941)
21
03
27 L
utzo
myia
(Psy
.) hi
rsut
a (M
anga
beira
1942
)6
11
215
21
116
2038
2728
Lut
zom
yia (P
sy.)
nocti
cola
(You
ng 19
73)
34
14
429
Lut
zom
yia (P
sy.)
pana
men
sis (S
hann
on 1
926)
c1
25
26
30 L
utzo
myia
(Psy
.) pa
raen
sis (C
osta
Lim
a 194
1)4
32
11
5374
5682
31 L
utzo
myia
(Psy
.) sq
uam
ivent
ris m
arip
aens
is
(Flo
ch &
Abo
nnen
c 194
6) d
18
114
714
12
132
13 (
3)64
153
7720
7 (5
6)32
Lut
zom
yia (S
ci.) f
luvia
tilis
(Flo
ch &
Abo
nnen
c 194
4)1
12
22
33 L
utzo
myia
(Sci.
) sor
delli
(Sha
nnon
& D
el Po
nte 1
927)
11
67
11
11
32
25
41
31
117
2434
Lut
zom
yia (T
ri.) b
rach
ypig
a (M
anga
beira
194
2)1
10
35 L
utzo
myia
(Tri.
) flo
chi (
Abo
nnen
c & C
hass
igne
t 194
8)1
10
36 L
utzo
myia
(Tri.
) ini
nii (
Floc
h & A
bonn
enc 1
943)
1520
3452
135
7168
23
839
51 (3
3)23
143
10 (1
0)3
12
320
823
2(13
6)37
Lut
zom
yia (T
ri.) u
biqu
italis
(Man
gabe
ira 1
942)
e22
108
65
156
22
32
32
41
310
624
38 L
utzo
myia
(Tri.
) try
chop
yga
(Flo
ch &
Abo
nnen
c 194
5)2
41
11
31
21
124
39 T
otal
4635
4779
197
148
132
1551
126
9310
291
8732
3718
5330
4426
2719
942
076
511
00
a: v
ecto
r of L
. (V.)
guy
anen
sis; b
: vec
tor o
f L. (
L.) a
maz
onen
sis;c
: vec
tor o
f L. (
V.) b
razil
iensis
;d: v
ecto
r of L
. (V.)
naiff
i.
3838383838 Leishmaniasis vectors in French Guiana � Florence Fouque et al.
were also sent to the Laboratory of Parasitology of theFaculty of Medicine of Montpellier for identificationby molecular typing, which was carried out by system-atic sequencing of a part of the RNA pol II large sub-unitgene.
All sand flies collected were brought to the Labora-tory of Entomology and processed for identification.Processing and mounting of the sand flies males and fe-males was made according to the following protocol(Tran-Kiem 2000): the individuals were rinsed in dis-tilled water until flotation, then left 10 min in the warmedup Marc-André Liquid (distilled water 300 g, chloralhydrate 400 g, acetic acid 300 g) for lightening, thenrinsed twice with alcohol 90°, then mounted in a drop ofCreosote, the exceeding of Creosote was removed andthe Euparal mounting liquid was added. The species werethen identified firstly with the informatics� key (Lebbeet al. 1987) and confirmed with the available descrip-tions (Floch & Abonnenc 1952, Young & Duncan 1994).The Laboratory of Mycology of the Faculty of Pharmacyof Châtenay-Malabry (Paris IX) further confirmed thespecies identifications. The molecular confirmation ofthe Lutzomyia species was not performed due to the ab-sence of reliable methods for the sand flies species ofFrench Guiana.
RESULTS
A total of 14 collections were carried out and 2245sand flies were collected including 1265 females, and980 males, among which 1865 individuals (83%) wereidentified (1100 females and 765 males) (Table). A lotof individuals (17%) could not be identified because theywere either damaged during the transportation betweenthe field and the laboratory, damaged during the labora-tory process of lightening and mounting, or because thespecimens could not be attributed to a species descrip-tion. A total of 38 species were collected, which is closeto the 43 species reported during the last sand flies sur-
vey in French Guiana (Léger et al. 1977), and about halfof the 78 species previously described from FrenchGuiana (Floch & Abonnenc 1952, Oliveira et al. 2001).The most abundant species in our survey were Lu. (Tri.)ininii, Lu. (Psy.) squamiventris maripaensis and Lu.(Nys.) flaviscutellata and the 3 species were found har-boring Leishmania-like flagellates parasites (Table). Themost abundant species reported during the 1977 surveywere Lu. (Tri.) ubiquitalis Mangabeira followed by Lu.(Tri.) ininii, and Lu. (Psy.) squamiventris maripaensis.Thus, the overall results do not show any significantchanges in the species diversity and densities, exceptfor the high density of Lu. (Tri.) ubiquitalis collected inMaripasoula in 1977 (Léger et al. 1977). The collectionmade in Maripasoula during our survey resulted in 32individuals that could not be identified.
About half (534 females, i.e. 48.5%) of the sand fliesfemales collected were dissected in the field, examinedfrom the presence of Leishmania parasites and placedon culture when found positive. Five sand flies femaleswere found harboring Leishmania-like flagellates para-sites, with an overall infection rate of 0.84%. Four posi-tive sand flies could be identified. No Leishmania cul-ture was successful, but molecular typing on four of theseLeishmania field samples allowed the identification of2 Leishmania species. One female of Lu. (Nys.) fla-viscutellata was found harboring L. (V.) guyanensis andone female of Lu. (Psy.) squamiventris maripaensis wasfound harboring L. (V.) naiffi. The other two moleculartyping were significant of a Leishmania genus, but thespecies were not identified.
The highest sand flies density and species diversitywere found during high and medium rainfalls periods, innatural and forested areas (December 2001 at Nancibo).In the contrary, the lowest sand flies density were foundduring the dry season, in forested and savanna areas, sub-mitted to important ecological changes due to militaryactivities (Regina in August and October 2000), agricul-
Fig. 2: fluctuations in the densities of the sand flies collected in French Guiana between March 2000 and December 2001, in the disturbed forested areasof Regina, Saul, and Cacao (-!-) and in the savannah areas submitted to agricultural pressure of Montsinery and Sinnamary (-!-) with the monthlyrainfall fluctuations in the location of the collection. The numbers in parenthesis represent the infection rates of the infected species.
3939393939Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 102(1), February 2007
tural practices (Sinnamary in September 2001), and goldmining activities (Saül in December 2000) (Fig. 2). Theinfected sand flies females were collected in Octoberand November 2000 at Regina, in February and July 2001at Montsinery and in September 2001 at Sinnamary.
DISCUSSION
This is the first report of Lu. (Nys.) flaviscutellataharboring L. (V.) guyanensis. This species is also con-sidered a proven vector of L. (L.) amazonensis (Killick-Kendrick 1990). This positive sand fly was collected inRegina in November 2000, and the overall infection ratefor this species was 2.32%. During the same capture,only one female of Lu. (Nys.) umbratilis, the provenvector of L. (V.) guyanensis in French Guiana (Le Pont& Pajot 1980) was collected (Table). The second spe-cies of Leishmania identified, L. (V.) naiffi was found ina female of Lu. (Psy.) squamiventris maripaensis. Thissand fly species was reported as a vector of L. (V.) naiffiin Brazil (Naiff et al. 1991), but this is the first time thatLu. (Psy.) squamiventris maripaensis can be consid-ered a vector of L. (V.) naiffi in French Guiana, where theHCL species has already been reported in human cases(Basset et al. 2001, Pratlong et al. 2002). The positivefemale of Lu. (Psy.) squamiventris maripaensis wascollected in September 2001 in the savanna area ofSinnamary and the overall infection rate for this specieswas 1.78%. Two females of Lu. (Tri.) ininii were foundharboring unidentified Leishmania flagellates with anoverall infection rate of 1.47%, and this could be thefirst report of Lu. (Tri.) ininii as probable vector of HCL.
The results are showing a natural Leishmania circu-lation in French Guiana expanding all year long and wide-spread in the country, in particular in areas submitted toimportant ecological changes due to military activities(Regina) and agricultural practices (Montsinery andSinnamary). The infected sand flies were collected dur-ing periods with low rainfalls (October 2000 and Sep-tember 2001) and medium rainfalls (November 2000 andFebruary 2001). However, the highest infection rateswere found in September 2001 at Sinnamary (0.33) andNovember 2000 at Regina (0.14). Furthermore, whenthe densities of the potential vectors are observed, Lu.(Nys.) flaviscutellata was most abundant in November2000 (at Regina) and December 2001 (at Nancibo), Lu.(Tri.) ininii was must abundant in November 2000 (atRegina) and Lu. (Psy.) squamiventris maripaensis wasmost abundant in December 2001 (at Nancibo), confirm-ing a transmission season mostly at the end of the yearwhen the dry season is also ending and the rainfalls startto increase (Fig. 2). Some HCL foci could be detectedat Regina, Montsinery and Sinnamary, with not only re-port of clinical cases but also presence of infected sandflies.
Although, the annual HCL incidence do not show arecent increase in French Guiana because 2.3 cases per1000 inhabitant were reported in 1989 (Dedet 1990)against 1.5 to 2 cases per 1000 inhabitant reported be-tween 1986 and 2000 (Carme et al. 2001), the transmis-sion patterns are changing with an increase in the num-ber of Leishmania species diagnosed (Basset et al. 2001)
and the report of new sand flies vector. HCL is probablyendemic in the Amazonian region since the arrival of thefirst human beings (Andrade Fiho & Brazil 2003) andthis is demonstrated by the great diversity of HCL trans-mission found in this region. French Guiana is includedin the Amazon geographic area and the small number ofLeishmania parasites and sand flies vectors previouslyfound in this country may be due to a report of casesmostly for the population living along the littoral area.Actually, French Guiana is submitted to an importanthuman pressure of deforestation for gold mining, roadconstruction and agricultural practices. Consequently,the population exposed to the natural leishmaniasis for-ested cycles is increasing and the HCL transmission isgaining in diversity. The first report of three sand fliesspecies found infected with Leishmania parasites inFrench Guiana, Lu. (Nys.) flaviscutellata for L. (V.)guyanensis, Lu. (Psy.) squamiventris maripaensis forL. (V.) naiffi, and Lu. (Tri.) ininii for unknown Leishma-nia species is the major finding of this survey, and sug-gests that more extensive studies should be undertakenfor a better knowledge of HCL transmission patternsand prevention in the Amazonian ecosystem of FrenchGuiana.
ACKNOWLEDGEMENTS
To the technicians of the Centre National de Reference desLeishmania from CHU Montpellier for molecular typing of theLeishmania parasites. To the DASS-Guyane and the FrenchArmy Headquarters in French Guiana for providing informationon the cases as well as their help in some restricted forestedareas. To Dr Patrick Rabarison of the Laboratory of MedicalEntomology of the Pasteur Institute of French Guiana, Dr Jean-Louis Sarthou, former Director of the Pasteur Institute of FrenchGuiana for his support, Dr Jacques Morvan, Director of the Pas-teur Institute of French Guiana, and the Laboratory of Immunol-ogy of the Pasteur Institute of French Guiana for the tentativecultures of Leishmania and the support in the work.
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