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114
and Petrarca, 1973). The female mosquitoes sampled in
2000 were immediately killed and fixed in 70% ethanol,
while in 2001 and 2002 were kept under controlled conditions
(25-27°C, ~90% r.h.) for 5 days and then killed,
identified in accordance with keys proposed by Snow
(1987) and fixed in 70% ethanol. The presence of filarial
parasites in mosquitoes (grouped for species and sampling
area) was evaluated by PCR examination of the
specimens in pools (usually 10 specimens each for insects
collected in 2000 and 2001, and 5 each for those caught
in 2002). DNA extraction was performed separately on
the insect abdomen and thorax-head to discriminate
between Dirofilaria infected/infective specimens. Pooled
samples were analysed with specific ribosomal primers
named S2-S16. DNA sequencing confirmed species
assessment (MWG-Biotech.) and sequence comparison
was achieved by CLUSTAL W analysis (Thompson et al.,
1997). Minimum infection rates (MIRs) were calculated
Assuming the presence of only one positive
mosquito/positive pool, MIRs resulted of 2.67%
(19/713) in summer 2000, 3.29% (40/1,216) in summer
2001 and 3.64% (22/605) in summer 2002. All
studied areas harboured infected mosquitoes. Specific
primers and sequencing identified all filarial DNA as
belonging to D. immitis.
Discussion
Aedes albopictus was the most abundant species in controlled
sites. In summer 2000 PCR-based technologies
of 713 specimens, allowed the detection of 27.5% pools
infected by D. immitis but lacking of positive thoraxheads
didn’t allow defining the actual value of this mosquito
as a natural vector for D. immitis. Specimens collected
in 2001 and 2002 were kept for 5 days to allow
that the development is overcome or, in case of incom-
M. Pietrobelli - Importance of Aedes albopictus
by the standard formula: number of positive mosquito
pools/total number of mosquitoes tested x 100.
According to a binomial distribution of the parasites,
expected infection rates (P) were also evaluated and calculated
as follows: P=1- k
n/N, where n is the number of
negative pools, N is the number of tested pools and k is
the average number of specimens in each pool.
Results
A total of 2,721 specimens were caught in the whole
sampling period; as expected, human-attracted mosquitoes
were almost all (97.1%) Ae. albopictus
(2,534). Results concerning Ae. albopictus sampling
are reported in table 1.
Results of PCR analyses on pools of all collected Ae.
albopictus, MIRs observed and P values are shown in
table 2.
Table 1. Aedes albopictus females collected in summer 2000, 2001 and 2002 in three sampling sites of Padova town (Veneto
Region, Italy) on human bait, and exposition time to bites.
Year Psychiatric Hospital Urban Park Botanical Garden Total
2000
2001
2002
Mosquit.
(no.)
125
60
-
Exposition
to bites (hours)
4
4
-
Mosquit.
(no.)
283
8
-
Exposition
to bites (hours)
16
2
-
Mosquit.
(no.)
305
1,148
605
Exposition
to bites (hours)
10
24
14
Mosquit.
(no.)
713
1,216
605
Exposition
to bites (hours)
Total 185 8 291 18 2,058 48 2,534 74
Table 2. Minimum infection rates (MIRs) of Dirofilaria immitis evidenced by PCR and expected infection rates (P) in pools of Aedes
albopictus females caught while landing on man (Padova town, Veneto Region, Italy).
Year
2000
2001
2002
Specimens
(no.)
713
1,216
605
Pool size
(min. – max.)
8 - 12
1 - 11
4 - 5
A = abdomen; ATH = abdomen + thorax-head ; TH = thorax-head
Positive / tested
pools
19/69 (A = 19)
40/144 (A=24; ATH=8; TH=8)
22/123 (A=16; ATH=2; TH=4)
MIRs
2.67
3.29
3.64
30
30
14
P
(95% C.I.)
3.07 (1.99 – 4.71)
3.78 (2.81 – 5.06)
3.93 (2.61 – 5.93)
petent host, the microfilariae are expelled yielding a
negative PCR. In fact it has been shown that the insect
defensive mechanisms against dirofilariae are efficient
only on microfilariae recently ingested or penetrated in
primary cells of the Malpighian tubules. Similar results
were confirmed in Central Italy also for D. repens
(Cancrini et al, 2003 and 2007). Results prove the risk
for heartworm disease in the town of Padova and support
the hypothesis that the stable presence of Ae.
albopictus should increase the probability of transmission
of canine and human dirofilariosis in urban environment.
The circulation of filarial nematodes among
animals might be improved and enhanced and, considering
the aggressive anthropophylic behaviour of the
species (30-48 bites/hour) proven in Padova town,
transmission from animals to humans, enhanced.
The role of Ae. albopictus as an efficient vector could
change the epidemiology of Dirofilariosis, in particular