Phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern.

Phlebotomine sandflies transmit pathogens that affect humans and animals worldwide. We review the roles of phlebotomines in the spreading of leishmaniases, sandfly fever, summer meningitis, vesicular stomatitis, Chandipura virus encephalitis and Carrión's disease. Among over 800 species of sandfly recorded, 98 are proven or suspected vectors of human leishmaniases; these include 42 Phlebotomus species in the Old World and 56 Lutzomyia species in the New World (all: Diptera: Psychodidae). Based on incrimination criteria, we provide an updated list of proven or suspected vector species by endemic country where data are available. Increases in sandfly diffusion and density resulting from increases in breeding sites and blood sources, and the interruption of vector control activities contribute to the spreading of leishmaniasis in the settings of human migration, deforestation, urbanization and conflict. In addition, climatic changes can be expected to affect the density and dispersion of sandflies. Phlebovirus infections and diseases are present in large areas of the Old World, especially in the Mediterranean subregion, in which virus diversity has proven to be higher than initially suspected. Vesiculovirus diseases are important to livestock and humans in the southeastern U.S.A. and Latin America, and represent emerging human threats in parts of India. Carrión's disease, formerly restricted to regions of elevated altitude in Peru, Ecuador and Colombia, has shown recent expansion to non-endemic areas of the Amazon basin.

Aspects of the bionomics of Phlebotomus sergenti sandflies from an endemic area of anthroponotic cutaneous leishmaniasis in Aleppo Governorate, Syria.

Aspects of the bionomics of phlebotomine sandflies (Diptera: Psychodidae) were studied from June to November 2005 in three foci of anthroponotic cutaneous leishmaniasis (ACL) in Aleppo Governorate, Syria, where the agent Leishmania tropica (Wright) (Kinetoplastida: Trypanosomatidae) is transmitted by Phlebotomus sergenti Parrot. Syria has been designated by the World Health Organization as one of four countries in the Old World where cutaneous leishmaniasis is hyperendemic, but little is known about the biology of local vector populations. Standard collections by sticky traps showed two peaks in density, in June and late August. In total, 1840 sandflies were caught, comprising five species: Phlebotomus papatasi (Scopoli) (68.0%); P. sergenti (25.4%); Sergentomyia minuta (Rondani) (6.4%); Phlebotomus tobbi Adler & Theodor (0.1%), and Phlebotomus mascittii canaaniticus Adler & Theodor (0.1%). Similar numbers of P. sergenti were caught indoors (246 specimens) and outdoors (222), whereas P. papatasi was significantly more abundant indoors (1096 specimens) than outdoors (156) (chi(2) = 241, P < 0.01). In total, 212 blood-fed females were tested for host blood determination, of which 176 (83.0%) reacted with anti-species reagent. Results from 20 P. sergenti suggest that this species is an opportunistic feeder, imbibing human, ovine, avian, bovine and feline blood, although more bloodmeals were taken from humans and cattle than expected in relation to the relative proportions of potential hosts present (the forage ratio, FR). The bionomics of P. sergenti are discussed in relation to the inefficacy of control campaigns based on indoor spraying with residual insecticides that have been implemented by the Syrian Ministry of Health to control the epidemics of ACL in the Aleppo Governorate.

Seasonal phenology, host-blood feeding preferences and natural Leishmania infection of Phlebotomus perniciosus (Diptera, Psychodidae) in a high-endemic focus of canine leishmaniasis in Rome province, Italy.

A 2-year survey aimed to study seasonal phenology, host-blood feeding preferences and Leishmania infections of Phlebotomus perniciosus, was carried out in a high-endemic focus of canine leishmaniasis in Rome province, Italy. Sandfly densities were monitored by sticky traps, while CDC light traps and hand collections were used for individual sandfly analyses. Four species were identified, three belonging to Phlebotomus (P. perniciosus, Phlebotomus mascittii and Phlebotomus papatasi) and one to Sergentomyia (Sergentomyia minuta) genera. In sticky traps, P. perniciosus (53.5%) and S. minuta (46.0%) were the prevalent species, whereas specimens collected by CDC light traps and hand collections consisted mostly of P. perniciosus. The study showed a markedly different sandfly density between 2003 and 2002 collections (5024 specimens versus 644 specimens, respectively), which was attributed to different climatic conditions. In 2003 adults were active during 7 months (May-November) with a typical bimodal distribution, while in 2002 their activity was shorter (5 months, June-October) without clear density peaks. In this low-density year, the prevalence of P. perniciosus was significantly higher in domestic than in wild resting sites. Blood meal tests showed that P. perniciosus had no preferences for any of the two Leishmania susceptible hosts, being the forage ratio rate 0.7 and 1.0 for man and dog, respectively. A significant number of P. perniciosus was found with avian (60.3%) or ovine (24.2%) blood, that was in relation with the habitats where fed specimens were collected. The rate of specimens of P. perniciosus without blood and found positive for Leishmania DNA, detected by a sensitive nested-PCR technique, was much higher (27.6%) than the promastigote infection rate determined by microscopy in the dissected females (1.4%). The intense Leishmania transmission in the study area was confirmed by the high prevalence of anti-leishmanial antibodies in dogs (33.3%).

Blood-feeding preferences of Aedes albopictus (Diptera: Culicidae) in urban and rural settings within the province of Rome, Italy.

We here report the results of field trials carried out in Rome with the aim to obtain data on the feeding behaviour of Aedes albopictus, in relation to different availability and abundance of putative hosts. Human Blood Index values were found higher than 75% in urban areas, where humans represented the most abundant hosts, and lower than 60% in rural areas, where host alternative to humans were frequent. The overall results confirm the generalist feeding-behaviour shown by this species in its original range of distribution and highlighting its high potential as vector of human pathogens in urban areas of Italy.

Insecticide-treated bednets to prevent anthroponotic cutaneous leishmaniasis in Aleppo Governorate, Syria: results from two trials.

The increasing incidence of anthroponotic cutaneous leishmaniasis (ACL) in Syria, coupled with the inefficacy of residual insecticide spraying to control the disease, have led to the further evaluation of the preventive efficacy of insecticide-treated bednets (ITNs) through large-scale field studies. Two trials were performed in the historical ACL-endemic areas of the Aleppo Governorate. In 1997-1999, a matched-cluster randomized trial was conducted in five intervention (ITNs) and five control (untreated bednets) villages, which involved a population of 10354 in 1321 households. In 2001-2003, a second study was performed in four villages (a population of 9325 in 858 households), which allowed the evaluation of the impact of the interruption of ITN intervention on ACL incidence rates. Both studies not only confirmed the high efficacy of ITNs in preventing ACL during 1 year post-intervention [about 85% (95% CL 76-98%) in the 1997-1999 trial], but also suggested that the interruption of this control measure might restore the pre-intervention disease incidence within 1-2 years. These findings underline the need for a sustainable and durable implementation of ITN-based control of the disease.

Deltamethrin-impregnated collars for the control of canine leishmaniasis: evaluation of the protective effect and influence on the clinical outcome of Leishmania infection in kennelled stray dogs.

A 2-year field study on kennelled stray dogs living in a highly endemic area of leishmaniasis was designed to evaluate whether deltamethrin-impregnated collars (Scalibor) Protector Band) could confer protection against leishmaniasis in this peculiar setting, and to assess differences in clinical outcomes between collared and uncollared dogs. A cohort of 120 clinically healthy and Leishmania-seronegative dogs was enrolled, 50% of which were collared before the 2003 transmission season, and then re-collared before the subsequent season. Collared and uncollared animals were allowed to live with infected dogs in same groups within the kennel. Follow-up included serological (IFAT) assessment twice a year with parasitological Leishmania confirmation, and clinical evaluation performed every 3 months on seroconverted dogs from both groups. Collar losses during the two seasons were high (35%). About 50% of enrolled dogs were lost at follow-up because of death or they were moved to other locations. After the 2003 season, cross-sectional serological examinations tested positive in 5 out of 44 collared animals (11.4%) and in 14 out of 34 controls (41.2%), with 72.3% estimated protection (P<0.005). After the 2004 season, 7/31 seronegative collared dogs seroconverted (22.6%) compared with 7/17 seronegative controls (41.2%), with 45.1% protection (P=0.15). At the end of the study, the cumulative rate of protection was 50.8% (P=0.005). At the clinical evaluation of 21 seroconverted dogs from both groups, canine leishmaniasis signs were significantly more frequent (90% versus 36%, P=0.017) and rapidly progressive in uncollared than in collared dogs. Reasons for such partial clinical protection in collared dogs may be found in the vector anti-feeding effect of protector bands, resulting in a lower number of infectious bites and, probably, in the reduction of antigenic stimuli necessary to shift toward a non-protective immune response.

Agrometeorology and models for the parasite cycle forecast.

Insects are strongly influenced by meteorological variables in their natural environment. In agriculture, mathematical models have been developed to understand and forecast the cycle of pests based on climate data. By this manner, with the goal of reduce and rationalize plant chemical treatments, agrometeorological models have been realized to estimate the length and starting times of parasites phenological phases. In Sicily a new network of 95 GSM meteorological stations and a specific mathematical model for Aonidiella aurantii are used by Sicilian Agrometeorological Information System (SIAS) for the integrated pest management program of citrus orchards in the Island. As the plants parasites, vector borne diseases are influenced by climate in their appearance and abundance. In lights of the benefits that could derive from a model for the control of Leishmania vectors, SIAS experiences in modelling were used to develop a deductive model for Phlebotomus perniciosus which represents the major vector of human and canine leishmaniasis in Sicily.

Entomological and parasitological study on phlebotomine sandflies in central and northern Albania.

An entomological survey was carried out in two districts of central (Kruje) and northern (Lezhe) Albania. Six collecting sites, showing a variety of diurnal resting sites, were monitored for adult sandflies from June through October 2002. Flies were collected with CDC miniature light traps, sticky traps and mechanical or hand aspirators in peridomestic sites, in bedrooms and inside cow barns, chicken coops and pigpens. All collecting sites monitored were found positive for sandflies. A total of 849 specimens were caught (29.2% males) belonging to five Phlebotomus species. Phlebotomus neglectus (75.6%) was the most abundant species followed by P. perfiliewi (14.4%), P. papatasi (4.6%), P. tobbi (3.6%) and P. similis (1.8%). The first adult of P. neglectus appeared on June 11 and the last one was collected on October 16. The highest density for this species was observed at the end of July. A total of 111 blood-fed females were caught from the two areas studied. P. neglectus was the only species found blood fed in Lezhe and the same species was prevalent (56.1%) in Kruje followed by P. perfiliewi (30.3%), P. tobbi (10.6%); P. papatasi was represented by only two specimens. Blood meal origin was determined in 45/66 (68.2%) of the females tested from Kruie district. P. neglectus was found fed on four hosts, showing the following feeding patterns: cow (71.4%), dog (117.1%), chicken (5.7%) and human (5.7%); P. perfiliewi was found fed on cow (80.0%) and chicken (20.0%), P. tobbi on cow (50.0%), chicken (25.0%) and dog (25.0%). One specimen of P. papatasi was found fed on cow. When such prevalences were analysed by the available biomass for each host present at the collecting site, P. neglectus resulted to be an opportunistic feeder rather than exhibiting preferences for any specific animal. PCR analysis of 39 P. neglectus from the Lezhe district gave negative results for the presence of Leishmania DNA.

Phylogenetic analysis of Phlebotomus species belonging to the subgenus Larroussius (Diptera, psychodidae) by ITS2 rDNA sequences.

In the genealogy of Phlebotomus (Diptera: Psychodidae), morphological analyses have indicated that the subgenus Larroussius is a monophyletic group which is most closely related to the subgenera Transphlebotomus and Adlerius. We conducted a phylogenetic analysis of the relationships among six representative species of the subgenus Larroussius and one species representatitive of the Phlebotomus subgenus, assessing sequences of the Second Internal Transcribed Spacer (ITS2) of the ribosomal RNA (rRNA). Three of the species (P. perniciosus, P. ariasi and P. perfiliewi perfiliewi) were collected in different parts of the Mediterranean area. The trees estimated from parsimony and neighbour-joining analyses supported the monophyly of the Larroussius subgenus inferred from the morphological analysis. According to our data, P. ariasi may be a sister group to the rest of the Larroussius subgenus, although additional sequence data are needed to confirm this observation. Our results suggest that P. perniciosus and P. longicuspis are distinct species, in spite of the fact that there are only slight morphological differences. The strict congruence between the phylogeny of the Larroussius subgenus inferred from the ITS2 sequences and that based on morphological studies further confirmed the ability of the spacer sequence to identify recently-derived affiliations.

Growth of two phleboviruses after experimental infection of their suspected sand fly vector, Phlebotomus perniciosus (Diptera: Psychodidae).

Phlebotomus perniciosus were infected by intrathoracic inoculation and membrane feeding techniques with two phleboviruses (Toscana and Arbia) isolated in Italy from this sand fly species. Low levels of multiplication of both viruses were detected after intrathoracic inoculation of the sand flies. Only some insects were found infected after oral ingestion of the two viruses. The percentage of flies infected orally was related to the amount of virus ingested. Toscana virus was transovarially transmitted to two larvae of the F1 progeny of orally infected sand flies. No signs of infection were observed after oral infection when unnatural virus-vector combinations were tested, e.g., Toscana virus-P. papatasi or Naples sand fly fever virus-P. perniciosus. The virus concentrations recorded in P. perniciosus experimentally infected with both Toscana and Arbia viruses were similar to those found in naturally-infected sand flies.

Experimental studies on the maintenance of Toscana and Arbia viruses (Bunyaviridae: Phlebovirus).

Transovarial transmission (TOT) of Toscana (TOS) and Arbia (ARB) viruses in a laboratory colony of Phlebotomus perniciosus is reported. Toscana and ARB viruses were maintained in P. perniciosus females, initially infected by intrathoracic inoculation, for 2 and 3 consecutive generations respectively. TOT was demonstrated in F1 (75%) and F2 (67%) generation adults for TOS and F1 (47%), F2 (37%), and F3 (34%) generation adults for ARB virus. The progressive decline of virus infection rates in each generation suggests that these agents cannot be maintained indefinitely by TOT. No infection was observed in F1 progeny after female parents were fed through membranes with either virus. Transovarially infected females were able to transmit TOS virus by bite to a susceptible vertebrate. Venereal infection of P. perniciosus females mated to males transovarially infected with TOS virus was seen.

Leishmaniasis in Tuscany (Italy): (I) An investigation on phlebotomine sandflies in Grosseto Province.

A survey of phlebotomines has been made in the area of the Province of Grosseto (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. perfiliewi was the only species collected; in the third group, P. perfiliewi was very abundant (98.0%), S. minuta was vert 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 the Monte Argentario and P. perfiliewi 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.

Laboratory tests of three repellents against Phlebotomus perniciosus (Diptera:Psychodidae).

Three topical repellents were evaluated against Phlebotomus perniciosus using dose-response techniques. A total of 1800 laboratory reared nulliparous females of P. perniciosus, 2 to 15 days old, and 21 human volunteers were used. The effective dose (ED50 and ED90) for Deet, Indalone and MGK 11 has been determined. Indalone and MGK 11 were more effective than standard Deet. The data obtained indicate that P. perniciosus is highly sensitive to the repellents compared with other blood-sucking arthropods.

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

One out of 213 Phlebotomus perfiliewi caught from an endemic cutaneous leishmaniasis focus in Abruzzi region (Italy) was naturally infected with promastigotes. The parasites were 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. perfiliewi in the transmission of cutaneous leishmaniasis due to L. infantum s.l. in the focus is discussed.

Studies on Mediterranean leishmaniasis: V.A. preliminary epidemiological survey of human leishmaniasis in Tuscany.

Leishmanin skin testing was performed on 1,285 people in the Tuscany region of Italy. In a known endemic area for leishmaniasis there was an increasing positivity with age, while in another area the curve was bell-shaped suggesting an interruption of transmission in the recent past. In all areas the positivity rate for males was higher than for females, but it was not possible to distinguish between areas of cutaneous and visceral disease.

Leishmaniasis in Tuscany (Italy): (IV) An analysis of all recorded human cases.

Recent investigations on the distribution of human and canine leishmaniasis, and on the vectors and wild reservoirs of the disease in foci in Tuscany have stimulated a retrospective evaluation of human leishmaniasis of the whole region. From a study of the literature, of statistical reports (ISTAT) and of unpublished information, 77 cases of visceral leishmaniasis and 97 of cutaneous leishmaniasis have been analysed. From the case reports of both forms it appears that: (a) a higher incidence of cases occurred in restricted areas; (b) visceral and cutaneous leishmaniasis have been hypoendemic in Tuscany since the years of the earlier reports (1933 and 1936, respectively); (c) the number of infantile visceral cases was much higher in the period from 1933 to 1955 (84.5% of subjects in the 0-10 years age group) than in the subsequent years, 1956-75 (50.0%). The possible significance of the infantile and adult forms of visceral leishmaniasis in Tuscany is discussed, and the local data are compared with those of other regions.

Laboratory transmission of Leishmania infantum to Rattus rattus by the bite of experimentally infected Phlebotomus perniciosus.

The role of the sandfly species Phlebotomus perniciosus Parrot in laboratory transmission of Leishmania infantum was investigated. One of the wild reservoirs of the disease, the black rat (Rattus rattus), was used as vertebrate host. The parasite was transmitted by the bite of experimentally infected P. perniciosus to the black rat which, six months later, was infective to the same sandfly species. These experimental data confirm the role of P. perniciosus in the transmission of L. infantum, as suggested by earlier studies based on epidemiological data.

Leishmaniasis in Tuscany (Italy): VII. Studies on the role of the black rat, Rattus rattus, in the epidemiology of visceral leishmaniasis.

The recent finding that four Leishmania isolates from Rattus rattus are enzymologically identical to L. infantum, aetiological agent of human and canine visceral leishmaniasis in the Mediterranean area, has stimulated further research on the role of R. rattus in the epidemiology of visceral leishmaniasis in Tuscany, Italy. The present work has led to the following results: (a) R. rattus experimentally infected with L. infantum displayed a general resistance to the parasite, even though it appeared that a very low number of amastigotes persisted in the animal for a long period; (b) Phlebotomus perniciosus and P. perfiliewi induced to feed on rats, with either a massive visceral infection or a subpatent immunodepressed infection, became infected, though in different proportions; (c) P. perniciosus and P. perfiliewi were readily attracted to, and fed on, the black rat in nature. These findings, together with those obtained earlier, strongly suggest that the black rat may be one of the wild reservoirs of L. infantum in the areas under study.

Natural infections of phlebotomine sandflies with Trypanosomatidae in central and south Italy.

In a survey of Leishmania infections of phlebotomine sandflies caught in central and south Italy in 1986, zymodeme MON-1 (Montpellier 1) of Leishmania infantum was isolated from Phlebotomus perniciosus and L. tarentolae [= Trypanosoma platydactyli?] was isolated from Sergentomyia minuta, providing for mainland Italy the first direct proof of these long-suspected parasite-vector associations. The roles of P. perfiliewi and P. perniciosus in the transmission of Leishmania spp. in Italy are discussed.

Recrudescence of visceral leishmaniasis in Albania: retrospective analysis of cases during 1997 to 2001 and results of an entomological survey carried out during 2001 in some districts.

The epidemiological status of zoonotic visceral leishmaniasis (VL) in Albania is almost unknown to international health organizations and to the scientific community. Results from a retrospective analysis of recent VL cases, and from an entomological survey carried out in summer 2001 are reported here. From January 1997 to December 2001, 867 parasitologically confirmed VL cases were recorded in 35 of 36 Albanian districts with an average of 173 cases/year and a cumulative morbidity of 2.8/10000 population. The temporal distribution of cases showed an increasing trend (from 144 to 209), indicating that cases have almost doubled during the past 10 years. A high proportion of the patients (67.6%) was children aged < 5 years. The entomological survey confirmed Phlebotomus neglectus as the probable vector, being the most abundant and widespread species. The increase in VL morbidity, 20-40-fold higher than in other southern European countries, could be attributed to increased susceptibility of infants to clinical disease or to variations in Leishmania infection associated with changes in canine reservoir and/or vector populations.