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.

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.

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%).

Immunolocalization of a lipase-like protein in the reproductive apparatus of female Phlebotomus papatasi, at various stages of the gonotrophic cycle.

In female phlebotomine sandflies, little is known about the reproductive accessory glands that presumably contribute to egg production and/or oviposition. The main protein secreted in the accessory glands of female Phlebotomus papatasi was recently characterised as a lipase-like protein, the first to be found in the female accessory glands of any insect. This protein, named PhpaLIP (for Phlebotomus papatasi lipase), has now been detected and localized in the reproductive tissues of female P. papatasi, at different stages of the gonotrophic cycle, using a polyclonal anti-PhpaLIP serum and both confocal scanning laser and immuno-electron microscopy. PhpaLIP appears to be always present in the accessory glands (with a secretory peak shortly before oviposition) but was also detected in the follicle cells of the ovarioles, within the developing vitelline envelope, and in the oviducts. The results are discussed in relation to the functions that PhpaLIP could have during the gonotrophic cycle, in the various reproductive structures of female P. papatasi.

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.

Lipase and antibacterial activities of a recombinant protein from the accessory glands of female Phlebotomus papatasi (Diptera: Psychodidae).

The reproductive accessory glands of the adult female have a functional versatility in insects, contributing to reproduction in various ways. The major protein secreted by the accessory glands of female Phlebotomus papatasi (Diptera, Psychodidae) has already been characterised and named PhpaLIP (for P. papatasi lipase) because, in terms of its amino-acid sequence, it is very similar to a wide range of vertebrate lipases. The gene coding for PhpaLIP has now been cloned into a pQE30 vector and expressed in Escherichia coli. When the recombinant PhpaLIP was tested in vitro, it was found to have not only lipase-like activity (when p-nitrophenyl caprylate was used as the substrate) but also specific antibacterial activity against some Gram-positive and Gram-negative bacteria. The possible physiological roles of PhpaLIP in P. papatasi are discussed, in the light of these results.

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.

About the presence of Phlebotomus sergenti Parrot, 1917 (Diptera: Psychodidae) in Eastern Sicily, Italy.

The note reports the data of a three-year sand fly investigation (1997-99) carried out in Eastern Sicily (Italy) with the aim to study the distribution of Phlebotomus sergenti. The survey involved a densely inhabited area at the foot of Mount Etna and the area of Iblei mounts. A total of 9,095 sand flies, of which 63.4% males, were captured. Five species belonging to the genus Phlebotomus (P. perniciosus, P. perfiliewi, P. neglectus, P. sergenti and P. papatasi) and one to the genus Sergentomyia (S. minuta) were identified. Both the prevalence and distribution of the species were different within the two areas studied. In Mount Etna area, P. perniciosus (77.7%) was the prevalent species followed by S. minuta (19.8%), P. sergenti (2.0%), P. neglectus (0.3%) and P. papatasi (0.2%). While in Iblei mounts region S. minuta (84.5%) showed the highest prevalence, followed by P. perniciosus (14.4%), P. perfiliewi (0.9%) and P. neglectus (0.1%). Here, P. sergenti was a very rare species (< 0.02). P. sergenti was mostly associated to domestic habitats of peri-urban and urban zones located between two and 750 m a.s.l. The density values of P. sergenti, expressed as number of specimens/m2 of sticky trap, were between 0.3 and 5.5 with the highest value in the hilly collecting sites. The low observed abundance of P. sergenti does not allow to draw any prediction on the role that the species could play in the transmission of leishmaniasis in Sicily.

[Monitoring of canine leishmaniasis in northern Italy: an update from a scientific network]. / Monitoraggio della leishmaniosi canina in nord Italia: aggiornamenti da un network scientifico.

Canine leishmaniasis (CanL) due to Leishmania infantum is a disease of great veterinary importance and a serious public health problem. In humans, L. infantum causes visceral (VL) and cutaneous leishmaniasis (CL) and the distribution of VL overlaps that of CanL. Currently, VL is considered by WHO as an emerging zoonosis in southern Europe. The dog is the only domestic reservoir of the infection and phlebotomine sandflies are the only proven vectors of leishmaniasis for dogs and humans. CanL is endemic in Italy, particularly in central and southern regions, including islands. Until 1983, all regions of northern Italy but Liguria and some territories of Emilia Romagna were considered free from CanL. From early '90s new stable foci of CanL have appeared, most of them located within classical endemic areas including territories of Emilia Romagna, Tuscany, Umbria, Marche, and Abruzzi regions. But the most relevant aspect, from an epidemiological point of view, has been the appearance of stable CanL foci in northern Italy, namely in Veneto and Piedmont regions. In these two foci, entomological surveys showed the presence of P. perniciosus and of a second phlebotomine vector, P. neglectus, which may have played a role in the CanL diffusion in some parts of northern Italy. Furthermore, in these areas, autochthonous human VL cases have occurred. There is therefore a realistic risk that CanL infection could rapidly spread through northern latitudes and a surveillance activity is strongly needed. For this reason, in October 2002, thanks to the collaboration and support of Intervet Italia, the network "LeishMap" was created, with the main purpose of monitoring the spread of CanL and vectors in northern Italy. LeishMap consists of scientific and sanitary institutions with proven experience both in field surveys and diagnostic methodologies on CanL and phlebotomine vector. It is organised in 4 Operational Units (OU), represented by researchers of the Veterinary Faculties of the University of Bologna, Padua, Milan and Turin, under the scientific coordination of the MIPI Department, ISS of Rome and with the collaboration of private and public veterinarians operating in the regions under study. During the first year of activity, each OU was involved in the serological and entomological surveillance of several territories in the respective regions, where recent autochthonous CanL cases were registered. The studies have involved five regions, namely Valle D'Aosta, Piedmont, Lombardia, Veneto, Trentino-Alto Adige and Emilia Romagna. In the Symposium 6 of this Congress we report detailed results of a retrospective analysis of data concerning CanL and vectors in northern Italy till 2002 and the preliminary results of 2003 on the seroprevalence rates observed in foci studied and on the entomological surveys carried out. In summary, the results outlined that already known foci of CanL are expanding from the original sites. Several new foci have been identified and many others are at high risk of evolving toward a stable endemicity. P. perniciosus has been found in all but one the suspected new foci. In Emilia Romagna region P. perfiliewi was identified in 2 areas and in one was the only species present. The occurrence of P. neglectus was confirmed in three regions, Veneto, Lombardia and Piedmont. In conclusion, from the 2002-2003 LeishMap activities it appears that further monitoring activities are necessary to identify new endemic foci of CanL, this representing the prerequisite for the implementation of programs for leishmaniasis control in northern Italy.

[Prevention and control of leishmaniasis vectors: current approaches]. / Prevenzione e controllo dei vettori di leishmaniosi: attuali metodologie.

Phlebotomine sandflies (Diptera: Psychodidae) are the suspected or proven vectors of Leishmania spp. in at least 88 countries, including over 40 Phlebotomus species in the Old World and a further 30 belonging to the genus Lutzomyia in the New World. In recent years, both cutaneous (CL) and zoonotic visceral leishmaniasis (ZVL) have become increasingly prevalent in urban areas, including large Latin American cities. A similar trend has been recorded in all Mediterranean areas during the last decade. Based on mathematical models, insecticidal control of sandflies appears to represent a more effective way of reducing Leishmania infantum transmission than the present strategy of culling infected dogs in Latin America as well as being more acceptable to the human population. Since man is a dead-end host of most Leishmania species, treatment of existing human cases generally does not affect transmission. Interruption of the cycle by vector control may offer a cheaper, more practical solution to treatment and improved knowledge of the alternatives available could lead to preventative measures being undertaken in more leishmaniasis foci. In this note a review of current knowledge on sandfly control is presented. Different measures to control phlebotomine sandflies, including residual spraying of dwellings and animal shelters, insecticide treated nets, application of repellents/insecticides to skin or to fabrics and impregnated dog collars are discussed. Although effective in urban areas with high concentrations of sandflies, residual spraying of insecticides is no often longer tenable in most situations. In rural areas where dwellings are more dispersed and surrounded by large, untargeted "reservoir" populations of sandflies, residual spraying of houses may be both impractical for logistic reasons and ineffective. Actually, this control measure depends on the availability of a suitable public health infrastructure, including adequate supplies of insecticide, spraying equipment and trained personnel. Ideally such personnel should be trained in insecticide application, monitoring techniques and interpretation of sampling data, as well as safety techniques. To date reports of resistance refer to one insecticide (DDT) in only three species (Phlebotomus papatasi, P. argentipes and Sergentomyia shorti) in one country (India), although there are reports of increased tolerance to this compound in several countries. Fortunately the insects remain susceptible to all the major insecticidal groups. Impregnated bednets may offer the best solution in rural areas where transmission is largely intradomiciliary. This measure has the advantage that it can be employed at the individual household level and affords collateral benefits such as privacy and control of other biting insects such as mosquitoes, fleas and bedbugs. Sandfly larvae are generally difficult to find in nature so control measures that act specifically against immatures are not feasible, although the effectiveness of a few biological and chemical agents has been demonstrated in laboratory evaluations. In ZVL foci, where dogs are the unique domestic reservoir, a reduction in Leishmania transmission would be expected if we could combine an effective mass treatment of infected dogs with a protection of both healthy and infected dogs from the sandfly bites. Laboratory and field evaluations have shown that impregnated dog collars and topical application of insecticides could protect dogs from most sandfly bites by means of both anti-feeding and killing effect of the pirethorids used.

Host preferences of phlebotomine sand flies at a hypoendemic focus of canine leishmaniasis in central Italy.

A survey was carried out on phlebotomine sand flies and their feeding habits at a hypoendemic focus of Leishmania infantum in Macerata province, central Italy. During two consecutive years (2000-2001), 1465 sand fly specimens (42.5% of which were males) were collected from a variety of diurnal resting sites in the municipality of Camerino. The most prevalent species was Phlebotomus perniciosus (76.6%), followed by P. papatasi (10.4%), Sergentomyia minuta (9.1%), Phlebotomus perfiliewi (3.3%) and P. mascittii (0.5%). Among the 842 females collected, 578 (68.6%) were blood-fed. Based on the results of blood meal analyses, P. perniciosus fed on man, dogs, equines, sheep and birds; P. perfiliewi on dogs, equines, sheep and birds; P. papatasi on dogs, equines and birds. Two specimens of P. mascittii fed on equines. Forage ratios (FRs) and host selectivity indices gave different results for the large domestic animals. More than 95% of the specimens collected inside a stable, dog kennel, sheep pen and chicken house were found to have fed on the animals housed in the respective shelters. In addition, at one collecting site where almost all the hosts mentioned above were present simultaneously, both P. perniciosus and P. perfiliewi were found to have fed on all five species, indicating that host choice was probably related to its availability (i.e. number and size) rather than specific attractiveness. The feeding habits of the two Leishmania vectors may have implications for the epidemiology of leishmaniasis in urban and peri-urban areas, where sand fly females deprived of other vertebrate hosts (particularly the larger species) may begin to bite humans and dogs more frequently.

A mammalian-like lipase gene is expressed in the female reproductive accessory glands of the sand fly Phlebotomus papatasi (Diptera, Psychodidae).

A lipase-like protein (PhpaLIP) was identified as the major protein component in the secretion of the female reproductive accessory glands of the sand fly Phlebotomus papatasi. The full-length cDNA encoding this protein was isolated and its nucleotide sequence determined. The deduced translational product of the gene contains a GFSFG motif, consistent with a GXSXG consensus, which is shared by most bacterial and eukaryotic hydrolases. Transcriptional analysis of the PhpaLIP gene showed that its expression is female-specific, and is also detectable in districts other than accessory glands, suggesting that it might play different functions. Taken together with the observation of sequence similarity shared by PhpaLIP and mammalian lipases, the demonstration of the presence of lipase activity in the accessory gland secretion suggests a possible biological role of PhpaLIP gene product.

Control of phlebotomine sandflies.

Phlebotomine sandflies (Diptera: Psychodidae) transmit many zoonotic diseases (arboviruses, bartonelloses and especially leishmaniases) of importance to human health in at least 80 countries. Measures used to control adult sandflies (Lutzomyia and Phlebotomus) include the use of insecticides (mostly pyrethroids) for residual spraying of dwellings and animal shelters, space-spraying, insecticide-treated nets, impregnated dog-collars and personal protection through application of repellents/insecticides to skin or fabrics. Because the breeding-sites of sandflies are generally unknown, control measures that act specifically against immatures are not feasible, although the effectiveness of a few biological and chemical agents has been demonstrated in laboratory evaluations. Reports of insecticide-resistance refer to only three sandfly species (P. papatasi, P. argentipes and S. shorttii) against DDT in one country (India), although there are reports of DDT-tolerance in several countries. Current knowledge of sandfly susceptibility to various insecticides is summarized. Constraints and advantages of different compounds, formulations and delivery methods for sandfly control under different environmental conditions 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.

A stable focus of canine leishmaniosis in the Bologna Province, Italy.

During an epidemiological survey carried out for two consecutive years (2001-2002), autochthonous cases of canine leishmaniosis (CanL) were reported in Communes of the Bologna Province (Emilia-Romagna Region, northern Italy), involved in the past (1971-1972) in a severe outbreak of human visceral leishmaniosis (VL). Serological controls, carried out by immunofluorescence antibody test on a sample of owned dogs, detected a mean prevalence of 2.5% in the first year in 4 Communes, and of 11.2% in the second year in only one Commune, where an incidence value of 9.3% was assessed. The autochthonous origin of the infection was confirmed in 11 out of 13 positive animals in the first year and in 5 out of 6 new cases in the second year. In one case the parasitological examinations led to the isolation of leishmaniae characterized as Leishmania infantum zymodeme MON 1. Entomological surveys carried out during two sandfly seasons in one of the areas concerned by the VL outbreak showed the presence of Phlebotomus perfiliewi and, to a lesser extent, of P perniciosus, both proven vectors of L. infantum in Italy. The results obtained seem to suggest the presence of a stable focus of CanL in the territory involved in the previous VL outbreak of 1971-1972, within which the infection in the canine population had been assessed only serologically. Such an epidemiological situation may be seen either as the persistence of an old focus or as a new imported one.

Recent findings of Phlebotomus neglectus Tonnoir, 1921 in Italy and its western limit of distribution.

During the sand fly season of 2001, sand flies were collected in two different regions of Italy to investigate the northern distribution of Phlebotomus neglectus. The study areas were two provinces of northern Italy, Ivrea and Verona, in Piedmont and Veneto regions respectively. Sticky traps and CDC miniature light traps were used both inside and outside domestic and peri-domestic environments. Sand flies were also collected in domestic resting sites with mouth and electric aspirators. Of 1,295 sand fly specimens collected, 855 were from the Veneto region and 440 were from the Piedmont region. Five species of the genera Phlebotomus and Sergentomyia were identified. S. minuta (66.3%) was the most abundant sand fly followed by P. perniciosus (22.2%), P. neglectus (10.3%) and P. papatasi (1.2%). Only one specimen of P. mascittii was caught in Piedmont region. P. neglectus was found in 9 out of 15 (60%) of the sites where sand flies were found with a prevalence of 1.5% to 45.8%; the highest value was recorded in Piedmont region. P. neglectus was mostly associated with peridomestic and domestic sites. The results confirm previous observations on the presence of P. neglectus in northern Italy and suggest that this species is probably widespread in the area. From a review of all previous published and unpublished records of P. neglectus, it appears that this species is present in only two areas of the country, namely in the regions of Friuli Venezia Giulia, Veneto and Piedmont in northern Italy and the regions of Calabria, Apulia and Sicily in southern Italy. The apparent absence of P. neglectus in Central Italy is discussed and the resulting settlement through two probable migration routes from the East is analysed.

Epidemiological and entomological survey in a focus of visceral leishmaniasis in Pap district (Fergana Valley) of Namangan region, Uzbekistan.

An active focus of human visceral leishmaniasis (VL) is present in Uzbekistan, Namangan region, Pap district, situated along the Fergana Valley at an altitude of 900-1300 m a.s.l. An overall total of 19 VL cases, all recorded in children less than 14 years of age, have been reported during the period of 1987-99 in three villages, namely Oltinkon (9 cases), Chodak (7 cases) and Guliston (3 cases). Clinical and epidemiological features suggest that the disease is zoonotic in nature and that it is possibly caused by Leishmania infantum, though no characterization of the causative agent was performed due to the failure in isolating the parasite. Currently, diagnosis is made exclusively on the basis of clinical manifestations followed by demonstration of parasites in bone marrow aspirates. Standardized monthly sandfly collections (July-September 1999) were made with sticky traps and hand captures from houses and peri-domestic areas. Twenty houses were monitored over two consecutive days every month in five villages scattered along the valley, three of which were the VL foci (Oltinkon, Chodak, Guliston) and the other two, Kandigan and Khonabad, were located at the top (>1300 m a.s.l.) and at the bottom of the valley (740 m a.s.l.), respectively. Among the sandfly species identified, Phlebotomus sergenti (46.0%) was the most prevalent species through the entire period, followed by P. papatasi (18.8%), P. longiductus (15.5%), P. alexandri (10.3%), P. angustus (6.6%), S. sumbarica (2.2%), P. keshishiani (0.6%) and S. grekovi (0.1%). The role of the suspected vector, P. longiductus in the transmission of VL cases in the Fergana focus is discussed in relation to its abundance and distribution in the valley.