The knowns and unknowns of the efficacy of neem oil (Azadirachta indica) used as a preventative measure against Leishmania sand fly vectors (Phlebotomus genus).

Since domestic dogs are the main reservoir hosts of Leishmania infantum throughout the world, they are the main focus in terms of controlling zoonotic visceral leishmaniosis. To protect dogs from leishmaniosis, chemical repellents of durable efficacy are available in the form of collars, spot-on and sprays. However, the negative effects of chemical pesticides on the environment are well established as they affect animals and plants. This phenomenon has created the need for safer and more environmentally friendly substitutes. Plant extract-based insecticides and/or repellents have therefore been increasingly used by pet owners and veterinarians. Several botanical products have been tested as insecticides and/or repellents against a variety of bloodsucking arthropods that transmit human diseases. Among the products tested against Leishmania vectors, neem oil containing azadirachtin is the most studied. This study reviews the scientific literature concerning the efficacy of neem oil (azadirachtin-based products) against phlebotomine sand fly bites. A questionnaire was also administered to assess Italian veterinarians' attitudes to the use of neem oil. The survey was anonymous and consisted of three closed-ended questions. According to the data reported in the literature, the efficacy of neem oil in reducing the risk of sand fly bites has been tested against Phlebotomus papatasi, Phlebotomus perniciosus, Phlebotomus argentipes, Phlebotomus orientalis and Phlebotomus bergeroti. The efficacy of the products tested was expressed in percentages, ranging from 74.9% to 100%. The protection time was only available for six out of eight studies, ranging from "only during the first hour" (minimum protection time) to "all night" (expected maximum protection time). As regards the attitude to recommending the use of neem oil, 208 veterinarians participated in the online survey. Of the 126 veterinarians who recommended natural products, 119 (94.44%) reported that they recommended the use of neem oil-based products. Considering the limited data on the duration of protection and the dose of the active ingredient, more studies are required on the efficacy of neem oil-based products in reducing the risk of contracting canine leishmaniosis. These studies should also refer specifically to the concentration of the active ingredient as well as the interval of administration. Until such results are available, the use of azadirachtin-based products as the only topical products for the prevention of leishmaniosis in dogs is not recommended.

Preventive measures of canine leishmaniosis in Italy: Attitudes of veterinarians based on a questionnaire.

There are several topical ectoparasiticides, drugs, supplements and vaccines that protect dogs against sand fly bites and/or reduce the risk of Leishmania infantum infection. This large variety of products and the absence of comparative studies mean that veterinarians are often faced with uncertainty. The aim of the study was to characterise the attitudes of veterinarians towards leishmaniosis prevention in client-owned dogs in Italy. A web-based questionnaire was prepared with 28 questions covering the working environment of veterinarians, advice given to owners regarding topical ectoparasiticides, advice on systemic prevention, and the use of diagnostic tests on which to base advice for prevention. The questionnaire was emailed to 9,426 veterinarians and answers were collected after 100 days. A total of 542 questionnaires were returned (response percentage 5.8 %): 54.8 % of the veterinarians considered their working area to be endemic, 29.3 % examined dogs from urban areas, and 42.3 % diagnosed ≥10 dogs/year with leishmaniosis. Those veterinarians who diagnosed ≥10 dogs/year with leishmaniosis were more likely to consider their working area to be endemic, whereas those who examined dogs mainly from urban areas were less likely to consider the area to be endemic. Veterinarians who considered the working area to be endemic were more prone to prescribe ectoparasiticides throughout the year, including collars and spot-on products and a combination of the two, vaccine alone or vaccine and domperidone combined, and used qualitative serological Leishmania tests. The attitude of veterinarians did not differ whether dogs were from urban or rural areas. In conclusion, veterinarians who consider their working area to be endemic or who frequently diagnose dogs with leishmaniosis tend to adopt more comprehensive strategies, and their attitudes are not influenced by the origin of the dogs: urban or rural. Overall, in Italy the attitudes of veterinarians in relation to canine leishmaniosis prevention appear to vary considerably.

The current status of phlebotomine sand flies in Albania and incrimination of Phlebotomus neglectus (Diptera, Psychodidae) as the main vector of Leishmania infantum.

The incidence of visceral leishmaniasis (VL) in Albania is higher than in other countries of southern Europe, however the role of local sand fly species in the transmission of Leishmania infantum was not addressed conclusively. In 2006, a country-wide collection of sand flies performed in 14 sites selected based on recent occurrence of VL cases showed that Phlebotomus neglectus was by far the most prevalent species (95.6%). Furthermore, 15% of pools made from 422 P. neglectus females tested positive for Leishmania sp. genomic DNA. In the same year, Culicoides trapping was performed for bluetongue disease surveillance in 91 sites of southern Albania, targeting livestock farms regardless recent occurrence of VL in the surveyed areas. In 35 sites where sand flies were collected along with midges, Phlebotomus perfiliewi was the most prevalent among the Phlebotomus species identified, however search for leishmanial DNA in females of this species was unsuccessful. In 2011, sand flies were trapped in 4 sites of north Albania characterized by high VL incidence, and females were dissected to search for Leishmania infections. Both P. neglectus and P. tobbi were collected at high densities. Two positive specimens were detected from a sample of 64 P. neglectus trapped in one site (3.1%). Parasites were successfully cultured from one specimen and characterized as belonging to Leishmania infantum zymodeme MON-1, the only zymodeme so far identified as the agent of human and canine leishmaniasis in the country. Altogether our studies indicate that P. neglectus is the main leishmaniasis vector in Albania.

Recent advances in phlebotomine sand fly research related to leishmaniasis control.

Phlebotomine sand flies are the subject of much research because of the role of their females as the only proven natural vectors of Leishmania species, the parasitic protozoans that are the causative agents of the neglected tropical disease leishmaniasis. Activity in this field was highlighted by the eighth International Symposium on Phlebotomine Sand flies (ISOPS) held in September 2014, which prompted this review focusing on vector control. Topics reviewed include: Taxonomy and phylogenetics, Vector competence, Genetics, genomics and transcriptomics, Eco-epidemiology, and Vector control. Research on sand flies as leishmaniasis vectors has revealed a diverse array of zoonotic and anthroponotic transmission cycles, mostly in subtropical and tropical regions of Africa, Asia and Latin America, but also in Mediterranean Europe. The challenge is to progress beyond descriptive eco-epidemiology, in order to separate vectors of biomedical importance from the sand fly species that are competent vectors but lack the vectorial capacity to cause much human disease. Transmission modelling is required to identify the vectors that are a public health priority, the ones that must be controlled as part of the integrated control of leishmaniasis. Effective modelling of transmission will require the use of entomological indices more precise than those usually reported in the leishmaniasis literature.

Persistence of phlebotomine Leishmania vectors in urban sites of Catania (Sicily, Italy).

BACKGROUND: Pioneering research on "Mediterranean Kala-Azar" carried out by Adler and Theodor early in the past century (~1930s) had identified Catania city (Sicily) as a major focus of the disease nowadays known as zoonotic visceral leishmaniasis (VL). Despite the fact that disease in both humans and dogs has continued to be highly prevalent in the Catania province up to the present times, research on Leishmania vectors in this urban focus dates back to that distant period. This study aimed to evaluate the persistence and current composition of the sand fly fauna in urban environments of Catania in recent years, 2006 and 2013. METHODS: In 2006 fifty-one suitable collecting sites were identified within 44 sub-units of a grid drawn to include the urban Catania area. In 2013 the survey was restricted to four of the most productive and representative sites resulting from the 2006 survey. In both periods 3 collections per month were performed using standard sticky traps set for 3 days in wall holes/cavities along public roads, from the end of April through December. RESULTS: 43/51 sites (84.3%) were found positive for sand flies. The 2006 collections accounted for a total of 4341 specimens including six species. Among competent Leishmania vector species, P. perniciosus was the most prevalent (36.5%) being identified in all sand fly-positive sites, with significant abundance in those of the old city centre. Other species of interest were P. sergenti (2.5%) and P. neglectus (1.5%). The 2013 survey produced 1130 sand flies, of which 39.5% were P. perniciosus, 1.6% P. sergenti and 0.7% P. neglectus. A search for Leishmania DNA in a small sample of 72 P. perniciosus females revealed 11% infection prevalence. CONCLUSIONS: Our findings from an old urban focus of leishmaniasis demonstrate that phlebotomine sand flies have adapted fairly well to the drastic environmental changes that have occurred in cities of the Western world in the past century and still represent a potential risk for Leishmania transmission.

Viral isolates of a novel putative phlebovirus in the Marche Region of Italy.

Thirty pools from 900 (540 females and 360 males) Phlebotomus perfiliewi sandflies collected during the summer of 2012 in the Fermo area (Marche Region, central Italy) were tested for the presence of Phleboviruses. A nested polymerase chain reaction was performed using degenerated primers amplifying a fragment of the polymerase gene (large segment) and a fragment of the nucleoprotein gene (small segment) of the genus Phlebovirus. One pool was positive for Toscana virus, as expected from results of studies in the area, and six pools were positive for a putative novel Phlebovirus. Virus isolation in Vero cells was performed. Minimum field infection rates/1,000 insects processed for the novel and Toscana viruses were 6.7 and 1.0, respectively. Phylogenetic analysis of the novel Phlebovirus, tentatively named Fermo virus, placed it in the Sandfly Fever Naples virus serocomplex.

Prognosis and monitoring of leishmaniasis in dogs: a working group report.

This review presents the consensus opinion of the Canine Leishmaniasis Working Group on the prognosis and monitoring of leishmaniasis in dogs. While the prognosis for both exposed and infected dogs is considered to be favourable, this changes if infection progresses to overt disease. For clinically affected animals undergoing treatment, the prognosis is dictated by the severity of the signs (and in particular the severity of renal dysfunction) when therapy is initiated; assessing the degree of proteinuria is useful in this context. Approximately 75% of dogs without evidence of renal involvement live for >4years if adequately treated. Monitoring the response to treatment includes ongoing clinical and clinicopathological assessment, as well as quantifying serological responses and the parasite load in the patient.

First surveys to investigate the presence of canine leishmaniasis and its phlebotomine vectors in Hungary.

Hungary is regarded as free of leishmaniasis because only a few imported cases have been reported. However, southern Hungary has a sub-Mediterranean climate, and so it was included in the EU FP6 EDEN project, which aimed to map the northern limits of canine leishmaniasis (CanL) in Europe. The numbers of traveling and imported dogs have increased in the last decade, raising concerns about the introduction of CanL caused by Leishmania infantum. Serum samples were collected from 725 dogs (22 localities, 6 counties) that had never traveled to endemic countries, as well as from other potential reservoir hosts (185 red foxes and 13 golden jackals). All sera were tested by the indirect fluorescent antibody test, but they were sero-negative using the OIE cut-off of 1:80 serum dilution except for those of two dogs resident since birth in southern Hungary. These had not received a blood transfusion, but the mode of transmission is unclear because no sandfly vectors were caught locally. From 2006 to 2009, phlebotomine sandflies were sampled in the summer months at 47 localities of 8 counties. They were trapped with castor-oil-impregnated sticky-paper, light, and CO(2)-baited traps. Small numbers of two vectors of Leishmania infantum were found. Phlebotomus neglectus occurred in three villages near to Croatia and one in north Hungary at latitude 47 °N, and Phlebotomus perfiliewi perfiliewi was trapped at two sites in a southeastern county close to the sites where it was first found in 1931-1932. Our report provides baseline data for future investigations into the northward spread of CanL into Hungary, which we conclude has yet to occur.

Longitudinal study on the detection of canine Leishmania infections by conjunctival swab analysis and correlation with entomological parameters.

A longitudinal study was carried out on kennelled stray dogs in a canine leishmaniasis (CanL) endemic area, to evaluate early and late diagnostic performance of a non-invasive conjunctival swab (CS) nested (n)-PCR analysis for Leishmania detection in 2 cohorts of dogs, respectively. (A) Sixty-five IFAT- and CS n-PCR-negative dogs exposed to, and followed up once or twice a month during a full sand fly season (July-November 2008). In parallel, a sand fly survey was performed on site using standard sticky traps set twice a month, for a cumulative surface of 63 m(2). (B) Seventeen IFAT- and CS n-PCR-negative dogs found positive in July 2008 at the peripheral blood buffy-coat (BC) n-PCR. These dogs were examined again by BC n-PCR in September and November 2008, and before the subsequent transmission season (May 2009) along with CS n-PCR and IFAT. None of the cohort (A) dogs converted to positive CS n-PCR during the transmission season. Although approximately 2500 phlebotomine specimens were collected with peaks of 100-147 specimens/m(2) sticky trap, the cumulative density of the only proven CanL vector in the area (Phlebotomus perniciosus) was found to be very low (0.5/m(2)). All cohort (B) dogs remained substantially seronegative; BC n-PCR showed an intermittent positive trend during the period surveyed, resulting in 82% conversions to negative by the end of the study, in contrast with 71% conversions to positive at the CS n-PCR analysis. In conclusion, while CS n-PCR was not found effective for the early detection of Leishmania contacts in dogs exposed to a low pressure of vectorial transmission, this assay showed to slowly convert to positive in a high rate of dogs, in the absence of seroconversion. CS n-PCR technique can be a suitable marker for assessing Leishmania exposure in dogs as a non-invasive alternative to current serological and molecular tools.

Host-feeding patterns of Aedes albopictus (Diptera: Culicidae) in urban and rural contexts within Rome province, Italy.

Knowledge of the frequency of contact between a mosquito species and its different hosts is essential to understand the role of each vector species in the transmission of diseases to humans and/or animals. However, no data are so far available on the feeding habits of Aedes albopictus in Italy or in other recently colonized temperate regions of Europe, due to difficulties in collecting blood-fed females of this diurnal and exophilic species. We analyzed Ae. albopictus host-feeding patterns in two urban and two rural sites within the area of Rome (Italy). Ae. albopictus was collected using sticky-traps and the blood-meal origin of 303 females was determined by direct dot-ELISA. The blood-fed sample, although representing only 4% of the total Ae. albopictus collected, demonstrates the useful application of sticky-trap in studying the feeding behavior of the species. The human blood index was significantly different among sites, ranging from 79-96% in urban sites to 23-55% in rural sites, where horses and bovines represented the most bitten hosts. The results obtained confirm the plastic feeding behavior shown by Ae. albopictus in its original range of distribution and highlights the high potential of this species as a vector of human pathogens in urban areas of Italy, where both humans and the mosquito itself may reach very high densities.

Risk assessment for canine leishmaniasis spreading in the north of Italy.

The incidence of zoonotic visceral leishmaniasis has not only been recognized but is, in fact, increasing in territories of northern continental Italy previously regarded as non-endemic. Recent findings of sporadic autochthonous canine infections and the presence of phlebotomine vectors in some provinces of north-eastern Italy have stimulated risk assessment for the spreading of leishmaniasis in the autonomous province of Bolzano-South Tyrol, the northernmost territory of the Italian eastern Alps. In July 2008, 61 phlebotomine sand flies (Diptera, Psychodidae) were caught and identified as Phlebotomus perniciosus and Sergentomyia minuta. This is the first record in South Tyrol of P. perniciosus, the most competent vector of Leishmania infantum in Mediterranean countries. Leishmania serology on local dogs kept in kennels gave negative results, while only imported canine leishmaniasis cases were reported by local veterinaries through a questionnaire survey. Bio-geographic aspects and epidemiological consequences are analyzed in relation with the risk of leishmaniasis introduction into the area.

Socio-demographic and climatic factors as correlates of Mediterranean spotted fever (MSF) in northern Sardinia.

Mediterranean spotted fever (MSF) is endemic in the Mediterranean area. We carried out a retrospective study to investigate the association between socio-demographic and climatic factors and MSF incidence in northern Sardinia. We found that maximum temperature levels during the previous summer were associated with increases in MSF incidence.

Molecular homogeneity in diverse geographical populations of Phlebotomus papatasi (Diptera, Psychodidae) inferred from ND4 mtDNA and ITS2 rDNA Epidemiological consequences.

An intraspecific study on Phlebotomus papatasi, the main proven vector of Leishmania major among the members of the subgenus Phlebotomus, was performed. The internal transcribed spacer 2 (ITS 2) of rDNA and the ND4 gene of mt DNA were sequenced from 26 populations from 18 countries (Albania, Algeria, Cyprus, Egypt, Greece, India, Iran, Israel, Italy, Lebanon, Morocco, Saudi Arabia, Spain, Syria, Tunisia, Turkey, Yugoslavia and Yemen), and compared. Samples also included three other species belonging to the subgenus Phlebotomus: P. duboscqi, a proven vector of L. major in the south of Sahara (three populations from Burkina Faso, Kenya and Senegal), P. bergeroti, a suspected vector of L. major (three populations from Oman Sultanate, Iran and Egypt), and one population of P. salehi from Iran. A phylogenetic study was carried out on the subgenus Phlebotomus. Our results confirm the validity of the morphologically characterized taxa. The position of P. salehi is doubtful. Variability in P. papatasi contrasts with that observed within other species having a wide distribution like P. (Paraphlebotomus) sergenti in the Old World or Lutzomyia (Lutzomyia) longipalpis in the New World. Consequently, it could be hypothesized that all populations of P. papatasi over its distribution area have similar vectorial capacities. The limits of the distribution area of L. major are correlated with the distribution of common rodents acting as hosts of the parasites.

The northward spread of leishmaniasis in Italy: evidence from retrospective and ongoing studies on the canine reservoir and phlebotomine vectors.

Visceral leishmaniasis (VL) incidence has been increased in Italy in humans and dogs since the 1990s, with new foci being detected within traditional boundaries of endemic transmission but also in northern regions previously regarded as non-endemic. To monitor the putative VL spreading, surveillance was implemented in northern continental Italy comprising: analysis of human cases recorded from 1990 through 2005; retrospective literature analysis of canine leishmaniasis (CanL) and phlebotomine sandfly records through 2002; prospective investigations in dogs from 2003 through 2005 and surveys on sandflies in 2003 and 2004. Two-hundred-thirty human cases (11% of Italian cases) were recorded. Their stratification by age and HIV status disclosed a sharp decrease of HIV/VL co-infections paralleled by concomitant increase of paediatric and HIV-negative adult patients during the study period. Four patients had no travel history. Seven leishmaniasis foci were retrospectively identified since 1990, whereas prospective investigations in dogs disclosed 47 autochthonous clinical cases and 106 autochthonous seropositives among 5442 dogs (2.1%) from 16 foci of six regions. Parasites were typed as Leishmania infantum MON-1. Four vector species were identified among 1696 Phlebotomus (Larroussius) collected specimens. Comparisons with historical data showed that P. perniciosus and P. neglectus have increased in density and expanded their geographic range in the study area. Northern continental Italy is now focally endemic for VL and a moderate risk for human disease does exist, although the intensity of transmission seems to be lower than in traditional settings of Mediterranean VL.

Mapping the main Leishmania phlebotomine vector in the endemic focus of the Mt. Vesuvius in southern Italy.

Geographical information systems and remote sensing were used to analyze the distribution of the Leishmania infantum-Phlebotomus perniciosus parasite-vector system in relation to environmental features of two opposite sides (coastal and Apennine) of Mt. Vesuvius, an area of intense transmission of human and canine leishmaniasis in southern Italy. Weekly phlebotomine collections were carried out during two consecutive warm seasons (2004- 2005) in 24 and 25 sites of the coastal and Apennine sides, respectively. Sandflies were caught using over one-thousand and seven hundred 20 x 20 cm-sticky traps placed in different environments. A total of 873 sandflies were collected, of which 284 (32.5%) were identified as P. perniciosus. The cumulative density (number of specimens/m2 of sticky trap/two nights) of this vector species was 3.9. P. perniciosus was significantly more abundant in the coastal side (5.8) as compared to the Apennine side (1.4). The main environmental differences between the two sides were the aspect (south-west for the coastal and north-east for the Apennine side) and land use. The predominance of green vegetated environments (forest, semi-natural and agricultural areas) in the coastal side, in contrast with the predominance of artificial surfaces (namely urban environment) in the Apennine side, could be responsible for the different P. perniciosus densities between the two surveyed areas.

Infection of sandflies by a cat naturally infected with Leishmania infantum.

Despite the recent reports of feline leishmaniosis from Southern Europe, cats are still regarded as unusual Leishmania hosts. A cat found chronically infected with Leishmania was submitted to xenodiagnosis. After being sedated, the animal was exposed to the bite of 100 laboratory-reared Phlebotomus perniciosus in a fine net cage for 90 min. Four out of 19 blood-fed sandflies (21%) showed motile promastigotes at the dissection. Parasites cultured from cat's lymph node and an infected fly were identical at PCR-RFLP genotyping and identified as Leishmania infantum MON-1, the main zymodeme responsible for human and canine leishmaniosis in Southern Europe. This is the first evidence of transmissibility of feline parasites to a proven vector, suggesting that cats may represent an additional domestic reservoir for L. infantum.

A review of leishmaniasis in Dalmatia (Croatia) and results from recent surveys on phlebotomine sandflies in three southern counties.

Zoonotic visceral leishmaniasis (VL) is a re-emerging disease in the Mediterranean area. A historical review on human and canine leishmaniasis in Croatia documents the presence of stable disease foci in coastal and insular territories of central and southern Dalmatia since the beginning of the 20th century. We report on a 3-year entomological investigation (2002-2004) aimed to study the current species composition and seasonality of phlebotomine sandflies in 10 localities of Dalmatia. Standard blacklight traps employed for sampling Culicoides midges in the frame of bluetongue disease surveillance were used for the first time in phlebotomine monitoring. A total of 2917 sandflies were collected and five species were identified, Phlebotomus tobbi, Phlebotomus neglectus, Phlebotomus perfiliewi, Phlebotomus mascittii and Sergentomyia minuta. Among the species, which may act as Leishmania infantum vectors, P. tobbi and P. neglectus were the most abundant.