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.

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.

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.

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.

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.

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.

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.

Short report: surveillance of Leishmania sp. among sand flies in Sicily (Italy) using a fluorogenic real-time polymerase chain reaction.

Leishmaniasis caused by Leishmania infantum is a complex zoonotic disease, resulting in cutaneous and visceral manifestations in both dogs and humans. The present study involved a published Taqman fluorogenic real-time polymerase chain reaction (PCR) assay for surveillance of Leishmania sp. parasites among sand flies trapped in two provinces in Sicily, Catania and Agrigento, during the summer and fall of 2003. Only male specimens were identified to species level, while females were used to evaluate Leishmania sp. infection by PCR testing. The two most prevalent sand fly species found were Phlebotomus perfiliewi and P. perniciosus. Of the female sand flies tested, 2.9% were positive for Leishmania sp. DNA by the PCR.

Three cases of ophthalmomyiasis externa by sheep botfly Oestrus ovis in Italy.

Human infection with the sheep nasal botfly Oestrus ovis is sporadic and is often the consequence of an accidental deposit of the larvae by an adult botfly in the eye. This infestation results in external ophthalmomyiasis that, although a very rare condition, is more common among people living close to farming communities. We report three cases of O. ovis infestation which occurred in Italy in a limited area of La Spezia province (Le Cinque Terre), Italy during summer 2004. None of the patients had contact with wild or farm animals.

[Evaluation of repellent and anti-feeding effect of garlic oil (Allium sativum) against the bite of phlebotomine sandflies Diptera: Psychodidae]. / Valutazione dell'effetto repellente ed anti-feeding dell'olio d'aglio (Allium sativum) nei confronti dei flebotomi (Diptera: Psychodidae).

The repellent and anti-feeding effect of garlic oil was evaluated in laboratory conditions against the bite of Phlebotomus papatasi females. The effectiveness was evaluated by two different laboratory procedures: (i) topical application of garlic oil on five human volunteers, using the "standard cage test", and (ii) feeding sandflies on artificial membranes treated with the compound. Garlic oil showed a significant protection by topical application on the skin of volunteers, being the protection 97% and 40%, respectively at 1% and 0.005% dilution. Garlic oil showed also an anti-feeding effect when tested on the artificial membrane feeding apparatus; the anti-feeding effect was dose dependent, being 100% at 1%. The results of the present study confirm previous observations on the repellent effect of garlic oil against haematophagous arthropods.

Laboratory study to investigate the efficacy of cyfluthrin EW treated nets against phlebotomine sandflies.

Toxic and feeding inhibition effect of cyfluthrin treated nets was assessed and compared with the activity of deltamethrin impregnated nets, before and after 2 and 4 washes against Phlebotomus papatasi. Impregnated cyfluthrin nets (50, 25, 12.5 mg active ingredient (a.i.)/m2) caused knock down and mortality rates higher than 90%, being of 100% at the highest dose, while the nets impregnated with deltamethrin (25, 12.5, 6.25 mg a.i./m2) showed low mortality rates (13.3-43.5%). Also after washing cyfluthrin at the dose of 50 mg a.i./m2 provided higher activity than deltamethrin (25 mg a.i./m2), being mortality rates within 24 hs of 66.7 and 48.9% for cyfluthrin and 38.0 and 2.1% for deltamethrin, respectively after 2 and 4 washes. Before washing, cyfluthrin (50 mg a.i./m2) significantly prevented blood feeding, with respect to both untreated (88.6%) and deltamethrin impregnated nets (56.8%). Deltamethrin (25 mg a.i./m2) showed low feeding inhibition (48.4%), although it was significantly higher than control. The washing of cyfluthrin impregnated nets (50 mg a.i./m2) produced a decrease of the feeding inhibition, being the reduction of 26.6% and 41.3%, respectively after 2 and 4 washes. Two washes of deltamethrin impregnated nets (25 mg a.i./m2) yielded a very high reduction of antifeeding inhibition (68.8%). These laboratory results if confirmed by field evaluation indicate cyfluthrin as good candidate for treatment of mosquito nets to be used against phlebotomine sandflies.

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.

ITS 2 sequences heterogeneity in Phlebotomus sergenti and Phlebotomus similis (Diptera, Psychodidae): possible consequences in their ability to transmit Leishmania tropica.

An intraspecific study on Phlebotomus sergenti, the main and only proven vector of Leishmania tropica among the members of the subgenus Paraphlebotomus was performed. The internal transcribed spacer 2 (ITS2) sequences of 12 populations from 10 countries (Cyprus, Egypt, Italy, Lebanon, Morocco, Pakistan, Portugal, Spain, Syria, and Turkey) were compared. Samples also included three species closely related to P. sergenti: Phlebotomus similis (three populations from Greece and Malta), Phlebotomus jacusieli and Phlebotomus kazeruni. Our results confirm the validity of the taxa morphologically characterised, and imply the revision of their distribution areas, which are explained through biogeographical events. At the Miocene time, a migration route, north of the Paratethys sea would have been followed by P. similis to colonise the north of the Caucasus, Crimea, Balkans including Greece and its islands, and western Turkey. Phlebotomus sergenti would have followed an Asiatic dispersion as well as a western migration route south of the Tethys sea to colonise North Africa and western Europe. This hypothesis seems to be well supported by high degree of variation observed in the present study, which is not related to colonisation or to intra-populational variation. Two groups can be individualised, one oriental and one western in connection with ecology, host preferences and distribution of L. tropica. We hypothesise that they could be correlated with differences in vectorial capacities.

Cutaneous leishmaniasis vector control perspectives using lambdacyhalothrin residual house spraying in El Ingenio, Miranda State, Venezuela.

An indoor vector control trial was carried out between December 1996 and February 1997, with the aim of reducing the population densities of Lutzomyia ovallesi, the proven vector of cutaneous leishmaniasis (CL) in El Ingenio, Miranda State, Venezuela. Houses were matched according to their structure ("bahareque" [mud and straw], concrete, and wood) and randomly assigned to a control group (n = 19) or a treated group (n = 20) that was sprayed with 25 mg/m(2) of lambdacyhalothrin. This dose was selected on the basis of a laboratory susceptibility test of Lu. ovallesi to pyrethroids. Sandfly abundance was measured using CDC light traps 7-79 d post-intervention, by which time the sandfly population in control houses had declined to very low levels, at what was the end of the transmission season. Catches of total females, fed females, and males were significantly lower in sprayed than in control houses immediately after treatment. Catches of total females and males in sprayed houses recovered, reaching the level of control houses after 7 and 11 weeks, respectively. Bioassays following WHO protocols using contact cones on a sandfly laboratory colony showed a short residual effect of the insecticide explaining these results. In contrast, there was no tendency for catches of fed females in the sprayed houses to recover during the course of the trial. Previous studies of adult population dynamics showed a short season of high abundance. Combined with these results, we believe that 2 indoor sprayings with lambdacyhalothrin, the first at the beginning of November and the second at the beginning of January, would considerably reduce the Lu. ovallesi population, and control CL transmission in this and other foci where this sandfly is considered to be the main vector.

[The pigeon tick, Argas reflexus, and hazard for human health]. / La zecca del piccione Argas reflexus (Acari: Argasidae) ed i rischi per la salute umana.

The massive presence of the pigeon (Columba livia f. domestica) in urban areas not only is cause of serious aesthetic damages as decline of squares, of churches, of statues and buildings, but often creates many hygienic and medical problems. The pigeon, in fact, can transmit a great number of pathogenic agents and also be infested by several ectoparasites. Among these, Argas reflexus (Acari: Argasidae), the well known pigeon tick, is the most important ectoparasite that can involve also human health. Buildings infested by A. reflexus and attacks by this tick species to man are frequently reported, with an increasing interest for human health. In fact the pigeon tick bites can induce anaphylactic reactions for the presence of allergens, and as vector, the tick can also transmit some pathogens. This review deals with actual knowledge on the distribution, eco-biology, vector competence, prevention and control of A. reflexus in Italy.

Testing insecticide susceptibility of Phlebotomus perniciosus and P. papatasi (Diptera: Psychodidae) in Italy.

A study was undertaken to establish the susceptibility of two recently colonized sand fly species, Phlebotomus perniciosus and P. papatasi to some insecticides. A laboratory colony of P. papatasi, unexposed to insecticides for a period of ten years, was used as reference strain. Bioassay tests were carried out according to the WHO standard protocols based on the mortality rates recorded at different exposure times. Serial (from 1 to 60 min) exposures to DDT 2%, lambda-cyhalothrin 0.06% and permethrin 0.2% were tested on groups of 30 adult sugar-fed female sand flies, for a total of 7,470 specimens. Data were subjected to probit analysis and the susceptibility was calculated at LT50 and LT95 by comparison with the laboratory reference strain. The LT50 values for P. perniciosus were 19.9, 3.2 and 6.9 min, and for P. papatasi 18.0, 7.4 and 11.0 min, using DDT, lambda-cyhalothrin and permethrin, respectively. The results showed that two Italian populations of P. perniciosus and P. papatasi from Campania region and from Rome, respectively, are susceptible to the insecticides tested as compared with the reference strain used.