Experimental investigation of the susceptibility of Italian Culex pipiens mosquitoes to Zika virus infection.

We investigated the susceptibility of an Italian population of Culex pipiens mosquitoes to Zika virus (ZIKV) infection, tested in parallel with Aedes aegypti, as a positive control. We analysed mosquitoes at 0, 3, 7, 10, 14, 20 and 24 days after an infectious blood meal. Viral RNA was detected in the body of Cx. pipiens up to three days post-infection, but not at later time points. Our results indicate that Cx. pipiens is not susceptible to ZIKV infection.

Cyanobacteria as a Source for Novel Anti-Leukemic Compounds.

Cyanobacteria are an inspiring source of bioactive secondary metabolites. These bioactive agents are a diverse group of compounds which are varying in their bioactive targets, the mechanisms of action, and chemical structures. Cyanobacteria from various environments, especially marine benthic cyanobacteria, are found to be rich sources for the search for novel bioactive compounds. Several compounds with anticancer activities have been discovered from cyanobacteria and some of these have succeeded to enter the clinical trials. Varying anticancer agents are needed to overcome increasing challenges in cancer treatments. Different search methods are used to reveal anticancer compounds from natural products, but cell based methods are the most common. Cyanobacterial bioactive compounds as agents against acute myeloid leukemia are not well studied. Here we examined our new results combined with previous studies of anti-leukemic compounds from cyanobacteria with emphasis to reveal common features in strains producing such activity. We report that cyanobacteria harbor specific anti-leukemic compounds since several studied strains induced apoptosis against AML cells but were inactive against non-malignant cells like hepatocytes. We noted that particularly benthic strains from the Baltic Sea, such as Anabaena sp., were especially potential AML apoptosis inducers. Taken together, this review and re-analysis of data demonstrates the power of maintaining large culture collections for the search for novel bioactivities, and also how anti-AML activity in cyanobacteria can be revealed by relatively simple and low-cost assays.

Evaluation of vector competence for West Nile virus in Italian Stegomyia albopicta (=Aedes albopictus) mosquitoes.

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle that involves wild birds as reservoir hosts. The virus is responsible for outbreaks of viral encephalitis in humans and horses. In Europe, Culex pipiens (Diptera: Culicidae) is considered to be the main vector of WNV, but other species such as Stegomyia albopicta (=Aedes albopictus) (Diptera: Culicidae) may also act as competent vectors of this virus. Since 2008 human cases of WNV disease have been reported in northeast Italy. In 2011, new areas of southern Italy became involved and a first outbreak of WNV lineage 1 occurred on the island of Sardinia. On the assumption that a potential involvement of St. albopicta in WNV transmission cannot be excluded, and in order to evaluate the competence of this species for the virus, an experimental infection of an St. albopicta laboratory colony, established from mosquitoes collected in Sardinia, was carried out. The results were compared with those obtained in a colony of the main vector Cx. pipiens. The study showed St. albopicta collected on Sardinia to be susceptible to WNV infection, which suggests this Italian mosquito species is able to act as a possible secondary vector, particularly in urban areas where the species reaches high levels of seasonal abundance.

New Italian guidelines for malaria prophylaxis in travellers to endemic areas.

BACKGROUND AND METHODS: As a consequence of the rapid evolution of malaria prophylaxis recommendations throughout the world, the Italian Society of Tropical Medicine (SIMET-Società Italiana di Medicina Tropicale) has set up a working group in charge of preparing a new national guideline. Other scientific societies interested in the topic were also involved in the project. RESULTS AND CONCLUSIONS: The group stated that awareness about malaria risk and characteristics, as well as protection from mosquito bites, are recommended for all travellers visiting malaria-endemic countries. The risk and benefit of malaria chemoprophylaxis must be carefully balanced before prescribing drugs: the disease-related risk must outweigh the possibility of drugs' side effects. As a general rule, malaria pills are the first choice for travellers to high-risk areas, such as sub-Saharan Africa, Eastern India, Myanmar, Eastern Indonesia, Papua New Guinea and, with some limitations, South-East Asia, and the Amazon part of Venezuela, Guyana and French Guyana. However, several other factors, such as itinerary, season, duration of trip, availability of insect bite protection, pre-existing conditions and compliance, must be taken into account. In low-risk areas, stand-by emergency treatment is the first option. In minimal-risk areas and in Plasmodium vivax areas, a prompt diagnosis only is advised (Central America, South America outside the Amazon basin, Middle East, China, Thailand, Nepal). Recommendations may be modified when particular groups of travellers are concerned, such as long-term residents, visiting friends and relatives, patients with pre-existing conditions, pregnant women and children.

Probable autochthonous introduced malaria cases in Italy in 2009-2011 and the risk of local vector-borne transmission.

We describe two cases of probable autochthonous introduced Plasmodium vivax malaria that occurred in 2009 and 2011 in two sites of South-Central Italy. Although the sources of the infections were not detected, local transmission could not be disproved and therefore the cases were classified as autochthonous. Sporadic P. vivax cases transmitted by indigenous vectors may be considered possible in some areas of the country where vector abundance and environmental conditions are favourable to malaria transmission.

Impact of environmental changes and human-related factors on the potential malaria vector, Anopheles labranchiae (Diptera: Culicidae), in Maremma, Central Italy.

The Maremma Plain (central Italy) was hyper-endemic for malaria until the mid-20th century, when a national campaign for malaria elimination drastically reduced the presence of the main vector Anopheles labranchiae Falleroni. However, the introduction of rice cultivation over 30 yr ago has led to an increase in the An. labranchiae population and concern over possible malaria reemergence. We studied the impact of anthropogenic environmental changes on the abundance and distribution of An. labranchiae in Maremma, focusing on rice fields, the main breeding sites. Adults and larvae were collected in three main areas with diverse ecological characteristics. Data were collected on human activity, land use, and seasonal climatic and demographic variations. We also interviewed residents and tourists regarding their knowledge of malaria. Our findings showed that the most important environmental changes have occurred along the coast; An. labranchiae foci are present throughout the area, with massive reproduction strictly related to rice cultivation in coastal areas. Although the abundance of this species has drastically decreased over the past 30 yr, it remains high and, together with climatic conditions and the potential introduction of gametocyte carriers, it may represent a threat for the occurrence of autochthonous malaria cases. Our findings suggest the need for the continuous monitoring of An. labranchiae in the study area. In addition to entomological surveillance, more detailed knowledge of human-induced environmental changes is needed, so as to have a more complete database that can be used for vector-control plans and for properly managing emergencies related to autochthonous introduced cases.

Aedes albopictus in Rome: results and perspectives after 10 years of monitoring.

In 1997, Aedes albopictus (Skuse 1894) was detected in Rome in two opposite areas of the city. In the following 2 years, the species quickly spread. In 2000, scattered foci of the species were reported in the whole urban area and in the outskirts of the capital city. In Rome, Ae. albopictus seems to have found optimal environmental conditions to proliferate and to overwinter through and without diapausing eggs. In ten years Ae. albopictus has colonized the whole urban area through three phases: first massive spread, following maintenance of infestation, and colonization of alternative winter breeding sites with favorable climatic conditions. Data collected during the 2007 show that rainfall is no longer the most important factor for the development of the species, with respect to the past. In fact Ae. albopictus probably has found new alternative larval breeding sites through the colonization of small water collections refilled periodically by human activities. During 2007-2008 winter season, in order to evaluate the species adaptability, a study of eggs hatching and length of larval cycle at low temperatures, was carried out in laboratory and in simulated field conditions. Data and results are showed and discussed also by the light of existing literature.

An overview of the lesson learned in almost 20 years of fight against the "tiger" mosquito.

Since its introduction in Italy in 1990, Aedes albopictus has spread quickly across the country, being at present reported in scattered foci in all regions below 600 m of altitude. The most important items of the lesson learned in almost 20 years of fight against the "Tiger" in Italy are here reported and discussed.

[Assessment of malaria resurgence risk in Morocco. Study of the vectorial capacity of Anopheles labranchiae in a rice cultivation area in the north of the country]. / Risque de réémergence du paludisme au Maroc. Etude de la capacité vectorielle d'Anopheles labranchiae dans une zone rizicole au nord du pays.

To assess the malaria reintroduction risk in Morocco, we analyzed the malariogenic potential of a rice cultivation area in the north of the country. Our results showed that the receptivity of this area is very high during all the period of the rice cultivation, from May to October, the vectorial capacity of An. labranchiae, malaria vector in Morocco, is considerably high during the summer which corresponds to the rice cultivation period. The risk of autochthonous malaria resumption is important because of the possible presence of gametocytes carriers in the last malaria focus which is bordering the study area. The risk of a tropical malaria introduction is unimportant seen the low vulnerability of the area and the uncertain competence of its vectors considered. However, this risk must be considered with a more attention.

Infection with chikungunya virus in Italy: an outbreak in a temperate region.

BACKGROUND: Chikungunya virus (CHIKV), which is transmitted by Aedes spp mosquitoes, has recently caused several outbreaks on islands in the Indian Ocean and on the Indian subcontinent. We report on an outbreak in Italy. METHODS: After reports of a large number of cases of febrile illness of unknown origin in two contiguous villages in northeastern Italy, an outbreak investigation was done to identify the primary source of infection and modes of transmission. An active surveillance system was also implemented. The clinical case definition was presentation with fever and joint pain. Blood samples were gathered and analysed by PCR and serological assays to identify the causal agent. Locally captured mosquitoes were also tested by PCR. Phylogenetic analysis of the CHIKV E1 region was done. FINDINGS: Analysis of samples from human beings and from mosquitoes showed that the outbreak was caused by CHIKV. We identified 205 cases of infection with CHIKV between July 4 and Sept 27, 2007. The presumed index case was a man from India who developed symptoms while visiting relatives in one of the villages. Phylogenetic analysis showed a high similarity between the strains found in Italy and those identified during an earlier outbreak on islands in the Indian Ocean. The disease was fairly mild in nearly all cases, with only one reported death. INTERPRETATION: This outbreak of CHIKV disease in a non-tropical area was to some extent unexpected and emphasises the need for preparedness and response to emerging infectious threats in the era of globalisation.

Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in central Italy.

To identify the natural vectors of Dirofilaria repens Railliet et Henry, entomological samplings were carried out in four sites within the Lazio region, foci of canine subcutaneous dirofilariasis. Collections were made in 2002-2003 by means of dog-baited and miniature Centers for Disease Control and Prevention traps as well as on humans. Microscopy identified 1576 attracted mosquito females as belonging to six species, but molecular diagnostics detected filarial DNA only in Culex pipiens L. and Aedes albopictus (Skuse, 1894). Dirofilaria immitis Leidy DNA, D. repens DNA, or both were found in the head and thorax of both mosquitoes. The simultaneous presence of vectors showing diurnal and nocturnal activity patterns is of concern for animal and human health. The finding of D. immitis DNA in mosquitoes in areas where only D. repens was been recovered in dogs also demonstrates that this filarial parasite circulates among carnivores (wild or domesticated pets).

High levels of human chitotriosidase hinder the formation of peritrophic membrane in anopheline vectors.

In the Anopheles midgut, Plasmodium falciparum produces a specific chitinase able to penetrate the blood meal surrounding the chitin-containing peritrophic membrane (PM). High levels of an analogous chitinase, chitotriosidase (CHIT), may be found in human blood, being the markers of macrophage activation. To verify the hypothesis that CHIT present in malaria patient blood could help parasite to overcome PM, we carried out a bioassay by feeding Anopheles stephensi females on an artificial apparatus that contained human blood from four different sources and with different chitinase concentrations: (1) healthy donors, as negative controls; (2) patients with malaria; (3) patients with Gaucher disease; and (4) whole blood enriched with commercial P. falciparum chitinase, as positive controls. After 16, 20 and 24 h of bloodfeeding, mosquitoes were dissected to extract the midgut and assess the effect of the different chitinases on membrane structure. Optical microscopy showed that formation of PM was clearly complete after 16 h in the posterior midgut from Anopheles already fed with healthy donor bloods. By contrast, PM formation was visible after 16 h in the posterior midgut of mosquitoes fed with malaria and Gaucher patient bloods but appeared clearly damaged at 20 and 24 h. At the same time, the PM formation was almost completely inhibited in the midgut of Anopheles fed with P. falciparum chitinase-enriched bloods. These alterations were clearly confirmed by transmission electronic microscopy. In the present paper, we demonstrate that human CHIT from different sources is active on anophelines' PM.

Laboratory evaluation of the bioinsecticide Spinosad for mosquito control.

Spinosad, a naturally occurring product of the fermentation of the bacterium Saccharopolyspora spinosa, is a highly effective bioinsecticide against a broad range of agriculturally important insect pests, and this agent has an excellent environmental and mammalian toxicological profile. In this study the efficacy of a Spinosad-based product (Laser 4.8% emulsifiable concentrate) was evaluated in laboratory bioassays against laboratory-reared mosquito strains of 3 species of medical importance: Aedes aegypti, Anopheles stephensi, and Culex pipiens. Spinosad was particularly effective against larval Aedes and Culex, with a less marked activity against anophelines (24-h median lethal concentration = 0.0096, 0.0064, and 0.039 mg/liter, respectively), showing a persistence of the insecticide action of about 6 wk in laboratory containers. The activity of the Spinosad-based product against adult mosquitoes (toxicity by ingestion and a possible irritant or repellent effect on gravid females) also was evaluated. Results are discussed and compared with those available in the literature.

Potential vectors of West Nile virus following an equine disease outbreak in Italy.

In the late summer of 1998, an outbreak of equine encephalomyelitis due to West Nile virus (WNV) occurred in the Tuscany region of central Italy. The disease was detected in 14 race horses from nine localities in four Provinces: Firenze, Lucca, Pisa and Pistoia. The outbreak area included Fucecchio wetlands (1800 ha), the largest inland marsh in Italy, and the adjacent hilly Cerbaie woodlands with farms breeding horses. To detect potential vectors of WNV, entomological surveys of Fucecchio and Cerbaie were undertaken during 1999-2002 by collecting mosquito larvae from breeding sites and adult mosquitoes by several methods of sampling. Among 6023 mosquitoes (Diptera: Culicidae) collected, 11 species were identified: Aedes albopictus (Skuse), Ae. vexans (Meigen), Anopheles atroparvus Van Thiel, An. maculipennis Meigen s.s., An. plumbeus Stephens, Culex impudicus Ficalbi, Cx. pipiens L., Culiseta longiareolata Macquart), Ochlerotatus caspius (Pallas), Oc. detritus (Haliday) and Oc. geniculatus (Olivier). In Fucecchio marshes, Cx. impudicus predominated with seasonal peak densities in spring and autumn: its greatest abundance during early spring coincides with arrival of migratory birds from Africa. In Cerbaie hills, Cx. pipiens predominated with peak population density in late summer. No viruses were isolated from 665 mosquitoes processed. These findings, plus other data on Italian mosquito bionomics, suggest a possible mode of WNV transmission involving the most abundant Culex in the Fucecchio-Cerbaie areas. Culex impudicus, being partly ornithophilic, might transmit WNV from migratory to non-migratory birds during springtime; Cx. pipiens, having a broader host range, would be more likely to transmit WNV from birds to horses and, perhaps, to humans by late summer.

[Note on Anopheles maculipennis complex in Morocco]. / Note sur le complexe Anopheles maculipennis au Maroc.

Anopheles belonging to Anopheles maculipennis complex, collected from February to June 2002 in eight provinces of Morocco (Khouribga, Taounate, Alhouceima, Chefchaouen, Fes, Khemisset, Kalaa Sraghna and Benslimane), were identified with characterization of the ribosomal DNA by PCR and ITS2 sequence analysis. The results of this study showed that all the identified specimens belong to the Anopheles labranchiae species.

First finding of Dirofilaria repens in a natural population of Aedes albopictus.

The invasive mosquito Aedes albopictus (Skuse) (Diptera: Culicidae) has become widespread in Italy during the past decade. Also Italy has foci of canine filariasis caused by Dirofilaria (Spirurida: Onchocercidae), due to subcutaneous D. repens Railliet & Henry as well as the dog heartworm D. immitis (Leidy) transmitted by various vector mosquitoes (Diptera: Culicidae). In 2002, at Fiumicino, west of Rome (Lazio Region), 17% of dogs were found to have D. repens microfilariae in peripheral blood. To evaluate the role of Ae. albopictus as a vector of Dirofilaria in this area, female mosquitoes were collected daily, June-October 2002, landing on dog or human bait in a rural house at Focene. Mosquitoes were maintained at 27 degrees C and 70% RH for 6 days, to allow development or purging of filaria larvae, then identified and frozen for subsequent molecular assay with filaria-specific ribosomal S2-S16 primers. To distinguish specimens harbouring infective L3 Dirofilaria larvae, DNA was extracted separately from the mosquito abdomen and head-thorax. Dirofilaria species were identified by sequencing, confirmed by polymerase chain reaction of positive specimens using primers specific for D. immitis and D. repens. Dirofilaria DNA was detected in 3/154 (2%) of Ae. albopictus females examined: D. repens DNA in head-thorax and abdomen of one collected 27th July; D. immitis in the abdomen of one collected 24th September; DNA of both D. immitis and D. repens in the head-thorax of one collected 11th October 2002. Thus Ae. albopictus is a potential vector of both Dirofilarias in Italy, representing risks for veterinary and human health.

Impact of the malaria control campaign (1993-1998) in the highlands of Madagascar: parasitological and entomological data.

Malaria transmission in the central highlands of Madagascar was interrupted in the 1960s by a national control program that used DDT indoor spraying and mass treatment with chloroquine. At the end of the 1980s in this region, epidemic malaria reappeared. Italian health authorities provided technical assistance to the National Malaria Control Program since the beginning of the resurgence of malaria in the central highlands. Yearly residual house spraying performed for 5 years (1993-1998) and the availability of antimalarial drugs reduced malaria transmission to very low levels, with improvement in parasitologic and entomologic indexes. A significant reduction of malaria prevalence was observed in the villages located at altitudes of 1,000-1,500 m, corresponding to the stratum of unstable malaria that was the main target of the antivector interventions. A significant reduction of malaria prevalence was also observed in the villages located at altitudes of 900-1,000 m, where malaria transmission is stable. The main vector Anopheles funestus was dramatically reduced in abundance and distribution in the sprayed areas.