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.

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.

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.

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.

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.

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.

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.

[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.

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).

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.

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 structure-antigenicity relationship of peptides from human immunodeficiency virus type 1 (HIV-1) p18 protein by circular dichroism.

The antigenicity of Human Immunodeficiency Virus type 1 (HIV-1) matrix p18 protein was evaluated by analyzing the specificity of anti-p18 antibodies elicited either in HIV-1 infected humans, or in HIV-1 infected or immunized chimpanzees, against a panel of long and short overlapping synthetic peptides [from 12 to 46 amino acid (aa) residues] covering the entire sequence of p18. The relationship between peptide structure and antigenicity was further investigated by probing the secondary structures of the peptides by circular dichroism. The results obtained clearly showed the immunodominance of the N-terminal region mimicked by peptide P1 (aa 2-45), which reacted with 52 and 100% of human and chimpanzee anti-p18 sera, respectively. In contrast smaller 15 aa long peptides C1, C2, C3, C4 and P3 which cover the entire sequence of immunodominant peptide P1, showed only weak or no reactivity. In contrast to widely accepted hypotheses, circular dichroism analysis of both small and large peptides secondary structures did not show any obvious correlation between antigenicity and the ability of peptides to adopt an ordered conformation.

Age-related prevalence of Plasmodium falciparum circumsporozoite antibody in an hyperendemic area of Madagascar, and its relationship with parasite prevalence.

An epidemiologic survey on malaria endemicity levels, including entomologic, parasitologic, and immunologic indicators, was carried out in a village of the Madagascar highlands (Analaroa) at the end of the 1990-1991 rainy season. The results indicate that malaria is hyperendemic and Anopheles funestus is the main vector in the area. The prevalence of parasitemia decreased with age from a maximum level of about 60% in children less than five years of age to a minimum of about 16% among those more than 29 years of age. The prevalence of Plasmodium falciparum circumsporozoite antibodies (Ab-Cs) increased with age from a minimum level of about 10% in children less than five years of age to a maximum of 71.7% among those more than 29 years of age. An inverse correlation was observed between P. falciparum prevalence and levels of Ab-Cs and parasite prevalence. The study confirmed that prevalence and Ab-Cs levels are reliable indicators of malaria endemicity in hyperendemic areas. Schoolchildren between five and 14 years of age are considered the most practical and susceptible group for this kind of epidemiologic study.

Plasmodium falciparum circumsporozoite antibody prevalence in Madagascar: a longitudinal study in three different epidemiologic areas.

A longitudinal study on the prevalence of Plasmodium falciparum circumsporozoite antibodies (Ab-CS) was carried out in 15 villages of three epidemiologic areas of Madagascar: the highlands, east coast, and the southwest region. A total of 3,967 blood samples were collected from November 1989 to April 1991 from cohorts of resident schoolchildren. The prevalence of Ab-CS in the examined population varied greatly according to the different ecoepidemiologic areas of the country. A correlation analysis was made between Ab-CS and P. falciparum parasite prevalence in the same population. High Ab-CS prevalence rates (25-75%) and levels (optical density = 0.28-0.76) were observed in the villages of the east coast (mesoendemic stable malaria). The Ab-CS prevalence rates varied from 0 to 37% in the highlands and southwest region villages (unstable malaria). The use of Ab-CS prevalence is proposed to be a useful and reliable seroepidemiologic marker of malaria endemicity in those areas of Madagascar in which malaria transmission is high and continues for more than four months a year.

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.

Avermectins, MK-933 and MK-936, for mosquito control.

Two avermectins were shown to be effective as mosquito larvicides or when fed to adult female mosquitoes in sucrose solution or in blood. Larval LC50 values of compounds MK-933 and MK-936, expressed as parts per billion, were found to be 3.94 and 2.42 for Culex pipiens, 5.85 and 2.90 for Anopheles stephensi and 23.4 and 10.4 for Aedes aegypti. When fed to adult females of An. stephensi, Ae. aegypti or Cx quinquefasciatus, MK-933 at 2.8mg active ingredient per litre in sucrose solution caused complete mortality within 60 hours. When female mosquitos engorged blood from mice injected subcutaneously 12 hours previously with MK-933 at 82 mg a.i./kg, mortality rates after 36 hours were 100% for An. stephensi, over 60% for Ae. aegypti and over 50% for Cx quinquefasciatus. Thus the systemic insecticidal effect was greater against anopheline than against culicine female mosquitoes.

Anopheles sacharovi (Diptera: Culicidae): a reemerging malaria vector in the Ararat Valley of Armenia.

In 1994, the first indigenous case of malaria since the 1960s was reported in Armenia, and the number of cases quickly increased in the ensuing years. In 1998, a roll-back malaria program was developed to eliminate the recently established foci of malaria infection and to prevent the reestablishment of malaria in Armenia. As part of this program, we carried out entomological surveys to identify the potential malaria vectors in the Ararat Valley, the area where most of the indigenous malaria cases had been reported. In particular, we attempted to ascertain the presence of Anopheles sacharovi Favre, which is historically the most important malaria vector in Armenia yet which had not been reported since 1965. In 1998-2000, we collected adult mosquitoes and larvae in the city of Masis and in three rural villages of the Ararat Valley. Species identification of the members of the Anopheles maculipennis complex was performed through egg and larval morphology, heteroduplex analysis, and sequencing of the second internal transcribed spacer of ribosomal DNA. Two species of the complex were identified: An. sacharovi, found in all of the study sites, andAn. maculipennis s.s. Meigen, the most common species in the area. The reemergence of An. sacharovi in the study sites shows that the receptivity for malaria is still high in the Ararat Valley and, likely, in other regions of Armenia.

Status of malaria vectors in Italy.

About 30 yr after malaria eradication, surveys to assess the presence and abundance of anopheline vectors were carried out in central and southern Italy and in the islands of Sardinia and Sicily from 1992 to 1994. Anopheles labranchiae Falleroni was present in scattered foci in all regions, except for Tuscany, where it breeds almost exclusively in rice fields (Grosseto Province). Most common breeding sites were rivers and streams, followed by ponds and ground pools. The highest adult density was found in Tuscany near rice fields and along the west coast of Calabria. Anophelines in Grosseto were abundant at human bait, with peaks of > 200 landings per human per night and vectorial capacity between 7.3 and 26 for Plasmodium falciparum and between 8.3 and 32.5 for Plasmodium vivax. Anopheles sacharovi Favre, a former malaria vector in Puglia and Sardinia, was not found in these regions. The other vector in southern Italy, Anopheles superpictus Grassi, was found at low densities on the western and eastern coasts of Calabria. All anopheline populations were fully susceptible to deltamethrin, malathion, and DDT but showed reduced susceptibility to permethrin and propoxur. These data are discussed in the light of a possible reintroduction of malaria into Italy.

Laboratory and field evaluation of metallic copper on Aedes albopictus (Diptera: Culicidae) larval development.

Laboratory bioassays and field trials were carried out to study the effect of metallic copper on the development of Aedes albopictus (Skuse). Multiwire electric cable was used as a source of metallic copper. Three different doses were used in laboratory tests (5, 10, and 20 g/liter) and two in field tests (20 and 40 g/liter). In the laboratory, 10 g/liter induced high mortality and a lack of development in Ae. albopictus larvae and doses of 20 g/liter completely inhibited development. Larval mortality was higher in earlier instars than in third through fourth instars and pupae. No effects were reported on egg hatching. Copper ion concentration in water increased up to 574 ppb for 5 g/liter dose, 710 ppb for 10 g/liter dose, and 1,210 ppb for 20 g/liter dose, within week 6. The increasing concentration of copper in water was correlated positively with the decreasing production of adults. Copper ions concentration < 500 ppb did not or only slightly affected larval development and mortality of Ae. albopictus in laboratory tests. Copper concentrations between 500 and 1,000 ppb delayed larval development and caused high mortality. Copper concentrations > 1,000 ppb inhibited larval development completely killing all the larvae. This last result has been achieved by the use of a 20 g/liter dose of metallic copper in water. Copper also affected adult weight. In field trials, 20 g/liter reduced the number of larvae in treated pots by 90%, and 40 g/liter completely prevented oviposition. Moreover, the persistence of the toxic action of metallic copper in the field lasted for several months.