Seasonal Dynamics of Phlebotomine Sand Fly Species Proven Vectors of Mediterranean Leishmaniasis Caused by Leishmania infantum.

BACKGROUND: The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011-2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported. METHODS/PRINCIPAL FINDINGS: A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am. CONCLUSIONS: Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories.

Experimental evaluation of sand fly collection and storage methods for the isolation and molecular detection of Phlebotomus-borne viruses.

BACKGROUND: Several viruses have been recently isolated from Mediterranean phlebotomine sand flies; some are known to cause human disease while some are new to science. To monitor the Phlebotomus-borne viruses spreading, field studies are in progress using different sand fly collection and storage methods. Two main sampling techniques consist of CDC light traps, an attraction method allowing collection of live insects in which the virus is presumed to be fairly preserved, and sticky traps, an interception method suitable to collect dead specimens in high numbers, with a risk for virus viability or integrity. Sand flies storage requires a "deep cold chain" or specimen preservation in ethanol. In the present study the influence of sand fly collection and storage methods on viral isolation and RNA detection performances was evaluated experimentally. METHODS: Specimens of laboratory-reared Phlebotomus perniciosus were artificially fed with blood containing Toscana virus (family Bunyaviridae, genus Phlebovirus). Various collection and storage conditions of blood-fed females were evaluated to mimic field procedures using single and pool samples. Isolation on VERO cell cultures, quantitative Real time-Retro-transcriptase (RT)-PCR and Nested-RT-PCR were performed according to techniques commonly used in surveillance studies. RESULTS: Live engorged sand flies stored immediately at -80 °C were the most suitable sample for phlebovirus identification by both virus isolation and RNA detection. The viral isolation rate remained very high (26/28) for single dead engorged females frozen after 1 day, while it was moderate (10/30) for specimens collected by sticky traps maintained up to 3 days at room temperature and then stored frozen without ethanol. Opposed to viral isolation, molecular RNA detection kept very high on dead sand flies collected by sticky traps when left at room temperature up to 6 days post blood meal and then stored frozen in presence (88/95) or absence (87/88) of ethanol. Data were confirmed using sand fly pools. CONCLUSIONS: While the collection and storage methods investigated had not much impact on the ability to detect viral RNA by molecular methods, they affected the capacity to recover viable viruses. Consequently, sand fly collection and handling procedures should be established in advance depending on the goal of the surveillance studies.

Preliminary investigation on tick fauna in the neighborhood of Tarquinia, Lazio, Italy.

INTRODUCTION: This study represents a preliminary inquiry on tick fauna composition carried out in Maremma, Central Italy, where very few data are available. MATERIALS AND METHODS: The study area was selected in the limestone hills surrounding Tarquinia town, on the base of suitable elements directly affecting the tick occurrence and the possible circulation of tick-borne pathogens, such as wild cattle rearing, kind of vegetation and human activities. Since a proper understanding of tick ecology is critical in predicting the risk of tick-borne pathogen transmission in a given area, a series of surveys was planned in such area in 2011. RESULTS: One hundred fifty-four ticks belonging to 4 genera and 6 species, namely Ixodes ricinus (n. = 109; 70%), Rhipicephalus bursa (n. = 18; 12%), Rhipicephalus turanicus (n. = 14; 9%), Rhipicephalus sanguineus (n. = 6; 4%), Hyalomma marginatum (n. = 4; 3%) and Dermacentor marginatus (n. = 3; 2%) were identified and reported for the area. DISCUSSION: The results of this acarological research represent a significant contribution to the knowledge of the tick fauna of rural areas in Northern Lazio Region, as first step toward a future molecular investigation on pathogen circulation.

Characterization of spotted fever group Rickettsiae in ticks from a city park of Rome, Italy.

BACKGROUND: Ticks are vectors and important reservoirs for microbial agents that cause disease in humans and animals. Among these pathogens, the members of Rickettsia species play an important role in public health. AIM AND METHODS: One hundred twenty-nine ticks belonging to four tick species (Ixodes ricinus, Rhipicephalus turanicus, Dermacentor marginatus, and Haemaphysalis punctata) were collected at different sites of the Insugherata Natural Reserve, localized in the urban area of Rome, Italy. Questing ticks were tested by PCR for Rickettsia spp., amplifying partial gene of ompA. RESULTS: Forty-six ticks were found to be infected with Rickettsia species. Five SFG rickettsiae were identified: three human pathogens Rickettsia conorii, Rickettsia massiliae and Rickettsia aeschlimannii, and two putative new strains Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2. The phylogenetic analysis of partial gene sequences of ompA, gltA, and 17-kd antigen showed that they clustered with several rickettsiae with unidentified pathogenicity. However, Rickettsia sp. strain RM1 and Rickettsia sp. strain RM2 clustered in a statistically supported clade with R. massiliae, and R. monacensis, respectively. CONCLUSION: Our findings suggest that Rickettsia species other than R. conorii are implicated in human disease in Italy.

Prevalence of tick-borne pathogens in an urban park in Rome, Italy.

INTRODUCTION: Limited information is available about the presence of tick-borne pathogens in urban parks in Italy. To fill this gap, ticks were collected in a public park in Rome over a 1-year period and screened by molecular methods for tick-borne pathogens. RESULTS AND CONCLUSION: The most abundant tick species were Rhipicephalus turanicus and Ixodes ricinus. The predominant pathogens detected were Borrelia. burgdorferi sensu lato (36%), Rickettsia spp. (36%), and Coxiella burnetii (22%). Among less frequently detected pathogens, Babesia microti was detected for the first time in Italy, with a prevalence of 4%. Neither Bartonella spp. nor Francisella tularensis were detected. With regard to co-infections, the most frequent double and triple infections involved Rickettsia spp., B. burgdorferi sl., and C. burnetii.. A positive correlation was detected between pathogens and I. ricinus. Further studies are needed in order to assess risk associated with tick-borne pathogens in urban areas.

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.

Detection of microbial agents in ticks collected from migratory birds in central Italy.

Tick species characterization and molecular studies were performed within ornithological surveys conducted during 2010 and 2011 in the Lazio Region of central Italy. A total of 137 ticks were collected from 41 migratory birds belonging to 17 species (four partial migrants and 13 long-distance migrants). Most ticks were nymphs, with a predominance of Hyalomma marginatum marginatum and H. m. rufipes, and a small portion of Ixodes and Amblyomma species. All tick species analyzed were infected, and the molecular pathogen recognition revealed the presence of Rickettsia aeschlimannii, Rickettsia africae, Erlichia spp., Coxiella burnetii, Borrelia burgdorferi sensu lato group, and Babesia microti, whereas no genomic DNA of Bartonella spp. or Francisella tularensis was detected. The results of the survey show that H. marginatum ticks appear to be a vector of microbial agents that may affect human and animal health and that migratory birds may be an important carrier of these ticks. Additional studies are needed to better investigate the role of migratory birds in the epidemiology of these pathogens.

Ecological niche modelling of potential West Nile virus vector mosquito species and their geographical association with equine epizootics in Italy.

In Italy, West Nile virus (WNV) equine outbreaks have occurred annually since 2008. Characterizing WNV vector habitat requirements allows for the identification of areas at risk of viral amplification and transmission. Maxent-based ecological niche models were developed using literature records of 13 potential WNV Italian vector mosquito species to predict their habitat suitability range and to investigate possible geographical associations with WNV equine outbreak occurrence in Italy from 2008 to 2010. The contribution of different environmental variables to the niche models was also assessed. Suitable habitats for Culex pipiens, Aedes albopictus, and Anopheles maculipennis were widely distributed; Culex modestus, Ochlerotatus geniculatus, Ochlerotatus caspius, Coquillettidia richiardii, Aedes vexans, and Anopheles plumbeus were concentrated in north-central Italy; Aedes cinereus, Culex theileri, Ochlerotatus dorsalis, and Culiseta longiareolata were restricted to coastal/southern areas. Elevation, temperature, and precipitation variables showed the highest predictive power. Host population and landscape variables provided minor contributions. WNV equine outbreaks had a significantly higher probability to occur in habitats suitable for Cx. modestus and Cx. pipiens, providing circumstantial evidence that the potential distribution of these two species coincides geographically with the observed distribution of the disease in equines.

Seasonal dynamics of tick species in an urban park of Rome.

Regular collections were obtained in the Natural Reserve of the Insugherata of Rome during 2011 in order to obtain the tick species composition and the respective seasonal dynamics of the area. A total of 325 ticks was collected in selected sites by means of drag sampling. Among the identified species, Rhipicephalus turanicus was the most abundant (72.3%), followed by Ixodes ricinus (19.7%), Dermacentor marginatus (6.5%), Haemaphysalis punctata (1.2%), and Rhipicephalus bursa (0.3%). R. turanicus occurred mainly in pastures, showing a mono-modal seasonal activity pattern from spring to early summer. Questing I. ricinus were prevalent in woodland from October to May, and the seasonal trend of specimens showed a weak peak in winter. Although adult D. marginatus exhibited seasonal dynamics similar to I. ricinus, with an activity period from October to April, this species occurred in a different environment (pasture) and with considerably lower densities. Haemaphysalis punctata and R. bursa were rare, with an apparent autumn and autumn-winter seasonal activity, respectively. While the species diversity recorded appears as an unequivocal consequence of the natural state of the park, the remarkable R. turanicus density could be a direct effect of the recent introduction of wild boar, as carriers, from the close Veio Park. The presence of the species, a proven vector of various diseases in humans and domestic animals, is discussed in the light of the possible risk of tick-bite exposure of park workers and visitors.

Environmental correlates of Crimean-Congo haemorrhagic fever incidence in Bulgaria.

BACKGROUND: Crimean-Congo Haemorrhagic Fever (CCHF) is a zoonotic viral disease transmitted by ixodid tick bites, mainly of Hyalomma spp., or through contact with blood/tissues from infected people or animals. CCHF is endemic in the Balkan area, including Bulgaria, where it causes both sporadic cases and community outbreaks. METHODS: We described trends of CCHF in Bulgaria between 1997 and 2009 and investigated the associations between CCHF incidence and a selection of environmental factors using a zero-inflated modelling approach. RESULTS: A total of 159 CCHF cases (38 women and 121 men) were identified between 1997 and 2009. The incidence was 0.13 cases per 100,000 population/year with a fatality rate of 26%. An epidemic peak was detected close to the Turkish border in the summer of 2002. Most cases were reported between April and September. Increasing mean temperature, Normalized Difference Vegetation Index (NDVI), savannah-type land coverage or habitat fragmentation increased significantly the incidence of CCHF in the CCHF-affected areas. Similar to that observed in Turkey, we found that areas with warmer temperatures in the autumn prior to the case-reporting year had an increased probability of reporting zero CCHF cases. CONCLUSIONS: We identified environmental correlates of CCHF incidence in Bulgaria that may support the prospective implementation of public health interventions.

A noteworthy record of Ornithodoros (Alectorobius) coniceps (Ixodida: Argasidae) from Central Italy.

The first record of Ornithodoros (Alectorobius) coniceps (Canestrini) was reported for Italy in 1877, inside the interstices of the ancient mosaics at S. Marco Basilica in Venice. Afterwards only few discoveries of the species are reported for Italy; the last record is dated back to 1984, in L'Aquila town (Abruzzo Region). The present study shows the data of a survey carried out as a result of a massive infestation by O. coniceps in an ancient villa in Anzio town (Latium region) recently restored. In the past decades the villa has been fallen into disrepair, becoming an occasional shelter for wild animals mainly pigeons, that colonized the whole building for generations. This case appears worthy of note because it is the first record of this species after more than 25 years in Italy. A total of 136 specimens were collected by three methods: manual, mechanical aspirators and Wilson traps. Wilson trapping indicates positive O. coniceps tropism for CO(2).

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.

Ticks infesting livestock on farms in Western Sicily, Italy.

The results of a 2-year survey on ticks infesting livestock on farms in Western Sicily, Italy, are reported. Several dogs living in the same area were also examined for ticks. Moreover, free-living ticks from one cattle farm were caught by CO(2) traps and flagging. A total of 6208 specimens was collected belonging to 9 species: Rhipicephalus bursa (32.4%), Rhipicephalus turanicus (22.7%), Rhipicephalus sanguineus (19.3%), Hyalomma lusitanicum (12.0%), Haemaphysalis punctata (7.8%), Hyalomma marginatum (3.5%), Dermacentor marginatus (1.4%), Ixodes ricinus (0.8%) and Ixodes hexagonus (0.1%). The species belonging to the genus Rhipicephalus were present on all hosts and habitats monitored, R. bursa was found to be abundant on cattle (46.3%) and sheep (62.0%), R. sanguineus was prevalent on dogs (50.4%), and R. turanicus on donkeys (41.0%) and as free-living species (48.5%).

[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.