Morphological and molecular characterization of adults and larvae of Crassicauda spp. (Nematoda: Spirurida) from Mediterranean fin whales Balaenoptera physalus (Linnaeus, 1758).

Crassicauda boopis is known to infect the kidneys and vascular system of mysticetes included Balaenoptera physalus and has been recently reported in Mediterranean waters. Identification at the species level relies on the observation of morphological features of the adult parasites, but field conditions during necropsy and the massive reaction of the host's immune system often prevent optimal conservation of the extremities. Moreover, larval stages of Crassicauda have never been described and no sequences are available in public databases to help such identification. Adult and larvae of Crassicauda were isolated from four specimens of B. physalus and studied with morphological and molecular techniques. Specimens of C. anthonyi, C. grampicola and Crassicauda sp. isolated from Ziphius cavirostris, Grampus griseus, Stenella coeruleoalba and Tursiops truncatus respectively were studied as well. Sequences of nuclear markers 18S and ITS-2 and of mitochondrial gene cox1 were obtained and phylogenetic relationships within the genus Crassicauda were analysed. Analysis of the ITS2 grouped the different species in accordance with morphological identification, as already evidenced in literature for other Spirurida. A higher intra-specific variability was observed for the cox1 gene, for which two species (C. grampicola and C. anthonyi) did not appear as monophyletic in the tree. Well-developed non-attached larval specimens in the intestinal lumen of a whale calf were molecularly identified as C. boopis, allowing new insights on the life cycle of this species. This work broadens the genetic database on cetaceans parasites, allowing species identification even in challenging field conditions or in poor conservation of the samples; moreover, the first morphological description of C. boopis larvae is provided.

Importance of Aedes albopictus in veterinary medicine.

To assess the role of Aedes albopictus in transmission of filarial nematodes of veterinary importance, researches were carried out in different geographical areas. In Italy a first research was performed to study the susceptibility of Ae. albopictus to Dirofilaria repens, D. immitis and Setaria labiatopapillosa. The development of L3 larvae was longer than in other species of mosquitoes but Ae. albopictus could be a suitable vector of filariae. To understand the role of Ae. albopictus in the natural transmission of Dirofilaria and to assess the risk for animal and human health, in 2000, 2001 and 2002 another study was carried out in the town of Padua. A total of 2,534 Ae. albopictus were caught on human-attracted mosquitoes. Specific primers and sequencing identified filarial DNA as D. immitis; Ae. albopictus was proved a natural vector of D. immitis. Similar results were confirmed in Central Italy also for D. repens. The presence of Ae. albopictus increased the probability of transmission of canine and human dirofilariosis in urban environment and it could change the epidemiology of dirofilariosis, in particular for what concern the time of biting and the risk season. These aspects must be considered to outline a correct prophylaxis.

Animal babesiosis: an emerging zoonosis also in Italy?

In Italy, babesiosis is widespread in several Central and Southern Regions, but few data are available on its presence in most Italian areas. In 2004 a project was financed by the MIUR to investigate on the babesiosis epidemiology in vertebrate and invertebrate hosts, and on the transmission risk for humans in Central and Northern Regions of the country. Microscopy and/or molecular tools were applied to blood samples of wild animals, livestock and pets, and to 1,677 ticks collected on animals or in the environment, with the aim of detect babesial parasites. Moreover, serological tests were used to evaluate the circulation of these protozoa among animals and people at risk. Microscopy identified as positive 5.0% of the animals, mostly living in Central Regions, but also in Northern areas considered Babesia-free. Serology evidenced the same general trend. PCR detected "piroplasm" DNA in 13.8% of the animals, and sequencing identified babesial parasites in 101/233 samples. The ticks were identified as belonging to 12 species, mostly represented by Ixodes ricinus, Rhipicephalus sanguineus and Dermacentor marginatus. Molecular analyses evidenced babesial parasites in 3.8% of them; in Rh. sanguineus was also demonstrated the vertical transmission of Babesia canis canis. To date 30 human sera have been analysed: 3 showed antibodies to B. microti. Animal babesiosis is largely present among pets, wild and farm animals, whereas goats seem refractory to the infection. In wild ungulates have been found the B. divergens-like, and the Babesia EU1 strains (reported in Italy in humans). Our findings evidenced the low reliability of microscopy in epidemiological studies, and the need of new/improved immunological tests to face diagnostic problems. The monitoring of infected areas and infection rates, joined to appropriate control programs, seems necessary to avoid the transmission of babesiosis to humans.

Mosquitoes as vectors of Setaria labiatopapillosa.

An infected bovine-baited trap was utilised in summer 1994 to catch possible intermediate hosts of S. labiatopapillosa in northeastern Italy. Collections were made for 21 nights from 8.00 p.m. to 6.00 a.m. every 2 h and, after 12 September, every 30 min. Among the 16,159 mosquitoes sampled, 11,052 were freshly blood-fed. Most of the unfed females and a representative sample of those which had fed were identified as follows: Culex pipiens, Aedes caspius, A. vexans, Culiseta annulata, Anopheles maculipennis s.l., A. claviger and Coquillettidia richiardii. In spite of having the highest relative density, C. pipiens is the species which fed the least frequently and showed lower susceptibility and efficiency. The 2 Aedes species appear to act as vectors, particularly A. caspius, which proved to be the most efficient vector (K.I. = 0.8). A. claviger and A. maculipennis contribute to S. labiatopapillosa transmission, but their scarce presence reduces their epidemiological relevance. The other species identified showed a complete refractoriness to the infection. The risks for veterinary and medical health are discussed.

Aedes albopictus is a natural vector of Dirofilaria immitis in Italy.

Investigations were carried out in Padova town (Veneto region, NE Italy) to define the actual role of Aedes albopictus in the natural transmission of Dirofilaria nematodes, and to assess the risk that its presence might represent for veterinary and medical health. During summer 2000-2002 daytime captures of human-attracted mosquitoes were carried out in three areas of the town. The presence of filarial parasites in mosquitoes was evaluated by PCR, and sequencing confirmed species assessment. DNA extraction was performed separately on pools of the insect abdomen and thorax-head, to discriminate between Dirofilaria infected/infective specimens. A total of 2721 mosquitoes were caught and A. albopictus was the most abundant species (2534). Filarial DNA was found in 27.5% (19/69) of the abdomen pools formed with mosquitoes collected in summer 2000, and in 11.1% (16/144) and 4.9% (6/123) thorax-head pools coming from samplings 2001 and 2002, respectively. Filarial DNA was belonging to D. immitis and all studied areas harboured infective specimens. These results prove A. albopictus as natural vector of D. immitis in Italy. Moreover, they support the hypothesis that the presence of the mosquito could affect the transmission pattern of canine heartworm disease in urban environment and, considering the aggressive anthropophylic behaviour of the species (30-48 bites/h) proven in Padova town, could enhance the circulation of filarial nematodes from animals to humans.

Eimeria leuckarti (Flesh, 1883) Reichenow, 1940 from Equus caballus in Italy.

Fecal examinations of horses (Equus caballus) from studs in the province of Udine (Friuli Venezia Giulia region, east-northern Italy), carried out during two periods of the year 1993, revealed coccidian oocysts in six foals, two stallions and one mare. The species was identified as Eimeria leuckarti. This species was reported only once in Italy, more than 20 years ago (1972). For this reason, the prevalence of the parasite and the description of immature and sporulated oocysts are given.

[Bovine setariasis (Note I):trap for the capture of possible intermediate hosts of Setaria labiatopapillosa]. / Setariosi bovina (Nota I): trappola per la cattura dei possibili ospiti intermedi di Setaria labiatopapillosa.

The authors describe a trap utilized in the field to catch possible intermediate hosts of Setaria labiatopapillosa attracted by naturally infected bovine. The trap was bulky and stable but also decomposable and removable. It was tested in an area of Friuli Venezia Giulia (Udine province, NE Italy) during 21 nights in the summer 1994 (29/6-29/9) and allowed to catch 16159 mosquitoes and 218 other Diptera, showing a very good efficiency.

[Bovine setariasis in Friuli Venezia Giulia]. / Indagine sulla setariosi bovina in Friuli Venezia Giulia.

Blood samples from 407 bovines of "Azienda Marianis", a farm in Friuli Venezia Giulia, Italy, were examined for microfilariae; 143 (35.1%) were positive. A survey in other 23 farms of dairy cattle from 6 Communes of the Province of Udine revealed a prevalence of 11.28%. The prevalence was significantly correlated with the age of animals; in cow was lower than in young animals. Blood microfilariae (210) and adult females (15) were identified as Setaria labiatopapillosa.

Development of Dirofilaria and Setaria nematodes in Aedes albopictus.

The development of Dirofilaria repens, D immitis and Setaria labiatopapillosa up to L3 stage was documented in a laboratory colony of Aedes albopictus originating from specimens collected in Civitavecchia (Central Italy). The susceptibility of Ae albopictus and its plastic trophic habits, strongly suggest that this mosquito may contribute to the spreading of these nematodes in the country. It is particularly emphasized the danger for human health of an increased probability of transmission of Dirofilaria in urban areas.

[Monitoring of canine leishmaniasis in northern Italy: an update from a scientific network]. / Monitoraggio della leishmaniosi canina in nord Italia: aggiornamenti da un network scientifico.

Canine leishmaniasis (CanL) due to Leishmania infantum is a disease of great veterinary importance and a serious public health problem. In humans, L. infantum causes visceral (VL) and cutaneous leishmaniasis (CL) and the distribution of VL overlaps that of CanL. Currently, VL is considered by WHO as an emerging zoonosis in southern Europe. The dog is the only domestic reservoir of the infection and phlebotomine sandflies are the only proven vectors of leishmaniasis for dogs and humans. CanL is endemic in Italy, particularly in central and southern regions, including islands. Until 1983, all regions of northern Italy but Liguria and some territories of Emilia Romagna were considered free from CanL. From early '90s new stable foci of CanL have appeared, most of them located within classical endemic areas including territories of Emilia Romagna, Tuscany, Umbria, Marche, and Abruzzi regions. But the most relevant aspect, from an epidemiological point of view, has been the appearance of stable CanL foci in northern Italy, namely in Veneto and Piedmont regions. In these two foci, entomological surveys showed the presence of P. perniciosus and of a second phlebotomine vector, P. neglectus, which may have played a role in the CanL diffusion in some parts of northern Italy. Furthermore, in these areas, autochthonous human VL cases have occurred. There is therefore a realistic risk that CanL infection could rapidly spread through northern latitudes and a surveillance activity is strongly needed. For this reason, in October 2002, thanks to the collaboration and support of Intervet Italia, the network "LeishMap" was created, with the main purpose of monitoring the spread of CanL and vectors in northern Italy. LeishMap consists of scientific and sanitary institutions with proven experience both in field surveys and diagnostic methodologies on CanL and phlebotomine vector. It is organised in 4 Operational Units (OU), represented by researchers of the Veterinary Faculties of the University of Bologna, Padua, Milan and Turin, under the scientific coordination of the MIPI Department, ISS of Rome and with the collaboration of private and public veterinarians operating in the regions under study. During the first year of activity, each OU was involved in the serological and entomological surveillance of several territories in the respective regions, where recent autochthonous CanL cases were registered. The studies have involved five regions, namely Valle D'Aosta, Piedmont, Lombardia, Veneto, Trentino-Alto Adige and Emilia Romagna. In the Symposium 6 of this Congress we report detailed results of a retrospective analysis of data concerning CanL and vectors in northern Italy till 2002 and the preliminary results of 2003 on the seroprevalence rates observed in foci studied and on the entomological surveys carried out. In summary, the results outlined that already known foci of CanL are expanding from the original sites. Several new foci have been identified and many others are at high risk of evolving toward a stable endemicity. P. perniciosus has been found in all but one the suspected new foci. In Emilia Romagna region P. perfiliewi was identified in 2 areas and in one was the only species present. The occurrence of P. neglectus was confirmed in three regions, Veneto, Lombardia and Piedmont. In conclusion, from the 2002-2003 LeishMap activities it appears that further monitoring activities are necessary to identify new endemic foci of CanL, this representing the prerequisite for the implementation of programs for leishmaniasis control in northern Italy.

Nine more cases of human infections by Dirofilaria repens diagnosed in Italy by morphology and recombinant DNA technology.

Dirofilariosis is a mosquito-borne disease of carnivors due, in Italy, to Dirofilaria repens and D. immitis species which are sympatric in many areas. Nine more cases of human infection are here reported, detected in Italian patients living in northern regions (where D. immitis and D. repens are largely present), in Tuscany (where D. repens is more frequent than D. immitis), and in Sicily (where D. immitis is present only sporadically). The nematodes surgically removed (7 female and 2 male specimens) have been identified by a PCR-based method and by morphology as D. repens. Morphological, clinical, and epidemiological data of these human infections are discussed.

Comparative field study to evaluate the performance of three different traps for collecting sand flies in northeastern Italy.

Three standard methods for collecting sand flies (sticky trap, CDC light trap, and CO2 trap) were compared in a field study conducted from June to October, 2012, at a site located in the center of a newly established autochthonous focus of canine leishmaniasis in northeastern Italy. Six traps (two sticky traps, two CDC light traps, and two CO2 traps) were activated at the same time for a single night every two weeks during the season of sand fly activity. A total of 5,667 sand flies were collected and 2,213 identified, of which 82.1% were Phlebotomus perniciosus, 17.4% P. neglectus, 0.3% Sergentomya minuta, and 0.2% P. mascitti. The performances of all traps were influenced by their position inside the site, increasing with proximity to the animal shelters. CO2 traps were more attractive for females of P. perniciosus and P. neglectus. CDC light traps showed an intermediate efficiency and were more attractive for P. neglectus, compared to other two traps. Results suggest that in northern Italy the CO2 trap is a suitable sampling method for sand fly monitoring programs that include transmitted pathogen surveillance.

New insights into the epidemiology of bovine piroplasmoses in Italy.

Few studies have been published on bovine piroplasmoses in Italy, and therefore a clear picture of the epidemiology of these infections is difficult to obtain. Vertebrate and invertebrate hosts in Central and Northern Regions of Italy were investigated in 2005 and 2006, when microscopy, molecular tools and serological tests were applied to 468 blood samples drawn from cattle in order to evaluate the presence of these protozoa and identify possible risk factors. Ticks were also collected, identified and analyzed by molecular techniques. Microscopy identified 6.5% of the animals as positive, whereas PCR detected piroplasm DNA in 21.6%. BLAST analysis showed 67 amplicons (17.0%) referable to the Theileria sergenti/buffeli/orientalis group, 17 (4.3%) to Theileria annae, and 1 to Babesia divergens. Serology evidenced a prevalence of 45.4% for Babesia bovis, 17.4% for Babesia bigemina, and 34.9% for B. divergens. The 127 collected ticks were identified as belonging to 5 species, mostly represented by Rhipicephalus bursa, Hyalomma marginatum and Ixodes ricinus. Molecular analyses evidenced the presence of B. bovis and B. bigemina, in 3 and 5 ticks, respectively. Our findings suggest that different species of piroplasms are circulating in bovine populations in Central and Northern Italy, and provide new insights into the complex epidemiology of bovine piroplasmoses in Italy.

Canine piroplasmosis in Italy: epidemiological aspects in vertebrate and invertebrate hosts.

In Italy, canine piroplasmosis is believed to be widespread, but few data are available on its presence in most areas. In 2005 and 2006, vertebrate and invertebrate hosts were investigated in Central and Northern Regions of the Country. Microscopy on blood smears, molecular tools and serological tests were applied to 420 blood samples collected from dogs, in order to evaluate the presence of these protozoa and to identify possible risk factors. Moreover, ticks were analyzed by molecular techniques. Microscopy identified as positive 2.8% of the animals, all from Central Italy, and PCR detected 'piroplasm' DNA in 6.0%. Serology evidenced a mean prevalence of 34.0% with a decreasing trend from Central to Northern areas. The 507 collected ticks were identified as belonging to 8 species, mostly represented by Rhipicephalus sanguineus (n=376) and Ixodes ricinus (n=58). Molecular analyses evidenced the presence of babesial parasites (Babesia canis canis, B. canis vogeli, B. microti-like) in 25 (4.9%) of them; in Rh. sanguineus there was also demonstration of the vertical transmission of B. canis canis. Statistical analysis identified 'kennel' as risk factor for Babesia infection. Our findings evidenced that different species of piroplasms potentially infectious for dogs are circulating in Italy, and that epidemiological aspects of these infections are more complex than expected. Vector importance of both Rh. sanguineus and I. ricinus is hypothesized, but further investigation is needed.

Distribution of Pholeter gastrophilus (Digenea) within the stomach of four odontocete species: the role of the diet and digestive physiology of hosts.

We compared the distribution of the digenean Pholeter gastrophilus in the stomach of 27 harbour porpoises, Phocoena phocoena, 27 striped dolphins, Stenella coeruleoalba, 18 bottlenose dolphins, Tursiops truncatus, and 100 long-finned pilot whales, Globicephala melas. The stomach of these species is composed of 4 chambers of different size, structure and function. In all species, P. gastrophilus was largely restricted to the glandular region of the stomach, but the parasite tended to favour the fundic chamber in bottlenose dolphins and harbour porpoises, the pyloric chamber in pilot whales, and none in striped dolphins. However, predictability at infrapopulation level was generally low, suggesting a weak preference of P. gastrophilus for any of the chambers. Three hypotheses were tested to investigate a common cause for the distribution of P. gastrophilus in all host species, namely, colonization of chambers was (1) sequential, (2) dependent on chamber size, or (3) dependent on the passage time of food through the whole stomach. The latter hypothesis was indirectly tested by assuming, based on previous evidence from other vertebrates, that the greater the size of the stomach and/or the energy content of prey, the greater the delay of food passage. We found no compelling evidence that chamber colonization was sequential, or related to chamber size in any species. However, the distribution of P. gastrophilus was significantly more anteriad when the host species had larger stomachs and, particularly, when hosts fed on prey with higher caloric content. Accordingly, the stomach distribution of P. gastrophilus at this scale seems to be passively driven by features of the diet and digestive physiology of each host species. This study provides a general framework to formulate null hypotheses in future studies on microhabitat choice by parasites.

Serological assays on eight cases of human dirofilariasis identified by morphology and DNA diagnostics.

Specimens of Dirofilaria sp. removed from eight Italian patients were identified as D. repens by morphology and confirmed as such by a PCR-based method of DNA analysis. Blood samples were also drawn from the patients so that two serological tests (ELISA and western blot), one based on the recognition of molecular markers recently identified in the somatic antigenic complex of D. repens, could be evaluated. The antigenic complex used in the ELISA only gave a weak sensitivity. However, the western-blot assays, based on the polypeptide molecular markers, were found to have greater sensitivity and should be useful in detecting human cases of dirofilariasis.

5 S ribosomal spacer sequences of some filarial parasites: comparative analysis and diagnostic applications.

We have amplified by PCR the sequences of the 5 S ribosomal spacer of Setaria labiatopapillosa and Foleyella furcata. After sequencing, these sequences have been compared with those of Dirofilaria immitis and Dirofilaria repens. Two major goals have been achieved: (i) the establishment of a multiplex PCR-based diagnostic assay, applicable to identify the four species in vertebrate and invertebrate hosts; (ii) the identification, in S. labiatopapillosa and F. furcata, of a canonical spliced leader 1 (SL1) sequence, so confirming that only D. repens, of the filarial parasites so far studied, shows a peculiar SL1 sequence. The PCR assay here developed and the analysis of the 5 S ribosomal spacer, can further improve both epidemiological and molecular analysis of these filarial species.