Gabriella Cancrini

Epilepsy, cysticercosis, and toxocariasis: a population-based case-control study in rural Bolivia.

Objective To assess the relationship between epilepsy and infection with Taenia solium and Toxocara canis with a case-control study, in the rural area of the Cordillera Province, Bolivia. Methods A preliminary two-phase door-to-door prevalence survey determined the prevalence of epilepsy and identified cases and control subjects. At least two control subjects per case were selected, matching on sex, age, and community of residence. Cases and control subjects were assessed serologically for antibodies against T. canis by ELISA and against T. solium by enzyme-linked immunoelectrotransfer blot (EITB). Results The prevalence survey found 130 confirmed cases of epilepsy, of which 113 were eligible for the case-control study (59 partial seizures and 54 generalized seizures). Two hundred thirty-three control subjects were selected. Multivariable analysis for a matched case-control study was carried out. There was an association between EITB positivity for T. solium and epilepsy with an OR of 1.85 (95% CI 0.99 to 3.4) for all cases. A stronger association was found in those with partial epilepsy with a late onset of disease (15 years and older), where the OR was 3.66 (95% CI 1.10 to 12.10). A positive association was also found with T. canis for all cases with an OR of 2.70 (95% CI 1.41 to 5.19). This increased for those with late-onset partial epilepsy to an OR of 18.22 (95% CI 2.10 to 158.10). Conclusion This finding suggests that both neurocysticercosis and toxocariasis may in part explain the higher prevalence of epilepsy, particularly partial epilepsy, in developing countries.

Polymerase chain reaction-identification of Dirofilaria repens and Dirofilaria immitis

On the basis of known DNA sequences of Dirofilaria repens and D. immitis we designed specific primers for the amplification by Polymerase Chain Reaction (PCR) of the DNA from the two species. The PCR-based identification was found to be unambiguous and allowed specific diagnosis of microfilariae in blood samples, of developing larvae in the mosquito vector and of immature adults in bioptic material, overcoming the serious constraints of the morphological separation of these filarial parasites at the pre-adult stages. The technique was found to be very sensitive and applicable to samples stored either dry or in various preservation media, with the exception of formalin. The reliable identification of D. repens and D. immitis from bioptic material is expected to greatly enhance the chances of detecting human infections and to further clarify the role of the two parasites as pathogens of man. The possibility of routine identification of developing larvae in the vector will substantially improve the perspectives for epidemiological investigations, particularly in Southern European regions, such as Italy, where the two nematode species are largely sympatric.

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

Abstract.  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°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.

Searching for Wolbachia (Rickettsiales: Rickettsiaceae) in Mosquitoes (Diptera: Culicidae): Large Polymerase Chain Reaction Survey and New Identifications

Abstract Bacteria of the genus Wolbachia constitute a group of intracellular and maternally inherited micro-organisms that are widespread in arthropods, inducing several reproductive disorders such as cytoplasmic incompatibility in their hosts. Considering relevant biological implications related to the presence of Wolbachia in several insect orders, for example its potential role as mechanism for rapid speciation and as vehicle to drive genetic markers in wild populations of vectors of medical and veterinary interest, we carried out an extensive polymerase chain reaction survey to detect Wolbachia in several species of mosquito belonging to genera involved in the transmission of pathogens. Five species out of 26 tested have shown to be infected; for four of them this is the first evidence of the Wolbachia infection. A phylogenetic analysis was also performed, positioning the five Wolbachia strains in the phyletic subdivision B.

Natural Vectors of Dirofilariasis in Rural and Urban Areas of the Tuscan Region, Central Italy

Abstract Entomological investigations by means of dog- and human-baited traps were carried out in summers 2000–2002 in urban and rural areas of the Tuscan region in central Italy. The aim of the study was to define the mosquito species involved in the transmission of Dirofilaria nematodes and to assess the risk that their presence might represent for animal and human health. Nocturnal fieldwork on host-seeking activity and feeding preferences was followed by microscopic identification of the mosquito species attracted and by molecular identification of Dirofilaria parasites in mosquitoes. In total, 3,611 mosquito females belonging to 12 species, largely represented by Culex pipiens L. and Aedes caspius (Pallas), were caught. Some females of each species collected fed on the dogs, indicating their possible role as an intermediate host, but filarial DNA was found only in Cx. pipiens, Anopheles maculipennis s.l. (Meigen), and Coquillettidia richiardii (Ficalbi). In rural environments, the DNA evidence indicated the presence of infective larvae of Dirofilaria immitis, whereas in urban areas, infective larvae of Dirofilaria repens were present. The role of Cx. pipiens as a vector for heartworm disease and subcutaneous infections in natural and artificial environments was confirmed, whereas Ae. caspius seemed refractory to the infection. The different role of the collected species is discussed. The vector competence of An. maculipennis and Cq. richiardii needs further investigation, because the importance of these species poorly represented, and the role of species such as Aedes albopictus (Skuse), characterized by a dominant diurnal activity pattern, has to be evaluated.

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

Abstract 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 1,576 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).

Endoparasites of red fox (Vulpes vulpes) in central italy

A parasitologic study on 129 red foxes (Vulpes vulpes) from Tuscany (central Italy) was carried out in 2004–2006. Five intestinal species were found at necropsy: Dipylidium caninum (prevalence 57.3%), Mesocestoides lineatus (45.4%), Uncinaria stenocephala (39.1%), Toxocara canis (9.1%), and Toxascaris leonina (5.4%). Other parasites not associated with the intestine included Crenosoma vulpis (14.7%), Capillaria aerophila (7.0%), Angiostrongylus vasorum (7.0%), and filarial parasites (17.8%). Coprologic tests were less sensitive and less specific in identifying parasites than direct examinations at necropsy. Trichinella larvae were not found in muscles submitted to artificial digestion. By immunologic assay, antigens of Echinococcus spp. were detected in fecal samples of 20 foxes, but results could not be confirmed by fecal examination or molecular tests.

Molecular identification of Wolbachia from the filarial nematode Mansonella ozzardi

Abstract Mansonella ozzardi, a filarial parasite of humans in Latin America, has been shown to harbour intracellular bacteria not yet identified. Here we show that these bacteria, like those of other filarial nematodes, belong to the genus Wolbachia (alpha 2 Proteobacteria; Rickettsiales). Their unambiguous placement in the Wolbachia group was shown by 16S rDNA sequence analysis. However, the exact position of the Wolbachia from M. ozzardi relative to the other wolbachiae is not clear. Indeed, 16S rDNA sequence analysis places this bacterium at a deep branch in Wolbachia evolution. It is interesting that analysis of the 5S rDNA gene spacer of the nematode host also suggests that the genus Mansonella, together with the genus Loa, could represent a deep-branching lineage in filarial evolution.

Tick reservoirs for piroplasms in central and northern Italy.

Ticks, collected in central and northern Italy from pets, livestock, wild animals and the environment (n=2107), were identified by microscopy and processed by molecular diagnostics to determine the species that act as a reservoir for piroplasms. A total of 11 ixodid tick species were identified, with five of them proving to be piroplasm positive. Molecular diagnostics identified Theileria equi and eight Babesia species in 52 adult specimens, mostly (n=50) removed from piroplasm-free vertebrate hosts. Ixodes ricinus hosted the highest number of species, although the highest infection rate was recorded in Hyalomma marginatum (9.1%), followed by I. ricinus (5.1%), Dermacentor marginatus (5%), Rhipicephalus turanicus (3.1%) and R. sanguineus (1.2%). Novel tick/pathogen associations were detected, suggesting that certain tick species (such as Hy. marginatum, R. sanguineus and I. ricinus) are vector of more piroplasm species than previously thought. Trans-stadial maintenance of the piroplasms was observed in each positive tick species; vertical transmission of B. canis canis was demonstrated in R. sanguineus. Finally, the detection of Babesia sp., B. microti-like species and B. rodhaini, phylogenetically related to zoonotic species, suggests that the human population could be at risk of infection in the studied area.

The first report of hepatozoon canis identified in vulpes vulpes and Ticks from Italy

This is the first report on the presence of Hepatozoon canis in Vulpes vulpes in Italy. During the years 2005 and 2006, a total of 119 foxes were collected and their spleen tissues were screened by microscopy, polymerase chain reaction, and sequencing. In the same area, 290 ticks were picked off from dogs or collected from the environment. Microscopy detected inclusion bodies regarded as belonging to the genus Hepatozoon in four samples, whereas molecular diagnostics evidenced 16 foxes (13.4%) and 6 ticks (2.1%) positive to H. canis. The H. canis isolates we found in foxes, compared with the strains we previously detected in dogs from the same area and with the strains found in foxes from other European countries, show a certain genetic heterogeneity. In fact, seven isolates cluster with the Italian dog strain and nine isolates cluster with the fox strain found in Spain and Slovakia; moreover, the dogs strain is closely related to one ticks isolate, and the strain found in three Rhipicephalus sanguineus and in one Ixodes ricinus collected from the environment cluster with the aforementioned Spanish and Slovak fox strains. Our findings confirm the importance of R. sanguineus as final host and suggest that I. ricinus might also be implicated in parasite transmission, explaining in that way the occurrence of hepatozoonosis in areas considered R. sanguineus-free. The peridomestic habits of V. vulpes and the increasing global temperature are expected to amplify the impact of this vector-borne disease and to enforce the transmission of Hepatozoon to domestic animals.