Alessio Giannelli

Morphological and genetic diversity of Rhipicephalus sanguineus sensu lato from the New and Old Worlds.

BackgroundThe taxonomic status of the brown dog tick (Rhipicephalus sanguineus sensu stricto), which has long been regarded as the most widespread tick worldwide and a vector of many pathogens to dogs and humans, is currently under dispute.MethodsWe conducted a comprehensive morphological and genetic study of 278 representative specimens, which belonged to different species (i.e., Rhipicephalus bursa, R. guilhoni, R. microplus, R. muhsamae, R. pusillus, R. sanguineus sensu lato, and R. turanicus) collected from Europe, Asia, Americas, and Oceania. After detailed morphological examination, ticks were molecularly processed for the analysis of partial mitochondrial (16S rDNA, 12S rDNA, and cox 1) gene sequences.ResultsIn addition to R. sanguineus s.l. and R. turanicus, three different operational taxonomic units (namely, R. sp. I, R. sp. II, and R. sp. III) were found on dogs. These operational taxonomical units were morphologically and genetically different from R. sanguineus s.l. and R. turanicus. Ticks identified as R. sanguineus s.l., which corresponds to the so-called “tropical species” (=northern lineage), were found in all continents and genetically it represents a sister group of R. guilhoni. R. turanicus was found on a wide range of hosts in Italy and also on dogs in Greece.ConclusionsThe tropical species and the temperate species (=southern lineage) are paraphyletic groups. The occurrence of R. turanicus in the Mediterranean region is confirmed. A consensual re-description of R. sanguineus s.s. and R. turanicus will be necessary to solve the taxonomic problems within the so-called R. sanguineus group.

Ticks infesting humans in Italy and associated pathogens.

BackgroundTicks may transmit a large variety of pathogens, which cause illnesses in animals and humans, commonly referred to as to tick-borne diseases (TBDs). The incidence of human TBDs in Italy is underestimated because of poor surveillance and the scant amount of studies available.MethodsSamples (n = 561) were collected from humans in four main geographical areas of Italy (i.e., northwestern, northeastern, southern Italy, and Sicily), which represent a variety of environments. After being morphologically identified, ticks were molecularly tested with selected protocols for the presence of pathogens of the genera Rickettsia, Babesia, Theileria, Candidatus Neoehrlichia mikurensis, Borrelia and Anaplasma.ResultsTicks belonged to 16 species of the genera Argas, Dermacentor, Haemaphysalis, Hyalomma, Ixodes and Rhipicephalus, with Ixodes ricinus (59.5%) being the species most frequently retrieved, followed by Rhipicephalus sanguineus sensu lato (21.4%). Nymphs were the life stage most frequently retrieved (41%), followed by adult females (34.6%). The overall positivity to any pathogen detected was 18%. Detected microorganisms were Rickettsia spp. (17.0%), Anaplasma phagocytophilum (0.8%), Borrelia afzelii (0.5%), Borrelia valaisiana (0.3%), C. N. mikurensis (0.5%) and Babesia venatorum (0.6%).ConclusionsResults indicate that people living in the Italian peninsula are at risk of being bitten by different tick species, which may transmit a plethora of TBD causing pathogens and that co-infections may also occur.

Evidence for direct transmission of the cat lungworm Troglostrongylus brevior (Strongylida: Crenosomatidae).

Metastrongyloids of cats are emerging pathogens that may cause fatal broncho-pulmonary disease. Infestation of definitive hosts occurs after ingestion of intermediate or paratenic hosts. Among metastrongyloids of cats, Troglostrongylus brevior and Troglostrongylus subcrenatus (Strongylida: Crenosomatidae) have recently been described as agents of severe broncho-pulmonary disease. Here, we provide, for the first time, observational evidence suggesting the direct transmission of T. brevior from queen cat to suckling kittens. This new knowledge will have a significant impact on current scientific information of this parasite and shed new light into the biology and epidemiology of metastrongyloid nematodes.

Molecular detection of tick-borne pathogens in Rhipicephalus sanguineus group ticks.

The Rhipicephalus sanguineus group includes some of the most widespread dog ticks, whose identification currently represents a difficult task due to the morphological similarities of these ixodid species. Recently, following the morphological and molecular characterization of tick specimens collected from dogs in all continents, Rhipicephalus sanguineus sensu lato (s.l.), Rhipicephalus turanicus and three different operational taxonomic units (namely Rhipicephalus sp. I-III) have been identified. Therefore, a comprehensive molecular study has been herein carried out to detect selected canine vector-borne pathogens (i.e., Anaplasma platys, Cercopithifilaria spp., Ehrlichia canis and Hepatozoon canis) in ticks belonging to the R. sanguineus group. A total of 204 tick specimens from dogs were examined and identified as R. sanguineus s.l. (n=81), R. turanicus (n=17), Rhipicephalus sp. I (n=66), Rhipicephalus sp. II (n=37), and Rhipicephalus sp. III (n=3). PCR assays were performed to detect mitochondrial and ribosomal target genes of Cercopithifilaria spp., A. platys, E. canis and H. canis. Out of 204 specimens examined, 2.5%, 7.4% and 21.6% scored positive to A. platys, H. canis and Cercopithifilaria spp., respectively. In addition, co-infections with two pathogens (i.e., Cercopithifilaria bainae and A. platys or H. canis) were detected in four tick specimens. This study suggests a relationship between ticks belonging to the R. sanguineus group and the geographical distribution of A. platys, H. canis and Cercopithifilaria spp. However, the role of each representative tick species within the R. sanguineus group in the transmission of these canine pathogens needs to be further investigated.

Development of the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior in Helix aspersa snails.

Aelurostrongylus abstrusus (Strongylida, Angiostrongylidae) and Troglostrongylus brevior (Strongylida, Crenosomatidae) are regarded as important lungworm species of domestic felids, with the latter considered an emerging threat in the Mediterranean region. The present study aimed to assess their concurrent development in the mollusc Helix aspersa (Pulmonata, Helicidae). Thirty snails were infested with 100 first-stage larvae (L1) of A. abstrusus and T. brevior, isolated from a naturally infested kitten. Larval development was checked by digesting five specimens at 2, 6 and 11 days post infestation. Larvae retrieved were morphologically described and their identification was confirmed by specific PCR and sequencing. All H. aspersa snails were positive for A. abstrusus and T. brevior, whose larval stages were simultaneously detected at each time point. In addition, snails were exposed to outdoor conditions and examined after overwintering, testing positive up to 120 days post infestation. Data herein presented suggest that A. abstrusus and T. brevior develop in H. aspersa snails and may eventually co-infest cats. Data on the morphology of both parasitic species in H. aspersa provide additional information on their development and identification, to better understand the population dynamics of these lungworms in receptive snails and paratenic hosts.

Zoonotic Onchocerca lupi Infection in Dogs, Greece and Portugal, 2011–2012

Onchocerca lupi infection is reported primarily in symptomatic dogs. We aimed to determine the infection in dogs from areas of Greece and Portugal with reported cases. Of 107 dogs, 9 (8%) were skin snip–positive for the parasite. DNA sequences of parasites in specimens from distinct dog populations differed genetically from thoses in GenBank.

Simultaneous detection of the feline lungworms Troglostrongylus brevior and Aelurostrongylus abstrusus by a newly developed duplex-PCR.

In addition to Aelurostrongylus abstrusus (Strongylida: Angiostrongylidae), referred to as the feline lungworm, Troglostrongylus brevior (Strongylida: Crenosomatidae) has recently been identified as an agent of bronco-pulmonary infestations in cats. These two parasites have a similar biology, share ecological niches, potentially co-infesting cats, but are difficult to be differentiated due to the morphological similarities of their first-stage larvae (L1). This paper describes a molecular tool, based on single-step duplex polymerase chain reaction (duplex-PCR) on the ribosomal internal transcribed spacer 2 region (ITS-2) for the simultaneous detection and differentiation of T. brevior and A. abstrusus. L1 of both species were collected from faecal samples, morphologically identified, and single larval specimens isolated. An aliquot of faeces was used as a test sample for a case of mixed natural infestation. The duplex-PCR was performed using species-specific forward primer sets for the ITS-2 region (i.e., A. abstrusus: 220bp; T. brevior: 370bp). The detection limit of the molecular assay was also assessed by serial dilutions of DNA from single larvae of both species (from ≈ 4.0 to 4.0 × 10(-5) μg/μl). The duplex-PCR carried out on individual DNA samples was able to detect as low as 5.2 × 10(-3) μg/μl of DNA for A. abstrusus, 4.9 × 10(-3)μg/μl for T. brevior, and as low as 4.0 × 10(-3) μg/μl for samples containing both species. Species-specific bands of the expected sizes and two bands were simultaneously amplified from the faecal sample containing both species. The phylogenetic analyses of the ITS-2 sequences here examined and those available for other metastrongyloids were concordant in clustering them with those of other Troglostrongylus brevior and A. abstrusus sequences available in GenBank database. This molecular approach proved to be effective and highly sensitive for the simultaneous detection of the two lungworms species and it might be used for molecular epidemiological studies and for monitoring therapeutic protocols.

Release of Lungworm Larvae from Snails in the Environment: Potential for Alternative Transmission Pathways

Background Gastropod-borne parasites may cause debilitating clinical conditions in animals and humans following the consumption of infected intermediate or paratenic hosts. However, the ingestion of fresh vegetables contaminated by snail mucus and/or water has also been proposed as a source of the infection for some zoonotic metastrongyloids (e.g., Angiostrongylus cantonensis). In the meantime, the feline lungworms Aelurostrongylus abstrusus and Troglostrongylus brevior are increasingly spreading among cat populations, along with their gastropod intermediate hosts. The aim of this study was to assess the potential of alternative transmission pathways for A. abstrusus and T. brevior L3 via the mucus of infected Helix aspersa snails and the water where gastropods died. In addition, the histological examination of snail specimens provided information on the larval localization and inflammatory reactions in the intermediate host. Methodology/Principal Findings Twenty-four specimens of H. aspersa received ~500 L1 of A. abstrusus and T. brevior, and were assigned to six study groups. Snails were subjected to different mechanical and chemical stimuli throughout 20 days in order to elicit the production of mucus. At the end of the study, gastropods were submerged in tap water and the sediment was observed for lungworm larvae for three consecutive days. Finally, snails were artificially digested and recovered larvae were counted and morphologically and molecularly identified. The anatomical localization of A. abstrusus and T. brevior larvae within snail tissues was investigated by histology. L3 were detected in the snail mucus (i.e., 37 A. abstrusus and 19 T. brevior) and in the sediment of submerged specimens (172 A. abstrusus and 39 T. brevior). Following the artificial digestion of H. aspersa snails, a mean number of 127.8 A. abstrusus and 60.3 T. brevior larvae were recovered. The number of snail sections positive for A. abstrusus was higher than those for T. brevior. Conclusions Results of this study indicate that A. abstrusus and T. brevior infective L3 are shed in the mucus of H. aspersa or in water where infected gastropods had died submerged. Both elimination pathways may represent alternative route(s) of environmental contamination and source of the infection for these nematodes under field conditions and may significantly affect the epidemiology of feline lungworms. Considering that snails may act as intermediate hosts for other metastrongyloid species, the environmental contamination by mucus-released larvae is discussed in a broader context.

Transstadial transmission of Hepatozoon canis from larvae to nymphs of Rhipicephalus sanguineus

Hepatozoon canis is an apicomplexan parasite of dogs, which is known to become infected by ingesting Rhipicephalus sanguineus adult ticks. To investigate the possibility of H. canis transovarial and transstadial transmission from larvae to nymphs, engorged adult female ticks were collected from a private animal shelter in southern Italy, where H. canis infection is highly prevalent. Female ticks (n=35) and egg batches were tested by PCR for H. canis. All eggs examined were PCR-negative whereas 88.6% of females from the environment tested positive. Additionally, fed larvae (n=120) from a dog naturally infected by H. canis were dissected at different time points post collection (i.e. 0, 10, 20 and 30 days). Molted nymphs dissected at 20 days post collection revealed immature oocysts displaying an amorphous central structure in 50% of the specimens, and oocysts containing sporocysts with sporozoites were found in 53.3% of the nymphs dissected at 30 days post collection. This study demonstrates that H. canis is not transmitted transovarially, but it is transmitted transstadially from larvae to nymphs of R. sanguineus and develops sporozoites in oocysts that may infect dogs.

Experimental evidence against transmission of Hepatozoon canis by Ixodes ricinus

Hepatozoon canis is among the most widespread tick-borne protozoa infecting domestic and wild carnivores. Its distribution is related to the occurrence of its major vector, the brown dog tick Rhipicephalus sanguineus. However, the role of Ixodes ricinus as a vector of H. canis has been hypothesized. In the present study, the development of H. canis was investigated in I. ricinus and R. sanguineus nymphs collected from a naturally infested dog. All I. ricinus ticks examined (n=133) were negative by cytological examination at days 20, 30, and 90 post collection, although H. canis DNA was detected in one nymph at day 20 and in 2 nymphs at day 30 post collection. On the other hand, H. canis sporogony was documented by cytology, and H. canis DNA was detected by PCR in R. sanguineus at day 30 post collection. These results indicate that H. canis sporogony does not occur in I. ricinus, but in R. sanguineus, suggesting that I. ricinus does not act as a vector of H. canis.