Valentina Chisu

Pathogens and symbionts in ticks: a survey on tick species distribution and presence of tick- transmitted micro-organisms in Sardinia, Italy

A total of 1485 adult ticks were collected from mammalian hosts in south-eastern Sardinia, Italy, during the years 2007-2008. Ticks were identified and tested by PCR analysis for presence of Rickettsia species of the spotted fever group, Ehrlichia canis, Anaplasma phagocytophilum, Coxiella burnetii, Bartonella species and Leishmania species. Among all tick species examined (Rhipicephalus sanguineus, Rhipicephalus turanicus, Rhipicephalus bursa, Rhipicephalus pusillus, Hyalomma marginatum marginatum, Haemaphysalis sulcata and Dermacentor marginatus), only Hyalomma marginatum marginatum produced negative results. A total of 22 pools belonging to the three tick species Rhipicephalus sanguineus (0.9 %), Rhipicephalus turanicus (4.5 %) and Rhipicephalus pusillus (100 %) were positive for Rickettsia species, while a total of five pools belonging to Rhipicephalus sanguineus (0.09 %), Haemaphysalis sulcata (16.7 %) and D. marginatus (7.8 %) were positive for E. canis. Five pools of Rhipicephalus turanicus (1.8 %) were positive for A. phagocytophilum. Positivity for C. burnetii was found in seven pools belonging to three tick species: Rhipicephalus sanguineus (0.5 %), Rhipicephalus turanicus (0.3 %) and Haemaphysalis sulcata (4.4 %). Finally, four pools belonging to Rhipicephalus sanguineus (0.09 %), Rhipicephalus turanicus (0.7 %) and Rhipicephalus bursa (1.1 %) were positive for Bartonella species. Leishmania species DNA was not detected in any of the tick pools examined. Data presented here increase our knowledge on tick-borne diseases in Sardinia, and provide a useful contribution to understanding their epidemiology.

Rickettsia slovaca from Dermacentor marginatus ticks in Sardinia, Italy.

Nineteen ticks belonging to the species Dermacentor marginatus, Rhipicephalus bursa, and Haemaphysalis sulcata were collected from wild animals (wild boar, deer, and mouflon) in south-western Sardinia, Italy. Five D. marginatus ticks from wild boar were PCR-positive when analyzed using gltA-specific and ompA-specific primers, leading to the identification and first isolation in cell culture of Rickettsia slovaca, the causative agent of tick-borne lymphadenopathy (TIBOLA), on the island of Sardinia. This study confirms the detection of a new tick-borne rickettsia that can be added to the others already known to be present in Sardinia (Rickettsia aeschlimannii, R. massiliae, and Candidatus Rickettsia barbariae). These data increase our knowledge of tick-borne rickettsioses in Sardinia and, more generally, in the Mediterranean basin.

Molecular characterization of Toxoplasma gondii Type II in sheep abortion in Sardinia, Italy.

During 2009–2010, 161 tissue samples (142 placentas, 16 brains, and 3 livers) from aborted ovine fetuses on Sardinia Island, Italy, were tested for toxoplasmosis. Organs that showed a positive result by nested polymerase chain reaction (PCR) targeting the ITS1 region for Toxoplasma gondii were also amplified with 11 genetic markers (SAG1, 5′-SAG2, 3′-SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) and then subjected to PCR/RFLP for genetic typing. T. gondii DNA was found in 5 placentas, 14 brains, and 2 livers by PCR analysis and all isolates displayed Type II alleles at all 11 loci with all 11 markers. The results indicate that the Type II T. gondii is associated with ovine abortion.

First detection of Ehrlichia canis in Rhipicephalus bursa ticks in Sardinia, Italy

Canine monocytic ehrlichiosis is a common and widespread disease. This disease is present in Sardinia year-round because the temperate weather on the island permits the survival of many types of tick vectors. A total of 35 ticks was collected from goats in south-eastern Sardinia, Italy, and tested for Ehrlichia DNA using polymerase chain reaction and DNA sequencing. This study provides the first evidence of the presence of Ehrlichia canis in Rhipicephalus bursa ticks. The data presented here increase our knowledge of tick-borne diseases in Sardinia and provide a useful contribution toward understanding their epidemiology. The role of R. bursa in the life cycle and transmission of E. canis needs however further investigation.

Detection of Rickettsia hoogstraalii, Rickettsia helvetica, Rickettsia massiliae, Rickettsia slovaca and Rickettsia aeschlimannii in ticks from Sardinia, Italy

Tick-borne diseases represent a large proportion of infectious diseases that have become a world health concern. The presence of Rickettsia spp. was evaluated by standard PCR and sequencing in 123 ticks collected from several mammals and vegetation in Sardinia, Italy. This study provides the first evidence of the presence of Rickettsia hoogstralii in Haemaphysalis punctata and Haemaphysalis sulcata ticks from mouflon and Rickettsia helvetica in Ixodes festai ticks from hedgehog. In addition, Rickettsia massiliae, Rickettsia slovaca and Rickettsia aeschlimannii were detected in Rhipicephalus sanguineus, Dermacentor marginatus and Hyalomma marginatum marginatum ticks from foxes, swine, wild boars, and mouflon. The data presented here increase our knowledge of tick-borne diseases in Sardinia and provide a useful contribution toward understanding their epidemiology.

Rickettsia conorii israelensis in Rhipicephalus sanguineus ticks, Sardinia, Italy

The presence of tick-borne Rickettsia spp. was examined by PCR using DNA samples extracted from 254 ticks collected from mammals originating from northern and eastern Sardinia, Italy. The spotted fever group rickettsial agent Rickettsia conorii israelensis was detected in 3 Rhipicephalus sanguineus ticks from a dog for the first time in this geographical area. In addition, Ri. massiliae, Ri. slovaca, and Ri. aeschlimannii were detected in Rh. turanicus, Rh. sanguineus, Dermacentor marginatus, and Hyalomma marginatum marginatum ticks from dogs, goats, wild boar, and horse. Moreover, Candidatus Rickettsia barbariae was detected in 2 Rh. turanicus ticks from goats. The detection of Ri. conorii israelensis, an emergent agent which causes Israeli spotted fever, increases our knowledge on tick-borne rickettsioses in Sardinia.

Molecular detection and groEL typing of Rickettsia aeschlimannii in Sardinian ticks

Rickettsia aeschlimannii is an emerging tick-borne pathogen of the spotted fever group Rickettsiae with considerable impact on both human and animal health. This study reports the molecular detection and groEL characterization of R. aeschlimannii in ticks collected from birds and ruminants in a typical Mediterranean environment. Phylogeny of R. aeschlimannii and species representative of the spotted fever and typhus groups based on the groEL gene is reconstructed for the first time. Results expand the knowledge on distribution and typing of emerging human tick-borne diseases in Sardinia and pave the way for future molecular epidemiology studies of zoonotic Rickettsiae.

A five-year survey of tick species and identification of tick-borne bacteria in Sardinia, Italy

Sardinia is a hotspot for studying tick-borne diseases in the Mediterranean region, where cases of notifiable tick-borne diseases are increasing. The aim of this study was to determine the presence of tick-borne bacteria of medical and veterinary importance in ixodid ticks collected from domestic and wild animals, humans, and vegetation from different collection sites in Sardinia. Using standard PCR and sequencing techniques, the presence of Rickettsia, Anaplasma, Ehrlichia, and Bartonella species, as well as Coxiella burnetii was evaluated. A total of 1619 ticks were morphologically identified as Rhipicephalus sanguineus sensu lato, R. bursa, R. annulatus, Dermacentor marginatus, Haemaphysalis punctata, Ha. sulcata, Hyalomma lusitanicum, H. marginatum, Ixodes festai (sometimes referred to erroneously as I. ventalloi), and Argas reflexus. Results indicated the presence of several circulating pathogens in Sardinian ticks. DNA of Rickettsia species was detected in 58 out of 1619 (4%) belonging to R. sanguineus s.l., D. marginatus, Ha. punctata, H. marginatum, and I. festai species. Ehrlichia canis DNA was detected in 33 out of 1619 ticks (2%) belonging to R. sanguineus s.l., R. bursa, and Ha. punctata species. A total of 61 out of 1619 (4%) ticks (R. sanguineus s. l., R. bursa, Ha. punctata, and I. festai) tested positive for Anaplasma spp. Coxiella burnetii was detected in 21 out of 1619 (1%) ticks belonging to R. sanguineus s.l., R. bursa, R. annulatus, and H. marginatum species. Five R. sanguineus s.l. and one R. bursa ticks were positive for the presence of Bartonella sp. 16S rRNA gene. Our findings expand the knowledge on tick-borne microorganism repertoires and tick distribution in Sardinia. Tick distribution should be monitored for effective control of these arthropods and the infections they transmit.

GroEL typing and phylogeny of Anaplasma species in ticks from domestic and wild vertebrates

Anaplasma species are globally distributed tick-borne bacteria causing a range of clinical conditions in domestic animals, wildlife, and human. Nevertheless, data on presence and distribution of Anaplasma strains in ticks are still lacking, especially in the Mediterranean region. This study reports the molecular identification, genetic characterization and phylogeny of Anaplasma strains of both veterinary and zoonotic importance in ticks collected from domestic and wild hosts sampled in a typical Mediterranean warm temperate region, the island of Sardinia. Results reveal the presence of A. ovis in Rhipicephalus bursa and R. sanguineus sensu lato ticks; A. platys-like was found in R. bursa ticks; while A. platys and A. phagocytophilum were detected in Hyalomma marginatum and H. lusitanicum ticks. Investigating the occurrence of different Anaplasma species in ticks from domestic and wild hosts improves the knowledge of tick-borne diseases in the Mediterranean area, and has implications in defining vector species distribution and risk assessment.

Molecular evidence of Chlamydiales in ticks from wild and domestic hosts in Sardinia, Italy

Ticks are well known to be important vectors for a wide range of bacteria, viruses and protozoa affecting human and animal health. Ixodid ticks are widely distributed in Sardinia, and an increasing number of tick-borne bacteria have been documented in the island. A growing number of evidence are supporting the hypothesis of alternative transmission routes for chlamydial bacteria such as the involvement of vectors. This study was conducted to provide possible molecular detection of members belonging to the Chlamydiales order in Sardinian ticks and to update information concerning the presence of new ectoparasite-borne bacteria in ticks collected from domestic and wild hosts in a typical Mediterranean environment. A total of 378 ticks were individually screened with a pan-Chlamydiales specific primers targeting the 16S rRNA gene. Chlamydiales DNA was detected in 28% of the total ticks analyzed. The analyses of sequences highlighted that Rhipicephalus sanguineus sensu lato, Rhipicephalus bursa, Rhipicephalus annulatus, Haemaphysalis sulcata, Haemaphysalis punctata and Dermacentor marginatus ticks exhibited DNA of Chlamydiaceae and Parachlamydiaceae members. Our results revealed that DNA of zoonotic microorganisms such as C. psittaci, C. abortus and the emerging pathogen Parachlamydia acanthamoebae are present in Sardinian ticks. Since routes of Chlamydia transmission are yet to be fully defined, the role of ticks as possible vectors for Chlamydiales remains the most challenging and interesting question to be addressed in future research. Continued monitoring of these pathogens in tick vectors is needed to provide strategies for controlling of possible chlamydial infections and disease outbreaks in the island.