Detection of Chlamydia psittaci in the Genital Tract of Horses and in Environmental Samples: A Pilot Study in Sardinia.

The members of the Chlamydiaceae family are important pathogens that infect a wide range of vertebrate hosts, including humans. Among them, Chlamydia psittaci, historically considered as an avian agent, has recently been identified in livestock, primarily sheep and cattle, but also in horses, with the infection being linked to reproductive disorders, such as abortion, absorption of embryos, stillbirth, and the birth of weak foals. Much less is known about chlamydial infections in the Sardinian equine population. This study aimed to identify the chlamydial diversity in genital samples from asymptomatic Sardinian horses. However, some horses had a previous history of reproductive disorders, i.e., abortion and infertility. A total of 60 horses (39 mares and 21 stallions) were opportunistically recruited from 17 equine farms in central-northern Sardinia. Vaginal and uterine swabs from mares and urethral swabs and seminal fluid from stallions were sampled for the presence of chlamydial DNA. Samples from environments where the horses lived were also tested for the detection of Chlamydia spp. Eight vaginal swabs (8/39; 20%), two uterine swabs (2/27; 7%), two seminal fluid samples (2/20; 10%), and one urethral swab (1/21; 4.7%) were found to be positive for Chlamydia spp. by PCR analysis. In addition, results from environmental samples showed the presence of Chlamydia spp. in three environmental swabs (3/8; 37.5%) and five water samples (5/16; 31.2%). Sequencing results revealed that strains here identified were 99-100% similar to members belonging to the Chlamydiaceae family, including C. abortus, C. psittaci, and uncultured Chlamydia genotypes. ompA species-specific PCR performed on samples was found to be positive after 16S rRNA amplification gave positive results for C. psittaci. These results reveal the first presence of C. psittaci in the genital tract of horses and in the environment in Sardinia and indicate that this pathogen could be the prevailing cause of infertility and abortion in the tested equines. However, these findings need further proof and highlight the importance of adopting a 'One Health' approach to control the presence of this zoonotic bacteria in domestic animals in order to understand its impact on people exposed to the infection risk.

Culicoides species responsible for the transmission of Epizootic Haemorrhagic Disease virus (EHDV) serotype 8 in Italy.

Epizootic haemorrhagic disease (EHD) is a viral disease transmitted by Culicoides biting midges that affects wild and domestic ruminants. The causative agent, EHD virus (EHDV), belongs to the family Sedoreoviridae, genus Orbivirus. The virus has never been reported in Europe until October 2022, when the virus was for the first time detected in Sicily and Sardinia. After the first clinical cases, an intensive entomological field activity was carried out in five EHD affected farms located in Sardinia, with the aim of assessing the EHDV vector competence in European species of Culicoides. EHDV­8 was detected in C. imicola, C. obsoletus/scoticus, C. newsteadi, C. pulicaris ss, and C. bysta. The first 4 species have also been demonstrated to be able to transmit bluetongue virus (BTV). According to these results, it is likely that EHDV­8, sharing the same transmission patterns of BTV, can also spread to Europe.

Pseudomonas protegens Affects Mosquito Survival and Development.

This study investigated the pathogenic potential of Pseudomonas protegens on mosquito larvae of the two species Culex pipiens and Aedes albopictus, representing major threats for disease transmission in the Mediterranean area and worldwide. The bacterium achieved to kill over 90% of the mosquito larvae within 72 h after exposition to a bacterial concentration of 100 million CFU/ml. These lethal effects were concentration dependent and a significantly higher susceptibility was associated with younger larvae of both mosquito species. Significant slowdown of immature (larval and pupal) development and decrease in adult emergence rate after treatment with sub-lethal doses of the bacterium were also detected. This study reports for the first time the insecticidal activity of a root-associated biocontrol bacterium against aquatic mosquito larvae.

Anaplasma Species in Ticks Infesting Mammals of Sardinia, Italy.

Ticks are hematophagous ectoparasites that are recognized for their ability to vector a wide variety of pathogens of viral, bacterial, protozoal, and helminthic nature to vertebrate hosts. Among the different diseases transmitted by ticks, also called "Tick-Borne Diseases" (TBD), many are zoonotic. Pathogens of the genus Anaplasma refer to obligate intracellular bacteria within the Rickettsiales order transmitted mainly through tick bites and considered as well-established threats to domestic animals, livestock, and humans, worldwide. In this retrospective study, 156 ticks collected from twenty goats, one marten, and one cattle from several Sardinian sites, were examined by molecular analyses to detect the presence of Anaplasma species. A total of 10 (10/156; 6.4%) ticks were shown to be Anaplasma-positive by PCR screening. After sequence analyses, A. phagocytophilum was detected in four Rhipicephalus sanguineus s.l. (3.3%) and four Rh. bursa (11%) ticks from goats, while one Rh. sanguineus s.l. (0.8%) and one Rh. bursa (2.8%) collected from the marten and cattle, respectively, exhibited 100% of identity with A. marginale strains. In this study, we provide the first description and molecular detection of A. marginale and A. phagocytophilum in ticks of the Rhiphicephalus genus in Sardinia. Considering the growing impact of tick-borne Anaplasma pathogens on human health, further studies are necessary to monitor the prevalence of these pathogens in Sardinia.

Molecular Investigation of Theileria and Babesia Species in Domestic Mammals from Sardinia, Italy.

Piroplasmoses are tick-borne diseases caused by hemoprotozoan parasites of veterinary and public health significance. This study focuses on the molecular identification and characterization of species belonging to the Theileria/Babesia genera in 152 blood samples, collected from 80 horses and 72 cattle from several farms in Sardinia, by targeting the 18S rRNA gene. The PCR results highlighted that 72% of the samples were positive for Theileria/Babesia spp., with a rate of infection of 68% and 75% for the horses and cattle, respectively. Sequencing and the BLASTn analysis showed that the 18S rRNA generated in this study has 99-100% homology with the B. bigemina, T. orientalis/sergenti/buffeli, T. equi and T. annulata strains isolated from different hosts worldwide. These findings improve the knowledge on Babesia and Theileria infections in domestic mammals and confirm the significant prevalence of piroplasmosis among subclinical and carrier animals throughout the island. Furthermore, the presence of T. annulata, reported for the first time in the study area, expands the repertoire of pathogens already detected in Sardinia. Our results gather updates on the diversity and distribution of piroplasms in Sardinia and suggest the need to develop procedures to improve animal and public health safety.

Experimental infection of Aedes (Stegomyia) albopictus and Culex pipiens mosquitoes with Bluetongue virus.

Bluetongue disease (BT), caused by Bluetongue virus (BTV), infects wild and domestic ruminants, causing severe economic damage in the cattle and sheep industry. Proven vectors of BTV are biting midges belonging to the Culicoides genus, but other arthropods are considered potential vectors, such as ticks, mosquitoes, wingless flies, and sand flies. The present study represents the first attempt to evaluate the vectorial capacity of Culex pipiens and Aedes albopictus for BTV. Mosquitoes were artificially fed with blood containing BTV serotype 1. Infection, dissemination and transmission rates were evaluated at 0, 3, 7, 14 and 21 days after an infected blood meal. Viral RNA was only detected up to 3 days post infection in the bodies of both species. This study indicates that the two Italian populations of Cx. pipiens and Ae. albopictus are not susceptible to BTV infection.

Coxiellaceae in Ticks from Human, Domestic and Wild Hosts from Sardinia, Italy: High Diversity of Coxiella-like Endosymbionts.

PURPOSE: Coxiella burnetii is known for its potential as veterinary and human bacterial pathogen. The bacteria have been described in ticks, but their role in transmission of Q fever in humans is considered low. Coxiella endosymbionts closely related to C. burnetii have been also isolated from an extensive range of tick species and evidence is growing that these endosymbionts could be linked to human bacteremia. The aim of this study was to get new information on the presence of Coxiella species in ticks infesting wild and domestic hosts in Sardinia, Italy. METHODS: Here, 138 ticks collected from the study area were analyzed for the presence of C. burnetii and Coxiella-like bacteria by polymerase chain reaction (PCR), sequencing and philogenetic analyses using a set of primers targeting the 16S rRNA gene. RESULTS: DNA of Coxiella species was detected in 69% of the total ticks examined. Based on phylogenetic analysis, the 16S rRNA Coxiella genotypes identified in this study grouped in strongly supported monophyletic clades with identified reference sequences of CLEs detected from Rhipicephalus, Dermacentor, Haemaphysalis and Ornithodoros species and with Coxiella burnetii strains isolated worldwide. CONCLUSION: This study reports the molecular detection of a high diversity of Coxiella-like bacteria in Sardinian ticks and confirms also the presence of C. burnetii in tick species previously identified in the island. The role that Coxiella-like endosymbionts play in Sardinian ticks and in their vertebrate hosts needs to be explored further.

Detection of potentially pathogenic bacteria from Ixodes ricinus carried by pets in Tuscany, Italy.

BACKGROUND: Ticks are vectors of disease-causing pathogens that pose a serious threat to animals and people. Dogs and cats are exposed to tick infestation in multiple ways and can easily transport infected ticks into domestic environments and potentially transfer them to people. Pet owners are at increased risk of picking up ticks from their pets and developing tickborne diseases. This study aims to detect the presence of pathogens of potential public health interest in ticks removed from cats and dogs in Tuscany, Italy. METHODS: The collected ticks were screened for the presence of protozoan (Theileria species and Babesia species) and bacterial (Rickettsia species, Anaplasma species, Ehrlichia species, Chlamydia species, Bartonella species and Coxiella burnetii) pathogens using PCR. RESULTS: PCR and sequencing analysis revealed that 3 per cent of the ticks were PCR-positive for the presence of Rickettsia helvetica DNA, 5 per cent of ticks were PCR-positive for Bartonella henselae DNA, and 46 per cent of ticks were PCR-positive for Chlamydia psittaci and Chlamydia abortus DNA. None of the examined ticks was PCR-positive for Theileria species, Babesia species, Anaplasma species, Ehrlichia canis or Coxiella burnetii DNA. CONCLUSION: The results of this preliminary study highlight the importance of monitoring companion animals as indicators to evaluate the health status of their owners. Preventive measures are necessary to limit the spread of zoonotic pathogens from companion animals to people within the home environment.

Coexistence of Tick-Borne Pathogens in Ticks Collected from their Hosts in Sardinia: an Update.

PURPOSE: In recent decades, the incidence and distribution of tick-borne diseases have increased worldwide, attracting the attention of both clinicians and veterinarians. In Sardinia, notifiable tick-borne diseases are spreading and Mediterranean spotted fever (MSF) rickettsiosis continues to be endemic with an incidence of 10/10,000 inhabitants per year. Furthermore, ticks can transfer more than one pathogen after a single blood meal from a coinfected host or after multiple feeding on different infected hosts. The aim of this study was to update information on ticks and tick-borne diseases, focusing also on the presence of coinfection in Sardinian ticks. METHODS: The presence of protozoan (Theileria and Babesia species) and bacterial pathogens (Rickettsia spp., Anaplasma spp., Ehrlichia canis, Chlamydia spp., Bartonella spp., and Coxiella burnetii) was evaluated in 230 ticks collected from different hosts in Sardinia. RESULTS: PCR and sequencing analyses highlighted that the 59% of ticks were infected with at least one pathogen while the 15% resulted in coinfection by double and triple pathogens. Among the double co-infections, those of E. canis/C. burnetii, Babesia sp. Anglona/Ch. psittaci and Babesia sp. Anglona/C. burnetii revealed a statistically significant index of coinfection. CONCLUSION: This study identifies new pathogens in Sardinian ticks and updates the information about tick-borne diseases in the island. We also provide new results on the presence of coinfections in collected ticks. The knowledge about the diversity of ticks and tick-borne diseases circulating in Sardinia is a necessary step toward implementing effective tick-borne disease prevention and control programs.

Predicting the success of an invader: Niche shift versus niche conservatism.

Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus. The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype-environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat-shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental-induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe.

First molecular detection of Francisella-like endosymbionts in Hyalomma and Rhipicephalus tick species collected from vertebrate hosts from Sardinia island, Italy.

Ticks are vectors of a wide variety of human and animal pathogens as well as non-pathogenic microorganisms acting as endosymbionts and whose role in ticks is still little known. Symbionts such as Francisella-like endosymbionts (FLEs) are members of Francisellaceae family with unknown pathogenicity, detected in both hard and soft ticks. A total of 236 ticks collected from several sites in Sardinia were screened for Francisella species by PCR using primers targeting a fragment of the 16S rRNA gene. DNA of Francisella was detected in 5.1% (12/236) of the ticks tested. Sequencing results revealed that seven Rhipicephalus sanguineus s.l., three Hyalomma marginatum, one Hy. lusitanicum, and one Rh. bursa ticks exhibited DNA with 99-100% similarity to Francisella-like endosymbionts isolated from different tick species all over the world. Further research is needed in order to better characterize FLE strains obtained in Sardinia and to better understand if their presence could be related to the infection with other zoonotic pathogens.

Comparative laboratory and field study of biorational insecticides for Culicoides biting midge management in larval developmental sites.

An appropriate management strategy of bluetongue vectors should include larvicidal treatments in their larval development sites utilizing active substances with low environmental impact. A selection of biorational insecticides with potential against dipteran larvae was assayed in the laboratory against field collected Culicoides larvae including C. cataneii, C. circumscriptus, and C. imicola, determining their median lethal concentrations in water and mud/water substrate. The efficacy of formulations containing the insect growth regulators pyriproxyfen and cyromazine, the botanical insecticide azadirachtin, and the entomopathogenic bacteria Bacillus thuringiensis israelensis and Brevibacillus laterosporus, was also assessed in field conditions in a comparative study conducted in sheep farm larval development sites, including treatments with the organophosphate temephos. Significant larvicidal properties were associated with the various insecticides evaluated in the laboratory assays and in field trials, although with different levels of effectiveness. While temephos was confirmed to be an effective broad spectrum larvicidal substance, B. laterosporus appeared to be the most effective among entomopathogens, while insect growth regulators combined a good efficacy to a long-lasting residual effect in the field. Everything considered, the use of these biorational insecticides alone or in combination with larval habitat manipulation techniques appears to be a promising method to complement integrated biting midge management programs.

Molecular characterization and phylogenetic analysis of Babesia and Theileria spp. in ticks from domestic and wild hosts in Sardinia.

Piroplasmoses are tick-borne protozoan diseases caused by hemoprotozoan parasites with considerable economic, veterinary and medical impact worldwide. Here, the presence and diversity of piroplasmids was investigated in ticks collected from domestic and wild hosts in a typical subtropical environment of Sardinia island by standard PCR, sequencing, and phylogenetic analyses. We demonstrate the presence of strains closely related to the Theileria buffeli/sergentii/orientalis complex in Rhipicephalus sanguineus s.l., Rh. bursa, Rh. annulatus, Hyalomma marginatum, Dermacentor marginatus and Haemaphysalis punctata ticks. A strain detected in two Rh. sanguineus s.l. ticks collected from dogs grouped with T. equi, the agent of equine piroplasmosis. T. ovis, the main etiological agent of ovine theileriosis, was detected in one Rh. bursa tick from a mouflon. Babesia bigemina, the causative agent of bovine babesiosis, was detected in two Rh. sanguineus s.l. ticks from dogs. Our findings expand the knowledge on the repertoire of tick-borne pathogens present in Mediterranean ticks. Further analyses are needed to determine the role of ticks in the biological or mechanical transmission of piroplasmoses in this area.

Bluetongue virus detection in new Culicoides species in Sardinia, Italy.

Bluetongue is an infectious disease transmitted by Culicoides biting midges. Culicoides imicola is considered the main vector in the Mediterranean basin but other species have been implicated in the Bluetongue virus (BTV) transmission. During 2017, BTV serotype 4 re-occurred in Sardinia causing outbreaks in sheep farms. A survey was carried out on affected farms with the aim to detect the virus in field-collected Culicoides Biting midges were morphologically identified, pooled and then assayed with a real time RT-PCR. To evaluate BTV dissemination, some Culicoides were dissected and head, thorax and abdomen were tested singly by PCR. A total of 173,738 Culicoides adults were collected. Viral RNA was detected in 68 out of 77 pools and all species analysed resulted positive. Detection of BTV in parous female body regions (head, thorax and abdomen) confirmed the full dissemination of BTV in all species analysed. During this study, the vector competence of C imicola, C newsteadi s.l. and Obsoletus complex was confirmed. The authors found two new Culicoides species BTV positive, C paolae never associated with BTV transmission and C circumscriptus only recently found BTV positive in Turkey, which could be considered potential vectors.

Tacking the vector of Xylella fastidiosa: geo-statistical analysis of long-term field observations on host plants influencing the distribution of Phylaenus spumarius nymphs.

The meadow froghopper, Philaenus spumarius L., is endemic in Italy and was not considered a harmful species until 2014, when the olive quick decline syndrome (OQDS) showed up in Apulia (southern Italy). It was immediately suspected and then verified as the main vector of Xylella fastidiosa, the bacterium responsible for the disease. Currently, EU Directives consider the fight against P. spumarius compulsory in member states and recommend Integrated Pest Management (IPM), both in uncultivated and cultivated infested areas, to minimise the environmental impact of chemical pesticides. This should be based on an improved knowledge of the vector with its seasonal trends and feeding habits linked to specific herbaceous species. In this context, our field study was aimed to improve the understanding of the vector nutritional behaviour, especially at its critical nymph stage, by monitoring its presence on different herbaceous target species, using its typical feeding foams as key indicator. The study area was in Lazio region (central Italy), dedicated to olive growing and still unaffected by the X. fastidiosa plague. Over two years, during the nymph development period, field data have been acquired over the test area and then analysed by coupling statistical (ANOVA), geographical information system (GIS) and geo-referenced field sampling approaches. Results highlighted that P. spumarius exhibits significant preferences for specific herbaceous plants, especially at its early development stages, detectable by tenuous spittle. This indicates female oviposition activity, which seems also not influenced by olive tree proximity. Furthermore, the non-host plant species identified here could be suitable for creating green barriers for limiting the vector diffusion to contiguous areas where sensible plantations are growing. In the final section, applied implications arising from the present findings for P. spumarius population management are discussed.

Entomological surveillance of Zika virus in Sardinia, Italy, 2016.

Zika Virus (ZIKV) is a RNA virus belonging to the genus Flavivirus, family Flaviviridae. This virus is transmitted through bite of Aedes mosquitoes, in particular Ae. aegypti. On February 1st 2016, the World Health Organization (WHO) has declared ZIKV a Public Health Emergency of International Concern. Successively, considering the establishment of Ae. albopictus, WHO has classified Italy as having a moderate likelihood of local transmission of ZIKV, preceded in Europe only by France. For this reason an entomological surveillance plan was been activated in Sardinia in 2016. BG Sentinel Mosquito Traps have been positioned in 29 sites, comprising urban areas and points of entry, as ports and airports. Mosquitoes were collected fortnightly from April to December. A total of 3,089 mosquitoes were collected belonging to 10 species. The most numerous species have been Cx. pipiens s.l. and Ae. albopictus. All mosquitoes sampled have been assayed by real time reverse transcriptase PCR for detection of ZIKV RNA. A total of 584 pool have been analyzed and have been reported no evidence of ZIKV. A permanent entomological surveillance should be implemented principally in the urban areas and points of entry, as ports and airports, because Ae. albopictus, susceptible to ZIKV, is established in Sardinia and also know the recent introduction of invasive mosquitoes species Ae. koericus and Ae. japonicus in Italy.

First molecular detection of the human pathogen Rickettsia raoultii and other spotted fever group rickettsiae in Ixodid ticks from wild and domestic mammals.

Tick-borne rickettsioses are recognized as emerging vector-borne infections capable of infecting both human and animal hosts worldwide. This study focuses on the detection and molecular identification of species belonging to the genus Rickettsia in ticks sampled from human, vegetation, and domestic and wild vertebrates in Sardinia. Ticks were tested by PCR targeting gltA, ompA, and ompB genes, followed by sequencing analysis. The results provide evidences of a great variety of Rickettsia species of the Spotted fever group in Ixodid ticks and allow establishing for the first time the presence of R. raoultii in Rhipicephalus sanguineus s.l. and Dermacentor marginatus ticks in Sardinia island. Rickettsia massiliae was detected on R. sanguineus s.l. and R. aeschlimannii in Hyalomma marginatum and Hy. lusitanicum ticks. In addition, eight D. marginatus ticks were positive for R. slovaca. This study provides further evidence that different Rickettsia species are widespread in Sardinian ticks and that detailed investigations are required to understand the role these tick species play on spotted fever group rickettsiae circulation. More studies will provide new background on molecular epidemiology of zoonotic rickettsiae, the geographical distribution of tick-transmitted rickettsial pathogens, and the involvement of vertebrate hosts in propagation and maintenance of these bacteria in nature.

Combined larvicidal and adulticidal treatments to control Culicoides biting midges (Diptera: Ceratopogonidae): Results of a pilot study.

Culicoides biting midges can transmit viruses such as bluetongue virus and Schmallenberg virus to ruminants and African horse sickness virus to equines. The control of Culicoides population can be based on physical and/or chemical methods. This study aims to evaluate the efficacy of larvicides and adulticides combined treatments to control the vector Culicoides populations. Field trials have been conducted during 2014 on seven farms in northern Sardinia (Italy). Three insecticide treatments (larvicides and adulticides) have been carried out on three farms, whereas four farms remained unsprayed as untreated controls. For the larval control two commercial formulations, based on diflubenzuron and Bacillus thuringiensis var. israelensis (H-14), have been used. Against adults a deltamethrin based product has been sprayed on the walls and roofs of animal shelters. Adult Culicoides have been collected on each farm twice a week with an Onderstepoort blacklight suction trap. After each treatment a significant reduction in the abundance of total Culicoides, C. imicola and C. newsteadi s.l., has been observed. The Obsoletus complex density has been affected by the first two treatments. Our results suggest the possibility to control the Culicoides populations with a combination of adulticide and larvicide treatments.

Retrospective analysis of Bluetongue farm risk profile definition, based on biology, farm management practices and climatic data.

Bluetongue (BT) is a vector-borne disease transmitted by species of Culicoides midges (Diptera: Ceratopogonidae). Many studies have contributed to clarifying various aspects of its aetiology, epidemiology and vector dynamic; however, BT remains a disease of epidemiological and economic importance that affects ruminants worldwide. Since 2000, the Sardinia region has been the most affected area of the Mediterranean basin. The region is characterised by wide pastoral areas for sheep and represents the most likely candidate region for the study of Bluetongue virus (BTV) distribution and prevalence in Italy. Furthermore, specific information on the farm level and epidemiological studies needs to be provided to increase the knowledge on the disease's spread and to provide valid mitigation strategies in Sardinia. This study conducted a punctual investigation into the spatial patterns of BTV transmission to define a risk profile for all Sardinian farmsby using a logistic multilevel mixed model that take into account agro-meteorological aspects, as well as farm characteristics and management. Data about animal density (i.e. sheep, goats and cattle), vaccination, previous outbreaks, altitude, land use, rainfall, evapotranspiration, water surface, and farm management practices (i.e. use of repellents, treatment against insect vectors, storage of animals in shelter overnight, cleaning, presence of mud and manure) were collected for 12,277 farms for the years 2011-2015. The logistic multilevel mixed model showed the fundamental role of climatic factors in disease development and the protective role of good management, vaccination, outbreak in the previous year and altitude. Regional BTV risk maps were developed, based on the predictor values of logistic model results, and updated every 10 days. These maps were used to identify, 20 days in advance, the areas at highest risk. The risk farm profile, as defined by the model, would provide specific information about the role of each factor for all Sardinian institutions involved in devising BT prevention and control strategies.

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.