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In Europe wildlife animals such as the red fox (Vulpes vulpes) are considered the main reservoir for Angiostrongylus vasorum as well as a potential threat for domestic dog infection. Though this parasite is endemic in fox populations, data on A. vasorum infection in wolves (Canis lupus italicus) are still scant, having only recently been described in Northwestern Spain, in Italy, in Croatia and in Slovakia. Based on the rising number of cases of canine lungworm infection in Central Italy (Abruzzo region), the aim of the present study was to investigate the infection by A. vasorum in fox and wolf populations sharing the same geographical area of dogs. From October 2008 to November 2019, A. vasorum specimens were collected, through routine post-mortem examination, from 56 carcasses (44 foxes and 12 wolves). Adult parasites were searched for in the right side of the heart and in pulmonary artery of all carcasses. First stage of larvae (L1) was searched in faeces using the Baermann technique and in lungs by tissue impressions. Overall, 230 adult specimens were collected and identified on a morphological basis. To confirm the morphological identification, 4 adult specimens (n = 3 from fox, n = 1 from wolf) were molecularly identified as A. vasorum by amplification of partial fragment of nuclear 18S rRNA (~1700 bp) genes. The anatomo-pathological and parasitological examinations indicated the presence of A. vasorum in 33 foxes (75%) and in 8 wolves (66.7%). The level of prevalence of infested wolves was higher than the previous one reported in other European countries. Interestingly, the prevalence of infection in foxes herein recorded was higher than that described in dogs (8.9%) living in the same geographical area. This result may confirm the hypothesis that the spread of canine angiostrongylosis is linked to fox populations infection., Graphical abstract Image 1, Highlights • Angiostrongylus vasorum was found in dead foxes and wolves in Chieti province, Italy. • The prevalence in foxes was higher than that described in dogs. • The prevalence in wolves was higher than the previous one reported in European countries. • The spread of canine angiostrongylosis is linked to fox populations infection. • The province of Chieti appears to be an enzootic focus for canine angiostrongylosis.

Wolbachia, a maternally transmitted Gram-negative endosymbiont of onchocercid nematodes and arthropods, has a role in the biology of their host; thus it has been exploited for the filariasis treatment in humans. To assess the presence and prevalence of this endosymbiont in reptiles and their ectoparasites, blood and tail tissue as well as ticks and mites collected from them were molecularly screened for Wolbachia DNA using two sets of primers targeting partial 16S rRNA and Wolbachia surface protein (wsp) genes. Positive samples were screened for the partial 12S rRNA and cytochrome c oxidase subunit 1 (cox1) genes for filarioids. Of the different species of lizards (Podarcis siculus, Podarcis muralis and Lacerta bilineata) and snakes (Elaphe quatuorlineata and Boa constrictor constrictor) screened from three collection sites, only P. siculus scored positive for Wolbachia 16S rRNA. Among ectoparasites collected from reptiles (Ixodes ricinus ticks and Neotrombicula autumnalis, Ophionyssus sauracum and Ophionyssus natricis mites), I. ricinus (n = 4; 2.8%; 95% CI, 0.9–7) from P. siculus, N. autumnalis (n = 2 each; 2.8%; 95% CI, 0.9–6.5) from P. siculus and P. muralis and O. natricis (n = 1; 14.3%; 95% CI, 0.7–55.4) from Boa constrictor constrictor scored positive for Wolbachia DNA. None of the positive Wolbachia samples scored positive for filarioids. This represents the first report of Wolbachia in reptilian hosts and their ectoparasites, which follows a single identification in the intestinal cells of a filarioid associated with a gecko. This data could contribute to better understand the reptile filarioid-Wolbachia association and to unveil the evolutionary pattern of Wolbachia in its filarial host.

Trypanosomes are haemoflagellate protozoa transmitted by blood-feeding arthropods causing infections in a wide range of mammals, including humans. Adult badgers (Meles meles, n = 2), displaying severe paralysis, ataxia and severe ectoparasite infestation, were rescued from a peri-urban area of Bari (southern Italy). Blood samples and ectoparasites were screened for Trypanosoma spp. by the combined PCR/sequencing approach, targeting a fragment of 18S rRNA gene. Smears of haemolymph, guts and salivary glands of the alive ticks were microscopically observed. No haematological alterations, except thrombocytopenia, were found. Trypomastigotes and epimastigotes were observed in the blood smears of both badgers and Trypanosoma pestanai was molecularly identified. Out of 33 ticks (i.e. n = 31 Ixodes canisuga, n = 2 Ixodes ricinus) and two fleas (Ctenocephalides felis), 11 specimens (n = 5 I. canisuga engorged nymphs, n = 4 engorged females and n = 2 I. ricinus engorged females) tested positive only for T. pestanai DNA. All smears from ticks were negative. The present study firstly revealed T. pestanai in Ixodidae and badgers from Italy, demonstrating the occurrence of the protozoan on the peninsula. Further studies are needed to clarify the occurrence of the only known vector of this parasite, Paraceras melis flea, as well as other putative arthropods involved in the transmission of T. pestanai.

To investigate the role of dipterans in the transmission of Onchocerca lupi and other zoonotic filarioids, samples were collected from different sites in Algarve, southern Portugal, morphologically identified and molecularly tested for filarioids. Culex sp. (72.8%) represented the predominant genus followed by Culicoides sp. (11.8%), Ochlerotatus sp. (9.7%), Culiseta sp. (4.5%), Aedes sp. (0.9%) and Anopheles sp. (0.3%). Nineteen (2.8%) specimens scored positive for filarioids, with Culex pipiens quinquefasciatus (2%) positive for Dirofilaria immitis (1.4%), Dirofilaria repens, Acanthocheilonema reconditum, Onchocerca lupi, unidentified species of Filarioidea (0.2%, each) and Onchocercidae (0.6%). Additionally, Culiseta longiareolata (6.5%), Ochlerotatus caspius (3%) and Culex laticinctus (0.2%) scored positive for unidentified Onchocercidae, A. reconditum and for O. lupi, respectively. This is the first report of the occurrence of DNA of O. lupi, D. repens and A. reconditum in Culex spp. in Portugal. Information regarding the vectors and the pathogens they transmit may help to adopt proper prophylactic and control measures., First report of the occurrence of the DNA of Onchocerca lupi, Dirofilaria repens and Acanthocheilonema reconditum in Culex spp. from Portugal.Unidentified species of Filarioidea and Onchocercidae were detected in mosquitoes from Portugal.The detection of Wolbachia supergroup E in Culex laticinctus positive for O. lupi is new to science and deserves further investigation.

Background Wolbachia is an obligate intracellular maternally transmitted, gram-negative bacterium which forms a spectrum of endosymbiotic relationships from parasitism to obligatory mutualism in a wide range of arthropods and onchocercid nematodes, respectively. In arthropods Wolbachia produces reproductive manipulations such as male killing, feminization, parthenogenesis and cytoplasmic incompatibility for its propagation and provides an additional fitness benefit for the host to protect against pathogens, whilst in onchocercid nematodes, apart from the mutual metabolic dependence, this bacterium is involved in moulting, embryogenesis, growth and survival of the host. Methods This review details the molecular data of Wolbachia and its effect on host biology, immunity, ecology and evolution, reproduction, endosymbiont-based treatment and control strategies exploited for filariasis. Relevant peer-reviewed scientic papers available in various authenticated scientific data bases were considered while writing the review. Conclusions The information presented provides an overview on Wolbachia biology and its use in the control and/or treatment of vectors, onchocercid nematodes and viral diseases of medical and veterinary importance. This offers the development of new approaches for the control of a variety of vector-borne diseases. Graphical Abstract

A study on the occurrence of Dirofilaria immitis and its vectors was carried out in order to assess the prevalence of the disease in dogs in previously non-endemic areas of southern Italy. Blood samples (n = 385) and mosquitoes (n = 1540) were collected in two dog shelters and analysed by Knott’s test and duplex real-time PCR, respectively. Dirofilaria immitis was the most prevalent filarioid (44.2%), while Culex pipiens was the most prevalent mosquito species (68.8%). This high prevalence of D. immitis infection confirms this location as one of the most hyperendemic foci of dirofilariosis in Europe.

Filarioids of the genus Cercopithifilaria (Spirurida, Onchocercidae) are parasites of wild and domestic animals in tropical and subtropical regions being transmitted by ixodid ticks. Though this filarioid species have been studied in canine and tick populations in Europe, data on their species diversity and geographical distribution in Greece is scant. Thus, the aims of this study were to investigate the presence of Cercopithifilaria spp. in dogs and ticks across Greece and to assess the possible risk factors. A total of 500 skin biopsies were collected from dogs, while 508 ticks were collected from 180 infested animals and examined. Sediments from skin biopsies were microscopically screened for detection of dermal microfilaria (mfs). Skin samples (n = 115) and tick specimens (n = 153) were molecularly subjected by PCR. Overall, 70 samples (14%) scored positive for mfs. Specifically, 68 samples (13.6%) were positive for Cercopithifilaria bainae and two (0.4%) were co-infected with C. bainae and Cercopithifilaria sp. II. Molecular analyses revealed that all sequences obtained belong to C. bainae. Haplotype I was the most frequent (92.6%), followed by haplotype XVIII (3%) and haplotypes II and IX (1.5%). Three new haplotypes of C. bainae, named XIX, XX, and XXI, were also identified. Among the risk factors examined, habitat, dog use, body weight, tick infestation history, and the use of acaricides were associated with the presence of C. bainae. The estimated prevalence of Cercopithifilaria spp. demonstrates that these filarioids are common in dogs and ticks in Greece. Finally, the identification of 7 haplotypes for C. bainae confirms their genetic variability.

Background Feline vector-borne pathogens (FeVBPs) have been increasingly investigated for their impact on cat health and their zoonotic potential. The aim of the present study was to assess the prevalence of FeVBPs and haemoplasmas in cats across Italy and to identify potential risk factors linked to their occurrence. Methods Blood samples from 958 owned cats living in the North (n = 556), Centre (n = 173) and South (n = 229) of Italy were tested for Babesia spp., Hepatozoon spp., Ehrlichia spp., Anaplasma spp. and filarioids by conventional PCR (cPCR) and for haemoplasmas and Bartonella spp. by SYBR green real-time PCR. Cats included in the study represent a sub-sample from a larger number of animals enrolled in a previous study, which were selected based on the geographical origin. Data on cats’ positivity for Leishmania infantum, feline leukaemia virus (FeLV) and for feline immunodeficiency virus (FIV), available from the previous study, were included and examined. Potential risk factors for pathogen infection were assessed in relationship to categorical variables including sex, geographical origin, breed, neutering status and age of cats. Results Out of the 958 cats, 194 (20.2%) were positive for at least one of the tested pathogens, 89 (16%) from the North, 32 (18.5%) from the Centre and 73 (31.9%) from the South of Italy. A high prevalence of FeVBPs was detected in male cats (n = 125, 27.8%), living in the southern part of the country (n = 73, 31.9%), younger than 18 months of age (n = 24, 22.4%) and not neutered (n = 39; 27.5%). In particular, 24 cats (2.5%) tested PCR-positive for Bartonella spp., of which 1.6% for B. henselae and 0.9% for B. clarridgeiae. A total of 111 cats scored PCR-positive for haemoplasmas (11.6%), specifically “Candidatus Mycoplasma haemominutum” (n = 95, 9.9%), M. haemofelis (n = 14, 1.5%) and “Candidatus Mycoplasma turicensis” (n = 2, 0.2%). Moreover, 39, 31 and 8 cats were positive for FeLV (4.1%), L. infantum (3.2%) and FIV (0.8%), respectively. Co-infections were registered for 19 (9.8%) cats. Conclusions These results confirm the occurrence of haemoplasmas and FeVBPs throughout Italy. Preventive measures to protect both animal and human health should be carried out also for owned cats, even if no health status of animals has been assessed in this study.

Background Leishmania tarentolae is a protozoan isolated from geckoes (Tarentola annularis, Tarentola mauritanica), which is considered non-pathogenic and is transmitted by herpetophilic Sergentomyia spp. sand flies. This species occurs in sympatry with Leishmania infantum in areas where canine leishmaniasis is endemic. In the present study, we investigated the circulation of L. tarentolae and L. infantum in sand flies, dogs and lizards in a dog shelter in southern Italy, where canine leishmaniasis by L. infantum is endemic. Methods Sheltered dogs (n = 100) negative for Leishmania spp. (March 2020) were screened by immunofluorescence antibody test (IFAT) using promastigotes of both species at two time points (June 2020 and March 2021). Whole blood from dogs, tissues of Podarcis siculus lizards (n = 28) and sand flies (n = 2306) were also sampled and tested by a duplex real-time PCR (dqPCR). Host blood meal was assessed in sand flies by PCR. Results Overall, 16 dogs became positive for L. infantum and/or L. tarentolae by IFAT at one or both sampling periods. One canine blood sample was positive for L. infantum, whilst two for L. tarentolae by dqPCR. At the cytology of lizard blood, Leishmania spp. amastigote-like forms were detected in erythrocytes. Twenty-two tissue samples, mostly lung (21.4%), scored molecularly positive for L. tarentolae, corresponding to 10 lizards (i.e., 35.7%). Of the female Sergentomyia minuta sampled (n = 1252), 158 scored positive for L. tarentolae, four for L. infantum, and one co-infected. Two Phlebotomus perniciosus (out of 29 females) were positive for L. tarentolae. Engorged S. minuta (n = 10) fed on humans, and one P. perniciosus, positive for L. tarentolae, on lagomorphs. Conclusions Dogs and lacertid lizards (Podarcis siculus) were herein found for the first time infected by L. tarentolae. The detection of both L. tarentolae and L. infantum in S. minuta and P. perniciosus suggests their sympatric circulation, with a potential overlap in vertebrate hosts. The interactions between L. tarentolae and L. infantum should be further investigated in both vectors and vertebrate hosts to understand the potential implications for the diagnosis and control of canine leishmaniasis in endemic areas. Graphical abstract

Visceral leishmaniasis (VL) caused by Leishmania infantum is endemic in the Mediterranean basin with most of the infected human patients remaining asymptomatic. Recently, the saurian-associated Leishmania tarentolae was detected in human blood donors and in sheltered dogs. The circulation of L. infantum and L. tarentolae was investigated in humans, dogs and cats living in the Pelagie islands (Sicily, Italy) by multiple serological and molecular testing. Human serum samples (n = 346) were tested to assess the exposure to L. infantum by immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) and to L. tarentolae by IFAT. Meanwhile, sera from dogs (n = 149) and cats (n = 32) were tested for both Leishmania species by IFAT and all blood samples, including those of humans, by specific sets of real time-PCR for L. infantum and L. tarentolae. The agreement between serological tests performed for human samples, and between serological and molecular diagnostic techniques for both human and animal samples were also assessed. Overall, 41 human samples (11.8%, 95% CI: 8.9–15.7) were positive to L. infantum (5.2%, 95% CI: 3.3–8.1), L. tarentolae (5.2%, 95% CI: 3.3–8.1) and to both species (1.4%, 95% CI: 0.6–3.3) by serology and/or molecular tests. A good agreement among the serological tests was determined. Both Leishmania spp. were serologically and/or molecularly detected in 39.6% dogs and 43.7% cats. In addition to L. infantum, also L. tarentolae circulates in human and animal populations, raising relevant public health implications. Further studies should investigate the potential beneficial effects of L. tarentolae in the protection against L. infantum infection., Author summary Leishmania infantum is the major agent of canine and human leishmaniasis being endemic throughout the Mediterranean basin, including Italy. The protozoan is transmitted through the bite of infected phlebotomine sand flies mainly represented by the species Phlebotomus perniciosus and Phlebotomus perfiliewi as proven vectors in Italy. The sand fly fauna in this country includes, among others Sergentomyia minuta, considered herpetophilic vectors of Leishmania tarentolae. This species non-pathogenic to mammalians has recently been detected in human blood donors and in sheltered dogs. Our data demonstrate the occurrence of both L. infantum and L. tarentolae in humans and animal population living in leishmaniasis endemic area opening new perspectives into the study of this human disease. Therefore, the ecology of L. tarentolae highlights the need of a more comprehensive study on the spread of this parasite and on its potential beneficial role in public health through a cross-protection that could occur against pathogenic Leishmania spp. when L. tarentolae occurs in sympatry.

Wolbachia, the endosymbiont of arthropods and onchocercid nematodes is present in many medically important insect species, being also considered for the indirect control of parasitic ones. Archaeopsylla erinacei is a flea species infesting hedgehogs acting as vector of Rickettsia felis, Bartonella henselae, and Rickettsia helvetica, thus having public health relevance. The Wolbachia surface protein (wsp) and 16S rRNA genes were used to determine the presence, prevalence and molecular typing of Wolbachia in this flea species collected in two regions of southern Italy. Of the 45 fleas tested (n = 16 males, 35.6%; n = 29 females, 64.4%), 43 (95.6%; 95% CI: 84.8–99.2) scored positive for Wolbachia, of which 15 (33.3%) and 28 (62.2%) were males and females, respectively. The sex-wise prevalence of this endosymbiont was almost equal in both sexes (males 93.8%; 95% CI: 69.5–99.7; females 96.7%; 95% CI: 83.1–99.8). Single locus sequence analysis (SLST) of Wolbachia revealed two sequence types for 16S rRNA gene, named as wAr_15227 and wAr_15234, which came from two different areas, equally distributed in male and female fleas, whilst only one sequence type was identified for wsp gene. The phylogenetic analysis placed the two 16S rRNA sequence types in paraphyletic clades belonging to the supergroup A and B, respectively. Whilst, the tree of wsp gene clustered the corresponding sequence in the same clade including those of Wolbachia supergroup A. In MLST analyses, both Wolbachia sequence types clustered in a monophyletic clade with Drosophila nikananu (wNik) and Drosophila sturtevanti (wStv) from supergroup A. ClonalFrame analysis revealed a recombination event in the wAr_15234 strain which came from Apulia region. Scientific knowledge of the presence/prevalence of Wolbachia among medically important fleas, may contribute to develop an alternative biological method for the vector control.

The fan mussel, Pinna nobilis (Linnaeus 1758), is an endemic bivalve of the Mediterranean basin, protected by international legislation as an endangered species. In the early summer of 2018, a mass mortality event (MME) of P. nobilis was recorded in the Gulf of Taranto (Southern Italy, Ionian Sea). Moribund specimens of P. nobilis were collected by scuba divers and processed by bacteriological, parasitological, histopathological and molecular analyses to investigate the causes of this MME. Different developmental stages (i.e., plasmodia, spores and sporocysts) of a presumptive haplosporidian parasite were observed during the histological analysis in the epithelium and in the lumen of the digestive tubules, where mature spores occurred either free or in sporocysts. The spores presented an operculum and an ovoid shape measuring 4.4 µm (±0.232) in length and 3.6 µm (±0.233) in width. BLAST analysis of an 18SrRNA sequence revealed a high nucleotide similarity (99%) with the reference sequence of Haplosporidium pinnae available in GenBank database. Phylogenetic analysis clustered the sequence of the pathogen in a paraphyletic clade with the reference sequence of H. pinnae, excluding other haplosporidians (i.e., Bonamia and Minchinia genera). Based on data reported, H. pinnae was the causative agent of MME in the populations of P. nobilis sampled in the Ionian Sea, where the conservation of this endangered species is heavily threatened by such a protozoan infection. Further investigations should contribute to knowledge about the life cycle of H. pinnae in order to reduce spread of the pathogen and to mitigate the burden of the disease where P. nobilis is facing the risk of extinction.

Fasciola hepatica is a trematode infecting ruminants worldwide, occasionally reported in a wide range of animal species, including humans. According to the WHO, fasciolosis is recognized as a re-emerging neglected tropical disease, responsible for endemic and epidemic outbreaks in humans. Although the main hosts of the parasite are represented by cattle, sheep and goats, wildlife may be involved in its circulation. Here we firstly report F. hepatica in a wild boar from Italy (southern area) and characterize it both morphologically and molecularly. The nad1 gene analysis of specimens analyzed, revealed a high genetic similarity with those of humans from Iran and Peru, as well as a close phylogenetic relationship to those in ruminants from Brazil, Ecuador and Egypt. Considering the increase in the wild boar populations in urban and peri-urban areas, a potential role of this ungulate in the circulation of this zoonotic trematode is suggested.

Wild and domestic carnivores share ectoparasites, although molecular evidence is lacking. The goals of this study were to describe tick and flea infestation in sympatric free-ranging dogs Canis lupus familiaris (Linnaeus, 1758) (Carnivora: Canidae) and Andean foxes Lycalopex culpaeus (Molina, 1782) (Carnivora: Canidae) and to determine whether interspecific transmission occurs. Fleas and ticks retrieved from 79 foxes and 111 dogs in the human-dominated landscapes of central Chile were identified and a subset of specimens characterized by PCR and amplicon sequencing. Each ectoparasite species was clearly associated with a host: abundance and occurrence of Rhipicephalus sanguineus (Latreille 1806) (Acari: Ixodidae) and Ctenocephalides spp. (Siphonaptera: Pulicidae) were significantly higher in dogs than in foxes, whereas the opposite was true for Amblyomma tigrinum (Koch, 1844) (Acari: Ixodidae) and Pulex irritans (Linnaeus, 1758) (Siphonaptera: Pulicidae). Genetic analyses of a subset of ectoparasites revealed that dogs and foxes shared a limited number of nucleotide sequence types, suggesting that the interspecific transmission of these ectoparasites happens infrequently. Data also indicated that the ecological association and biological cycles of ticks and fleas determine the ectoparasite fauna of sympatric carnivores. In conclusion, our study shows that cross-species transmission should be assessed at a molecular level.

We assessed the presence of Rickettsia spp., Coxiella burnetii and Anaplasma phagocytophilum in reptiles, their ectoparasites and in questing ticks collected in a nature preserve park in southern Italy, as well as in a peri-urban area in another region. We also investigated the exposure to these pathogens in forestry workers, farmers and livestock breeders living or working in the nature preserve park given the report of anecdotal cases of spotted fever rickettsioses. Rickettsia spp. were molecularly detected in Podarcis muralis and Podarcis siculus lizards (i.e., 3.1%), in Ixodes ricinus (up to 87.5%) and in Neotrombicula autumnalis (up to 8.3%) collected from them as well as in I. ricinus collected from the environment (up to 28.4%). Rickettsia monacensis was the most prevalent species followed by Rickettsia helvetica. An undescribed member of the family Anaplasmataceae was detected in 2.4% and 0.8% of the reptiles and ectoparasites, respectively. Sera from human subjects (n = 50) were serologically screened and antibodies to Rickettsia spp. (n = 4; 8%), C. burnetti (n = 8; 16%) and A. phagocytophilum (n = 11; 22%) were detected. Two ticks collected from two forestry workers were positive for spotted fever group (SFG) rickettsiae. Ixodes ricinus is involved in the transmission of SFG rickettsiae (R. monacensis and R. helvetica) in southern Europe and lizards could play a role in the sylvatic cycle of R. monacensis, as amplifying hosts. Meanwhile, N. autumnalis could be involved in the enzootic cycle of some SFG rickettsiae among these animals. People living or working in the southern Italian nature preserve park investigated are exposed to SFG rickettsiae, C. burnetii and A. phagocytophilum., Author summary Zoonotic pathogens such as Rickettsia spp., Coxiella burnetii and Anaplasma phagocytophilum are associated with ticks, lice, fleas and mites and may infect a wide range of vertebrate species. There are still many knowledge gaps concerning the arthropod vectors and amplifying hosts of these pathogens. Reptiles are known to host infected ticks with these zoonotic pathogens and eventually become infected themselves by these bacteria. Hence to address this question from a broad One-Health perspective, we assessed the molecular prevalence of Rickettsia spp., C. burnetii and A. phagocytophilum in reptiles, ticks and mites from both hosts and environment and seroprevalence in humans living or working near the same area. Rickettsia DNA was detected in lizards’ tails, Ixodes ricinus ticks and in Neotrombicula autumnalis mites collected from them and in questing ticks. Although DNA of C. burnetii and A. phagocytophilum was not detected in lizards and their ectoparasites, sera samples of human subjects tested positive for the antibodies against these zoonotic pathogens. These findings indicate the epidemiological role of lizards in spreading SFG rickettsiae as well as I. ricinus, and hence representing a potential public health concern in geographical areas where lizards, ticks and humans share the same environment.

Leishmanioses are vector-borne diseases caused by Leishmania spp., which are transmitted by phlebotomine sand flies (Diptera, Psychodidae). The recent reports in humans of Leishmania tarentolae, which is primarily found in cold-blooded animals, and Leishmania infantum in Sergentomyia minuta spurred us to develop an internal transcribed spacer 1-based duplex quantitative real-time PCR (dqPCR) assay for the detection and differentiation between these Leishmania spp. The specificity of dqPCR was assessed by processing DNA samples from Phlebotomus spp. (n=188) and Se. minuta (n=171) and from tissues (i.e., heart, liver, muscle, lungs, spleen, kidney, eggs) of Podarcis siculus (n=4) and Tarentola mauritanica (n=3). In the absence of naturally infected and/or co-infected lizards, DNA from cultured L. infantum and L. tarentolae were spiked into tissues of lizards and used as controls. The analytical sensitivity of the dqPCR, assessed using 10-fold serial dilutions of DNA from both Leishmania spp. and spiked DNA samples from lizards was 2.3 x 10-7 ng/2 µl for L. infantum and 2.1 x 10-7 ng/2 µl for L. tarentolae. With the spiked DNA samples, the dqPCR detected up to 2.6 x 10-6 ng/2 µl of L. infantum and up to 2.1 x 10-7 ng/2 µl of L. tarentolae. Of 359 phlebotomine sand flies tested, five (3.6%) and two (1.4%) Ph. perniciosus scored positive for L. infantum and L. tarentolae, respectively. Similarly, of 171 Se. minuta, 56 (32.7%) and six (3.5%) scored positive for L. tarentolae and L. infantum, respectively. Co-infection with both Leishmania spp. was detected in two Se. minuta (1.2%). Out of seven reptiles tested, four P. siculus were positive for L. tarentolae. The newly dqPCR herein described may represent an improvement in the diagnosis of L. infantum and L. tarentolae and may assist in identifying the role of lizards as reservoirs and Se. minuta as vector, for these Leishmania spp.

Made available in DSpace on 2021-06-25T10:14:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Trombidiformes and Mesostigmata mites, as well as Ixodida ticks, infest ectothermic tetrapods worldwide, potentially acting as vectors of bacteria, viruses and protozoa. The relationship among ectoparasites, transmitted pathogenic agents (e.g., Borrelia spp., Coxiella spp., Hepatozoon spp., and Rickettsia spp.) and ectothermic hosts has been scarcely investigated. This research focuses on a large collection of Brazilian herpetofauna screened for the presence of arthropod ectoparasites and vector-borne microbial agents. Reptiles (n = 121) and amphibians (n = 49) from various locations were infested by ectoparasites. Following genomic extraction, microbial agents were detected in 81 % of the Acari (i.e. n = 113 mites and n = 26 ticks). None of the mites, ticks and tissues from amphibians yielded positive results for any of the screened agents. Blood was collected from reptiles and processed through blood cytology and molecular analyses (n = 48). Of those, six snakes (12.5 %) showed intraerythrocytic alterations compatible with Hepatozoon spp. gamonts and Iridovirus inclusions. Hepatozoon spp. similar to Hepatozoon ayorgbor and Hepatozoon musa were molecularly identified from seven hosts, two mite and two tick species. Rickettsia spp. (e.g., Rickettsia amblyommatis, Rickettsia bellii-like, Rickettsia sp.) were detected molecularly from four mite species and Amblyomma rotundatum ticks. Phylogenetic analyses confirmed the molecular identification of the above-mentioned microbial agents of mites and ticks related to snakes and lizards. Overall, our findings highlighted that the Brazilian herpetofauna and its ectoparasites harbour potentially pathogenic agents, particularly from the northern and south-eastern regions. The detection of several species of spotted fever group Rickettsia pointed out the potential role of ectothermic hosts and related arthropod ectoparasites in the epidemiological cycle of these bacteria in Brazil. Department of Veterinary Medicine University of Bari Faculty of Veterinary Medicine University of São Paulo Zoological Collections Laboratory Butantan Institute Master's Program in Veterinary Medicine and Animal Welfare and Doctoral Program in One Health Santo Amaro University Deparment of Veterinary Medicine Anhembi Morumbi University Vila Tramontano Department of Agriculture Food and Environment University of Pisa, via Del Borghetto 80 Department of Veterinary Pathology Universidade Estadual Paulista Julio De Mesquita Filho (UNESP) Department of Pathobiology Faculty of Veterinary Science Bu-Ali Sina University Felestin Sq. Department of Veterinary Pathology Universidade Estadual Paulista Julio De Mesquita Filho (UNESP) CNPq: 142409/2015-4 FAPESP: 2010/51875-9 FAPESP: 2017/12910-2 CNPq: 440639/2015-8 CNPq: 454907/2014-1

The zoonotic Onchocerca lupi and tick-transmitted filarioids of the genus Cercopithifilaria remain less well known due to the difficulties in accessing to skin samples as target tissues. Here, we proposed a molecular approach reliying on multiplex qPCR assays that allow the rapid identification of filarioids from canine blood, skin, and tick samples. This includes two newly developed duplex qPCR tests, the first one targeting filarial and C. grassii DNA (CanFil-C. grassii). and the second qPCR assay designed for the detection of Cercopithifilaria bainae and Cercopithifilaria sp. II DNAs (C. bainae-C.spII). The third one is a triplex TaqMan cox 1 assay targeting DNA of blood microfilariae (e.g., Dirofilaria immitis, Dirofilaria repens and Acanthocheilonema reconditum). The novel duplex qPCRs developed were validated in silico and by screening of known DNA collection. The qPCR assays were also used for screening the blood and tick samples of 72 dogs from Algeria. This allowed the identification of canine filariasis infection with 100% of specificity and 89.47% and 100% of sensitivity from naturally infected blood and tick samples, respectively. The prevalences of 26.39% for D. immitis and 5.56% for both D. repens and A. reconditum were reported in blood and tick samples. Cercopithifilaria DNAs were detected only in tick samples, with a prevalence of 4.17% and 5.56% for C. bainae and Cercopithifilaria sp. II, respectively. Co-infections were diagnosed in 6.94% and 13.89% of blood and tick samples, respectively. Whereas all samples were negative for C. grassii DNA. The use of engorged ticks instead of blood and skin samples could be an easier option for the surveillance of all canine filarioids herein investigated. The multiplex qPCR assays herein validated were shown to be useful in the detection of filarial co-infections by overcoming sequencing of positive samples.

Lutzomyia migonei is incriminated as a vector of Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil. Recently, this phlebotomine sand fly species has been suggested as a vector for Leishmania infantum, which causes zoonotic visceral leishmaniasis. Considering the widespread distribution of Lu. migonei in South America, the existence of isolated populations has been hypothesized. Three Lu. migonei populations, two from north-eastern Brazil (Machados, Pernambuco State, and Baturité, Ceará State) and other from the south-eastern region (Niterói, Rio de Janeiro State) were analysed both morphologically and genetically. Though no significant morphological differences were found amongst the sand fly specimens analysed, discriminant analysis based on specific morphometric characters (i.e., length of wing, antennal segment 3 and coxite for males, and length of wing and antennal segment 3 for females), showed that specimens from Machados were closer to Baturité than to Niterói. The molecular analysis of cytochrome c oxidase subunit I gene sequences also supported this observation by the distinct separation of two monophyletic clades, grouping specimens from Machados and Baturité separately from those of Niterói. Our results suggest the existence of different populations within the distribution range of Lu. migonei. Whether these populations are reproductively isolated and/or present differences in terms of vector competence/capacity for L. braziliensis and L. infantum needs to be further investigated.

Ticks are known to transmit pathogens which threaten the health and welfare of companion animals and man globally. In the present study, mainly adult ticks were collected from dogs and cats presented at their local veterinary practice in Hungary, France, Italy, Belgium (dogs only) and Germany (cats only), and identified based on tick morphology. If more than one tick was collected from a host animal, ticks were pooled by tick species for DNA extraction and subsequent PCR examination for the presence of tick-borne pathogens. Out of 448 tick samples, 247 (95 from dogs and 152 from cats) were Ixodes ricinus, 26 (12 from dogs and 14 from cats) were I. hexagonus, 59 (43 from dogs and 16 from cats) were Dermacentor reticulatus and 116 (74 from dogs and 42 from cats) were Rhipicephalus sanguineus sensu lato (s.l.). In 17% of the I. ricinus samples Anaplasma phagocytophilum was found. Borrelia spp. were mainly identified in I. ricinus collected from cats (18%) and to a lesser extent in dog-sourced ticks (1%), with Borrelia afzelii (n = 11), B. garinii (n = 7), B. valaisiana (n = 5), B. lusitaniae (n = 3) and B. burgdorferi sensu stricto (n = 3) being identified. One I. hexagonus sample collected from a cat in France tested positive for B. afzelii. Babesia canis was detected in 20% of the D. reticulatus samples, mainly from Hungary. Rhipicephalus sanguineus s.l. was found positive for Hepatozoon canis (3%), A. platys (5%) and three Rickettsia species (7%; R. massiliae; R. raoultii and R. rhipicephali). Furthermore, a total of 66 R. sanguineus s.l. ticks were subjected to molecular analysis and were identified as R. sanguineus sp. II-temperate lineage, with seven haplotypes recorded. Amongst them, the most prevalent sequence types were haplotype XIII (n = 24; 69%) and haplotype XIV (n = 16; 52%) in France and in Italy, respectively, found both in cats and dogs. Although differences related to both country and host, were observed, the results of this study indicate that cats and dogs are exposed to tick-borne pathogen infected ticks, which may represent a medical risk to these host animals.

Ocular thelaziosis is caused by nematodes of the genus Thelazia (Spirurida, Thelaziidae), which inhabit the surface of the eyes and associated tissues. Thelazia callipaeda affects a range of mammal species, including humans, and in the last two decades has been reported in multiple European countries, being classified as an emergent vector-borne pathogen. In Portugal T. callipaeda is endemic in north-eastern areas, where it has been reported in domestic dogs, cats, red foxes (Vulpes vulpes) and wild European rabbits (Oryctolagus cuniculus). We report, for the first time, T. callipaeda in a beech marten (Martes foina) from Portugal and highlight the presence of haplotype 1 as the only one found in Europe, irrespective of the host species and geographical area of provenience.

Dirofilariosis by Dirofilaria immitis and Dirofilaria repens is endemic in dogs from countries of the Mediterranean basin. Both species may infect humans, with most of the infected patients remaining asymptomatic. Based on the recent description of the southernmost hyperendemic European focus of heartworm disease in dogs from the Pelagie archipelagos, we performed a serological and molecular survey in human population of that area. Human blood samples were collected in the islands of Linosa (n=101) and Lampedusa (n=296) and tested by ELISA and molecular test for the detection of D. immitis and D. repens. Samples were also screened for filarioid-associated endosymbionts, Wolbachia sp. The seroprevalence of D. immitis and D. repens was, respectively, 7.9% and 3.96% in Linosa, and 7.77% and 19.93% in Lampedusa. Out of 397 human blood samples tested molecularly, 4 scored positive (1%) for Dirofilaria spp. by qPCR (i.e., three for D. immitis and one for D. repens) and 6 (1.5%) for Wolbachia. Of the qPCR positive for Dirofilaria spp., only D. repens was amplified by cPCR and was positive for Wolbachia. In the phylogenetic analysis, the sequence of Wolbachia detected in D. repens positive samples clustered along with other C supergroup filarioids. Our results overlap with the recent prevalence data collected on dogs from the same area, where D. immitis is prevalent in Linosa and D. repens prevails in Lampedusa. Molecular detection of D. immitis in human blood is quite unusual considering that humans are dead-end hosts for dirofilarial infection and most of the human cases described so far in Europe were ascribed to D. repens. An integrative diagnostic approach using serum analysis and Wolbachia detection is also presented. In endemic areas for canine dirofilarioses humans are exposed to the infection, suggesting the importance of One Health approach in diagnosing, treating and controlling this zoonotic parasitosis.

International trade of animals may represent a gateway for the spreading of zoonotic pathogens and their vectors. Amongst animals, reptiles are commonly illegally imported worldwide, being Italy in the fifth position of importation of these animals. Thus, the current study analysed the pathogens associated with Hyalomma aegyptium ticks, which were collected from illegally imported tortoises from North Africa to Italy. All tick DNA samples were tested by conventional PCR for the presence of Anaplasma spp., Babesia spp., Borrelia spp., Coxiella burnetti, Ehrlichia spp., Hepatozoon spp., Rickettsia spp. and microfilariae of filarioids. Out of 22% (n=161) of ticks screened, 78.9% (n=127) were males and 21.1% (n=34) females. Among them, three male specimens collected from two different turtles (1.9%; 95% CI; 0.5-5.5) scored positive for Anaplasma spp./Ehrlichia spp., whereas all females were negative. BLAST analysis of the sequences obtained from positive samples revealed 99-99.3% nucleotide identity with the sequence of Ehrlichia ewingii available in GenBank. The finding of E. ewingii in ticks from imported reptiles warrants the need for imposing strict rules in the international trade of reptiles to effectively reduce the introduction of exotic pathogens and their vectors in new geographic areas.

Background Leishmaniosis caused by Leishmania infantum is one of the most important vector-borne diseases affecting animals and humans worldwide. Dogs are considered main reservoirs of the zoonotic forms, though in the last years the role of cats as reservoirs has been increasingly investigated. Feline leishmaniosis (FeL) occurs in endemic areas and no specific preventive measures have been investigated so far. In this study the efficacy of a 10% imidacloprid/4.5% flumethrin polymer matrix collar, licensed for tick and flea prevention, has been assessed against FeL in a longitudinal study on 204 privately owned cats from the Aeolian islands (Sicily), an area highly endemic for the disease. From March to May 2015 [Study Day 0 (SD 0)], cats negative for FeL were collared (G1, n = 104) or left untreated (G2, n = 100). Diagnosis consisted of serology and qPCR on blood and conjunctival swabs, which were collected at baseline (SD 0) and at the end of the study (SD 360). Interim clinical examinations were performed on SD 210 (when collars were replaced in G1) and SD 270. Results Of the 159 cats which completed the study, 5 in G1 and 20 in G2 were positive for L. infantum infection, in at least one of the diagnostic tests leading to a yearly crude incidence of 6.3% and 25.0% in G1 and G2, respectively (P = 0.0026). This translates into an efficacy of the collar of 75.0% in preventing feline Leishmania infection. The collar was generally well tolerated with no systemic adverse reactions and few local skin reactions were observed in the application area in four out of 104 treated cats (3.8%). Conclusions The 10% imidacloprid/4.5% flumethrin collar significantly reduced the risk of L. infantum infection in cats. To our knowledge, this is the first study in which a preventative strategy against feline Leishmania infection is assessed under natural conditions. These findings close a gap in veterinary medicine, in that they confirm this collar as a tool in reducing the risk of Leishmania infection in cats. Such a preventative tool could contribute to the reduction of the risk of the disease in animal and in human populations when included in integrated leishmaniosis control programmes.

Background With the aim to assess the occurrence of hard ticks and the pathogens they may carry in dogs from Greece, ixodid specimens (n = 757) were collected from 310 animals living in six provinces across the Greek peninsula. All ticks were morphologically identified, and genomic DNA was extracted from 344 (45.5%) representative specimens, according to their species, engorgement status and sampling area. The occurrence of Anaplasma spp., Ehrlichia spp., Hepatozoon spp., Rickettsia spp., Babesia spp., Theileria spp. and Cercopithifilaria spp. was assessed by conventional and quantitative real-time PCR. Results Overall, 150 dogs (48.4%) were infested by ticks, with Rhipicephalus sanguineus (sensu lato) being the most prevalent (70.1%), followed by Haemaphysalis parva (14.7%), Rhipicephalus turanicus (11.4%), and Haemaphysalis concinna (2.4%). Out of 344 specimens molecularly examined, 41 (11.1%) were positive for at least one microorganism (i.e. 5.5% for Cercopithifilaria bainae, 2.9% for Hepatozoon canis, 1.7% for Rickettsia hoogstraalii, 1.2% for Hepatozoon felis, 0.6% for Rickettsia massiliae, 0.6% for Theileria ovis, 0.3% for Anaplasma platys and 0.3% for Coxiella like-endosymbiont). Conclusions The results of this study show that different tick species parasitize dogs in Greece, carrying a range of microorganisms potentially pathogenic for dogs and humans. Consequently, control strategies against ticks are of great importance to prevent the risk of tick-borne diseases. The relationship between ticks infesting dogs and associated microorganisms is described according to collection site and dog lifestyle.

Leishmania infantum infection in wildlife is increasingly reported in Europe, but scant data are available in Italy so far. This study aimed to investigate the circulation of L. infantum among sylvatic hosts in Sicily (southern Italy), a highly endemic area for canine leishmaniosis, through serological and molecular tools. Target tissues (skin, spleen, lymph nodes) collected from 71 European rabbits, 2 European hares, 7 red foxes, 11 European wildcats and 1 pine marten, were qPCR analysed for the detection of L. infantum DNA. Additionally, 40 rabbits, older than one year, were serologically screened for specific anti-Leishmania antibodies. Leishmania infantum was molecularly diagnosed in 5.4% (n = 5) of the examined animals (3/71 European rabbits, 2/7 red foxes). In many of the qPCR positive animals (4/5), the parasite DNA was more prevalent in visceral than cutaneous tissues. None of the positive animal showed signs of disease and/or macroscopic alterations of organs; low parasitic burden in all positive tissue samples was also recorded. Only one rabbit serum (i.e., 2.5%) tested positive for anti-Leishmania antibodies. The seropositive rabbit was in good health status and no amastigotes were observed in lymph-node aspirate and blood smears. This study provides first evidence of L. infantum infection in wild animals from Sicily (southern Italy). Despite the low prevalence of infection here reported, the circulation of the Leishmania in wild reservoirs in Sicily remains worthy of future investigations for a better understanding of their role in the epidemiology of the disease as well as to fine-tune control strategies in the area.

The zoonotic protozoan parasites Toxoplasma gondii, Cryptosporidium parvum and Giardia duodenalis have been recorded worldwide in economically important edible shellfish, and are thus likely to represent a significant public health risk. Therefore, an innovative, user-friendly diagnostic tool is required in order to improve food safety control. The Q3 system is a miniaturized platform whose efficiency and applicability were investigated and compared with results obtained using standard Real-Time PCR. Tanks of saltwater containing acclimated Mytilus galloprovincialis, Ruditapes philippinarum and Ostrea edulis specimens were spiked with purified Cryptosporidium, Giardia and Toxoplasma cysts/oocysts at different concentrations (i.e., 10(3), 10(4) and 10(5)). We then collected 30 specimens for each shellfish species from each group at 24 h and 72 h post-contamination. After DNA extraction, we tested all samples by Real-Time-PCR and Q3, and evaluated the sensitivity, specificity, predictive values, repeatability and concordance between the two systems. Concordance between Real-Time-PCR and Q3 was very good (p

Though scantly investigated, Leishmania infantum infection and clinical cases of leishmaniasis in cats have been recently reported in several countries of the Mediterranean basin, with large variability in prevalence data. A major limitation in the comparability of the data available is attributed to the differences in diagnostic techniques employed and cat populations sampled. The aim of this study was to assess the prevalence of L. infantum infection in owned cats across Italy by serological and molecular tests and the identification of potential risk factors. Blood samples from 2,659 cats from northern (n = 1,543), central (n = 471) and southern (n = 645) Italy were tested for antibodies against L. infantum, by an immunofluorescence antibody test and for the parasites’ DNA, by real-time PCR. Samples were additionally screened for feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) proviral DNAs. An overall cumulative L. infantum prevalence of 3.9% was recorded by serology (3.3%) and/or qPCR (0.8%), with a higher rate (10.5%) in southern Italy. The risk of L. infantum infection in cats was significantly associated to the geographical areas (South vs North and Centre; p, Author summary Zoonotic visceral leishmaniasis is a potentially fatal parasitic disease, which is caused by Leishmania infantum. Its distribution is associated with the occurrence of the sand fly vectors and reservoir hosts. Since L. infantum infection can occur in cats with clinical or subclinical outcomes, the role of cats in the epidemiology of zoonotic visceral leishmaniasis needs to be thoroughly assessed. This study aimed to evaluate the prevalence and associated risk factors for infection with L. infantum in a large subset of cats across Italy, a known endemic area with records of human cases of visceral leishmaniasis. Serum and blood samples from 2,659 cats from northern (n = 1,543), central (n = 471) and southern (n = 645) Italy were tested for antibodies against L. infantum and parasites’ DNA, respectively. A cumulative L. infantum prevalence of 3.9% was recorded by serology (3.3%) and/or real-time PCR (0.8%). The risk of L. infantum infection in cats was associated to the geographical areas, age class, neutering status and feline immunodeficiency virus infection. These findings reveal that cats are exposed to and/or infected by this protozoan across the country, warranting further investigation to assess their role in the epidemiology of zoonotic visceral leishmaniasis to refine surveillance and prevention strategies against this veterinary and medically important ailment.

Research Areas: Parasitology ;Tropical Medicine Background: Canine and feline leishmanioses are increasingly reported worldwide and represent a threat to both animal and human health. Despite their relevance, data about leishmanioses in companion animals in the east-central part of the Arabian Peninsula are unavailable. Therefore, we investigated the occurrence of Leishmania spp. in dogs and cats from Qatar. Methods: From March 2016 to May 2018, 199 pets (120 dogs and 79 cats) living in Doha or its outskirts were included in this study. From each animal a blood sample was collected and tested for Leishmania spp. by quantitative real-time PCR. Results: Out of the 199 animals, four (2.0%) were positive for Leishmania spp., including three dogs (2.5%) and one cat (1.3%). Conclusions: All positive animals were born in Qatar and had not travelled overseas, suggesting that infection was locally acquired. Considering the occurrence of Leishmania spp. and its potential impact on the health of animals and humans, it is crucial to increase scientific knowledge in order to plan screening and regular prophylaxis against sand fly vectors to reduce the risk of infection. info:eu-repo/semantics/publishedVersion

Paternal leakage of mitochondrial DNA (mtDNA) and heteroplasmy have been recently described in several animal species. In arthropods, by searching in the Scopus database, we found only 23 documented cases of paternal leakage. Therefore, although arthropods represent a large fraction of animal biodiversity, this phenomenon has been investigated only in a paucity of species in this phylum, thus preventing a reliable estimate of its frequency. Here, we investigated the occurrence of paternal leakage and mtDNA heteroplasmy in ticks belonging to one of the most significant tick species complexes, the so-called Rhipicephalus sanguineussensu lato. By developing a multiplex allele-specific PCR assay targeting a fragment of the 12S rRNA ribosomal region of the mtDNA, we showed the occurrence of paternal leakage and mtDNA heteroplasmy in R. sanguineuss.l. ticks originated from experimental crosses, as well as in individuals collected from the field. Our results add a new evidence of paternal leakage in arthropods and document for the first time this phenomenon in ticks. Furthermore, they suggest the importance of using allele-specific assays when searching for paternal leakage and/or heteroplasmy, as standard sequencing methods may fail to detect the rare mtDNA molecules.

Leishmania infantum causes human and canine leishmaniosis. The parasite, transmitted by phlebotomine sand flies, infects species other than dogs and people, including wildlife, although their role as reservoirs of infection remains unknown for most species. Molecular typing of parasites to investigate genetic variability and evolutionary proximity can help understand transmission cycles and designing control strategies. We investigated Leishmania DNA variability in kinetoplast (kDNA) and internal transcribed spacer 2 (ITS2) sequences in asymptomatically infected wildlife (n = 58) and symptomatically and asymptomatically infected humans (n = 38) and dogs (n = 15) from south-east Spain, using single nucleotide polymorphisms (SNPs) and in silico restriction fragment length polymorphism (RFLP) analyses. All ITS2 sequences (n = 76) displayed a 99%-100% nucleotide identity with a L. infantum reference sequence, except one with a 98% identity to a reference Leishmania panamensis sequence, from an Ecuadorian patient. No heterogeneity was recorded in the 73 L. infantum ITS2 sequences except for one SNP in a human parasite sequence. In contrast, kDNA analysis of 44 L. infantum sequences revealed 11 SNP genotypes (nucleotide variability up to 4.3%) and four RFLP genotypes including B, F and newly described S and T genotypes. Genotype frequency was significantly greater in symptomatic compared to asymptomatic individuals. Both methods similarly grouped parasites as predominantly or exclusively found in humans, in dogs, in wildlife or in all three of them. Accordingly, the phylogenetic analysis of kDNA sequences revealed three main clusters, two as a paraphyletic human parasites clade and a third including dogs, people and wildlife parasites. Results suggest that Leishmania infantum genetics is complex even in small geographical areas and that, probably, several independent transmission cycles take place simultaneously including some connecting animals and humans. Investigating these transmission networks may be useful in understanding the transmission dynamics, infection risk and therefore in planning L. infantum control strategies.

Ehrlichia canis is among the most prevalent tick-borne pathogens infecting dogs worldwide, being primarily vectored by brown dog ticks, Rhipicephalus sanguineus sensu lato (s.l.). The genetic variability of E. canis has been assessed by analysis of different genes (e.g., disulfide bond formation protein gene, glycoprotein 19, tandem repeat protein 36 - TRP36) in the Americas, Africa, Asia, and in a single dog sample from Europe (i.e., Spain). This study was aimed to assess the variations in the TRP36 gene of E. canis detected in naturally infected canids and R. sanguineus s.l. ticks from different countries in Asia and Europe. DNA samples from dogs (n = 644), foxes (n = 146), and R. sanguineus s.l. ticks (n = 658) from Austria, Italy, Iran, Pakistan, India, Indonesia, Malaysia, the Philippines, Singapore, Thailand, Vietnam, and Taiwan were included in this study. Ehrlichia canis 16S rRNA positive samples (n = 115 from the previous studies; n = 14 from Austria in this study) were selected for molecular examination by analyses of TRP36 gene. Out of 129 E. canis 16S rRNA positive samples from dogs (n = 88), foxes (n = 7), and R. sanguineus s.l. ticks (n = 34), the TRP36 gene was successfully amplified from 52. The phylogenetic analysis of the TRP36 pre-repeat, tandem repeat, and post repeat regions showed that most samples were genetically close to the United States genogroup, whereas two samples from Austria and one from Pakistan clustered within the Taiwan genogroup. TRP36 sequences from all samples presented a high conserved nucleotide sequence in the tandem repeat region (from 6 to 20 copies), encoding for nine amino acids (i.e., TEDSVSAPA). Our results confirm the US genogroup as the most frequent group in dogs and ticks tested herein, whereas the Taiwan genogroup was present in a lower frequency. Besides, this study described for the first time the US genogroup in red foxes, thus revealing that these canids share identical strains with domestic dogs and R. sanguineus s.l. ticks.

Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis., Author summary The diagnosis of Onchocerca lupi (Spirurida: Onchocercidae), a zoonotic nematode of domestic animals, is currently based on microscopic examination of skin snip sediments and on the identification of adults embedded in ocular nodules or by molecular assays. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dogs is mandatory. In this context, combined immunoblotting and mass spectrometry-based analyses have been performed to identify two proteins, major antigen and paramyosin, of O. lupi. Peptides herein identified represent suitable candidate biomarker for the development of a specific diagnostic test for canine onchocercosis. An accurate, minimally invasive diagnostic method could prove useful for the control of the canine diseases, for establishing large sero-surveys, for mapping the distribution of the infection in endemic areas as well as in areas where information on the disease is not available and for the reduction of risks for human infection.

Cardiopulmonary infections by Angiostrongylus chabaudi affect domestic and wild felids but, due to limited information on the biology of this nematode, its pathogenicity remains unclear. This article describes the histopathological alterations associated with Angiostrongylus infection in a wildcat from Bulgaria, and reviews current literature on this feline angiostrongylid. Nematodes were isolated from lung lavage and faecal samples of a road killed wildcat in Southern Bulgaria. The morphological identification of parasite larvae as A. chabaudi was confirmed by molecular analysis of part of the 18S ribosomal RNA gene. Upon histopathological examination, severe granulomatous pneumonia, ranging from multifocal to coalescing, and pulmonary vascular lesions were observed. Extensive alveolar collapse, alveolar emphysematous changes, parenchymal haemorrhages and small artery wall hyperplasia were observed in the parenchyma adjacent to the granulomas. Histopathological examination revealed the presence of cross-sections of adult female parasites within the lumen of the pulmonary artery branches, the intima altered markedly by subendothelial proliferation and oedematous changes. This study compliments current knowledge of the pathogenesis of feline angiostrongylosis by A. chabaudi in wildcats, as well as of the distribution of this little-known parasite.

Ehrlichiosis and Q fever caused by the intracellular bacteria Ehrlichia canis and Coxiella burnetii, respectively, are tick-borne diseases with zoonotic potential and widespread geographical distribution. This study investigated the prevalence of both infections in wild mammals in southern Italy. Tissue samples obtained from the red fox (Vulpes vulpes), European badger (Meles meles), gray wolf (Canis lupus), beech marten (Martes foina), and crested porcupine (Hystrix cristata) were processed for molecular detection of both pathogens. E. canis was detected in 55 out of 105 (52 %) red foxes and three out of six gray wolves. Four sequence types were identified, three of which were found in the spleen and liver samples of red foxes and wolves, and one in the kidney of a red fox. None of the examined mammals was positive to C. burnetii type. This represents the first report of E. canis in free-ranging wolves worldwide, as well as the first evidence of this pathogen in red foxes in the peninsular Italy. Our results suggest that E. canis infection is common in free-ranging canids in southern Italy and that a sylvatic life cycle of this pathogen may occur.

Canine vector-borne diseases (CVBDs) pose a major health problem in dogs globally, with the potential to cause zoonoses, in particular in developing countries where scientific knowledge on the topic is minimal. Blood samples and ticks were collected from stray dogs in Tamil Nadu, South India to assess the prevalence of CVBD-causing pathogens (Anaplasma spp., Babesia spp., Ehrlichia spp., Hepatozoon spp., filarioids and Leishmania spp.). Of the 230 dogs examined, 229 (99.6%) were infested by ticks (mean intensity, 5.65) with Rhipicephalus sanguineus sensu lato and Rhipicephalus haemaphysaloides being morphologically identified in the 98.3% and 1.7% of the infested dogs, respectively. Overall, the 67.8% (n = 156) of dogs was positive for at least one pathogen with Hepatozoon canis being the most prevalent (37.8%) followed by Anaplasma platys (22.6%), Ehrlichia canis (16.1%) Babesia vogeli (10%), Anaplasma phagocytophilum (0.4%) and Babesia gibsoni (0.4%). Two filarioids (Dirofilaria sp. "hongkongensis" and Brugia malayi, 0.4%) were diagnosed in sampled animals. Co-infection with H. canis and A. platys was the most prevalent (8.3%, P = 0.00001), whilst all animals scored negative for Leishmania spp.. Out of 295 ticks analysed, 215 R. sanguineus s.l. (75.4%) and 8 R. haemaphysaloides (88.9%) were positive for at least one pathogen with H. canis as the predominant species (42.5%), followed by A. platys (33.8%), E. canis (16.9%), B. vogeli (3.8%) and A. phagocytophilum (0.3%). Fifty-six dogs (35.9%) harboured the same pathogen as the respective tick specimens, while 29 dogs (18.6%) had a different pathogen. Thirteen sequence types (STs) were identified for H. canis, with ST2 (49.4%) as the most representative in dogs and ST1 (73.5%) in ticks. Similarly, seven STs were found for Anaplasma spp. (i.e., five for A. platys, one for A. phagocytophilum and one for Anaplasma sp.), with ST2 as the most representative in dogs (70.6%) and ST3 (52.5%) in ticks for A. platys. Only one ST was identified for B. vogeli, B. gibsoni, E. canis, D. sp. "hongkongensis" and B. malayi. Regular surveillance and adoption of adequate treatment and control measures are needed to reduce the risk of disease-causing pathogens in stray dogs and of pathogens with zoonotic potential.

Cutaneous leishmaniasis is a disease caused by various Leishmania spp., which are transmitted by phlebotomine sand flies. Algeria is one of the most affected countries, with thousands of cutaneous leishmaniasis cases registered every year. From March to November of 2016 and 2017, sand flies were collected in 12 municipalities in Setif province, North-Eastern Algeria. Sand flies were identified and females were tested by PCR for detecting Leishmania DNA. Additionally, cutaneous leishmaniasis cases notified during the study period were analysed. Out of 1804 sand flies collected, 1737 were identified as belonging to seven species, with Phlebotomus perniciosus (76.2%), Ph. papatasi (16.7%) and Ph. sergenti (5.0%) being the most common species, representing together 97.9% of the collected specimens. The remaining specimens were identified as Sergentomyia minuta, Se. fallax, Ph. longicuspis and Ph. perfiliewi. The number of sand flies collected monthly was positively correlated with temperature. Out of 804 females tested, nine Ph. perniciosus (1.1%) scored positive for Leishmania infantum (n = 5), L. major (n = 3) and L. tropica (n = 1), respectively. During the study period, 34 cutaneous leishmaniasis cases were notified in Setif, of which 58.8% were patients residing in two urban and peri-urban municipalities and 41.2% in rural areas. The finding of Ph. perniciosus as the most abundant species in Setif suggests that this sand fly may be adapted to different biotopes in the North-East region of Algeria. The detection of different Leishmania spp. in Ph. perniciosus suggests a complex epidemiological picture of cutaneous leishmaniasis in Setif, with the involvement of different etiological agents and possibly with different reservoir hosts and vectors., Author summary Leishmaniasis is a group of neglected diseases, with more than 350 million people at risk and 2 million new cases every year. Leishmania parasites are transmitted by phlebotomine sand flies. Algeria is one of most affected countries, with thousands of cutaneous leishmaniasis cases registered every year. However, in spite of the scientific knowledge gained over the last decades, the understanding of the biology and the ecology of sand flies in some areas of Algeria is still fragmentary. The present study was conducted from 2016 to 2017 to assess the sand fly population in Setif and also the Leishmania spp. circulating in this province. Our data suggest a complex epidemiological picture of cutaneous leishmaniasis in Setif, with the involvement of different etiological agents and possibly with different reservoir hosts and vectors.

Background The brown dog tick Rhipicephalus sanguineus (sensu stricto) is reputed to be the most widespread tick of domestic dogs worldwide and has also been implicated in the transmission of many pathogens to dogs and humans. For more than two centuries, Rh. sanguineus (s.s.) was regarded as a single taxon, even considering its poor original description and the inexistence of a type specimen. However, genetic and crossbreeding experiments have indicated the existence of at least two distinct taxa within this name: the so-called “temperate” and “tropical” lineages of Rh. sanguineus (sensu lato). Recent genetic studies have also demonstrated the existence of additional lineages of Rh. sanguineus (s.l.) in Europe and Asia. Herein, we assessed the biological compatibility between two lineages of Rh. sanguineus (s.l.) found in southern Europe, namely Rhipicephalus sp. I (from Italy) and Rhipicephalus sp. II (from Portugal). Methods Ticks morphologically identified as Rh. sanguineus (s.l.) were collected in southern Portugal and southern Italy. Tick colonies were established and crossbreeding experiments conducted. Morphological, biological and genetic analyses were conducted. Results Crossbreeding experiments confirmed that ticks from the two studied lineages were able to mate and generate fertile hybrids. Hybrid adult ticks always presented the same genotype of the mother, confirming maternal inheritance of mtDNA. However, larvae and nymphs originated from Rhipicephalus sp. I females presented mtDNA genotype of either Rhipicephalus sp. I or Rhipicephalus sp. II, suggesting the occurrence of paternal inheritance or mitochondrial heteroplasmy. While biologically compatible, these lineages are distinct genetically and phenotypically. Conclusions The temperate lineages of Rh. sanguineus (s.l.) studied herein are biologically compatible and genetic data obtained from both pure and hybrid lines indicate the occurrence of paternal inheritance or mitochondrial heteroplasmy. This study opens new research avenues and raises question regarding the usefulness of genetic data and crossbreeding experiments as criteria for the definition of cryptic species in ticks. Electronic supplementary material The online version of this article (10.1186/s13071-018-2941-2) contains supplementary material, which is available to authorized users.

The ocular onchocercosis is caused by the zoonotic parasite Onchocerca lupi (Spirurida: Onchocercidae). A major hindrance to scientific progress is the absence of a reliable diagnostic test in affected individuals. Microscopic examination of skin snip sediments and the identification of adults embedded in ocular nodules are seldom performed and labour-intensive. A quantitative real-time PCR (qPCR) assay was herein standardized for the detection of O. lupi DNA and the results compared with microscopic examination and conventional PCR (cPCR). The specificity of qPCR and cPCR was assessed by processing the most common filarial nematodes infecting dogs, skin samples from O. lupi infected (n = 35 dogs) or uninfected animals (n = 21 dogs; n = 152 cats) and specimens of potential insect vector (n = 93 blackflies; n = 59 mosquitoes/midges). The analytical sensitivity of both assays was assessed using 10-fold serial dilutions of DNA from adult specimen and from a pool of microfilariae. The qPCR on skin samples revealed an analytical specificity of 100% and a sensitivity up to 8 x 10−1 fg/2μl O. lupi adult-DNA and up to 3.6 x 10−1 pg/2μl of mfs-DNA (corresponding to 1 x 10−2 mfs/2μl). Only 9.5% O. lupi-infected skin samples were positive for cPCR with a sensitivity of 8 x 10−1 pg/2μl of DNA. Out of 152 blackflies and mosquitoes/midges, eight specimens experimentally infected (n = 1 S. erythrocephalum; n = 1 S. ornatum; n = 6 Simulium sp.) were positive by qPCR. The qPCR assay herein standardized represents an important step forward in the diagnosis of zoonotic onchocercosis caused by O. lupi, especially for the detection and quantification of low number of mfs. This assay provides a fundamental contribution for the establishment of surveillance strategies aiming at assessing the presence of O. lupi in carnivores and in insect species acting as potential intermediate hosts. The O. lupi qPCR assay will enable disease progress monitoring as well as the diagnosis of apparently clinical healthy dogs and cats., Author summary The diagnosis of zoonotic ocular onchocercosis caused by Onchocerca lupi (Spirurida: Onchocercidae) is currently based on microscopic examination of skin snip sediments and on the identification of adults embedded in ocular nodules. These methods are labour-intensive and require multiple steps to achieve the diagnosis. In this context, a novel quantitative real-time PCR assay (qPCR) has been herein standardized and analytical specificity and sensitivity assessed. The results indicate that the qPCR assay could represent an important step forward in the diagnosis of onchocercosis, in carnivores and in insect species acting as potential intermediate hosts.

Phlebotomine sand flies (Diptera, Psychodidae) are vectors of Leishmania spp., among which Leishmania infantum is recognized as the main agent of human and canine leishmaniosis (CanL) in the Mediterranean area. In this study, females of Phlebotomus spp. (P. perniciosus, P. neglectus and P. papatasi) and Sergentomyia minuta were collected in a dog shelter of southern Italy, where CanL is endemic, and examined for Leishmania DNA. In total, 32 out of 56 of Phlebotomus spp. insects (57.1%) were found positive for L. infantum DNA by quantitative PCR (qPCR), with a mean parasite load of 1.9 × 103 promastigotes/ml among 23 positive P. perniciosus and 2.1 × 103 promastigotes/ml among five positive P. neglectus. Four P. papatasi, a species known to be refractory to L. infantum development, were also found positive. Among 216 S. minuta specimens examined, 25 (11.6%) scored positive for Leishmania tarentolae by conventional nested PCR; two (16.7%) of them were also positive for lizard blood, which is in agreement with the feeding preference of this phlebotomine species. Nine S. minuta (4.2%) were positive for L. infantum by qPCR, with a mean parasite load of 1.62 × 102 promastigotes/ml. The detection of L. infantum DNA in S. minuta may suggest that this species could acquire the protozoan, occasionally feeding on infected dogs. Further investigations need to clarify the potential role that S. minuta may have in the transmission of L. infantum to receptive mammal hosts.

This study was designed to evaluate the efficacy and safety of a novel free polyaggregated amphotericin B (FPA) formulation used to treat experimental canine leishmaniosis (CanL) caused by Leishmania infantum. Eight healthy beagles were intravenously challenged with 5×107 promastigotes per mL of L. infantum. One year after infection, they received an intravenous dose of FPA (5 mg/kg) every 2 weeks three times. Dogs were assessed monthly for clinical signs, serology, and parasite detection during a follow-up period of 6 months. Transient adverse effects (i.e., hypotension, diarrhea, bodyweight loss, fever, and asthenia) were observed within 24–48 hours after treatment in 4 animals. In three dogs mean clinical signs scores were reduced. Antibody titers measured by immunofluorescence antibody test (IFAT) had significantly diminished at the end of the study, although according to bone marrow smears and cultures a high percentage of dogs tested positive for the parasite at 6 months posttreatment (PT6). Real-time quantitative PCR (rtQ-PCR) on blood, bone marrow, and urine samples revealed the presence of parasitic DNA in all animals at PT6, although blood loads of the parasite were reduced. These findings indicate that FPA at the dosing regimen used did not achieve clinical or parasitological cure in dogs experimentally infected with L. infantum.

Background Vector-borne diseases (VBDs) have been increasingly reported in dogs and cats worldwide. However, no data are currently available regarding canine and feline VBDs in Qatar and limited information is available from other Persian Gulf countries. Methods Blood samples from 98 client-owned animals (i.e. 64 dogs and 34 cats) living in Doha (Qatar) were collected and the presence of genomic DNA of Anaplasma spp., Babesia spp., Dirofilaria spp., Ehrlichia spp., Hepatozoon spp., Mycoplasma spp. and Rickettsia spp. was assessed by polymerase chain reaction (PCR), real time-PCR (rt-PCR) and sequence analysis. Results Of the 64 dogs, 12 (18.8%) were infected with at least one pathogen (i.e. 7.8% with Mycoplasma spp., 4.7% with Babesia vogeli, 3.1% with Ehrlichia canis, and 1.6% with Anaplasma platys, Babesia gibsoni and Hepatozoon canis, each). One of the 12 dogs was co-infected with B. vogeli and E. canis. Of the 34 cats, seven (20.6%) animals were infected with at least one pathogen (i.e. 5.9% were positive for Mycoplasma spp., and 2.9% for Babesia felis, B. vogeli, E. canis, “Candidatus Mycoplasma haemominutum” and Mycoplasma haemofelis, each). No dogs or cats were positive for Dirofilaria spp. or Rickettsia spp. Conclusions Although the sample sizes of dogs and cats herein analysed was moderately small, data from this study report the occurrence of A. platys, B. vogeli, B. gibsoni, E. canis, H. canis and Mycoplasma spp. in domestic dogs and of B. felis, B. vogeli, “Candidatus M. haemominutum”, E. canis and M. haemofelis in domestic cats from Qatar. Further investigations along with prophylactic measures are strongly recommended in order to reduce the risk of dogs and cats acquiring VBDs in Qatar.

Babesia spp. are globally distributed tick-borne protozoan parasites that infect the red blood cells of a wide range of vertebrate hosts, including humans. Diagnosis of babesiosis is often impeded by the transient presence of the parasites in peripheral blood, as well as by their pleomorphic nature. Given the reports of an expanding and, in some cases, sympatric geographical distribution of Babesia canis and Babesia vogeli in dogs and associated vectors, in Europe, the development of time-efficient and cost-effective diagnostic tools to detect and differentiate these two species is warranted. In this study, we designed and developed a novel polymerase chain reaction (PCR) assay targeting the parasite cytochrome c oxidase subunit 1 (cox1) gene, for the simultaneous detection and differentiation of B. canis and B. vogeli. The analytical sensitivity of the PCR was evaluated using serial dilutions of genomic DNA extracted from individual and artificially mixed canine blood samples infected by B. canis (3×102 infected erythrocytes/ml, ie/ml) and B. vogeli (2.1×101 ie/ml). The analytical specificity of the assay was assessed using blood samples positive for Hepatozoon canis, Ehrlichia canis, Anaplasma platys, Babesia microti, Babesia rossi and Theileria annae (syn. Babesia vulpes). The clinical specificity of the PCR assay was evaluated on 147 blood samples from dogs and 128 tick specimens (Dermacentor reticulatus and Rhipicephalus sanguineus sensu lato). Species-specific bands of the expected sizes (i.e., 750bp for B. canis and 450bp for B. vogeli), and two bands in the mixed blood samples were obtained. The PCR assay developed herein detected a low number of infected erythrocytes (i.e., 3×10-2B. canis, 2.1×10-2B. vogeli ie/ml). Of the 147 blood samples, nine (6.1%) were positive for B. canis and six (4.1%) for B. vogeli, whereas only one tick (D. reticulatus) was positive for B. canis. This PCR assay represents a rapid and reliable tool for the diagnosis of B. canis and B. vogeli in areas where these species occur in sympatry, and will assist future epidemiological investigations aimed to track the expanding range of these parasites.

Background The taxonomic status of the brown dog tick Rhipicephalus sanguineus (sensu stricto) is a subject of on-going debate; there is a consensus that populations of this tick species should be referred to as R. sanguineus (sensu lato) until its taxonomic status is resolved. Recent genetic studies revealed the existence of more than one lineage of R. sanguineus (s.l.) in temperate countries. In this study, we assessed the genetic identity of ticks collected from rural dogs living in several areas located in all major geographical regions of Portugal. Methods A total of 347 ticks were collected from rural dogs living in different regions of Portugal. These ticks were morphologically identified and partial mitochondrial 16S rRNA gene sequences (~300 bp) were obtained from representative specimens. Results The ticks were morphologically identified as Ixodes ricinus (seven males and 27 females), Rhipicephalus bursa (one male), Rhipicephalus pusillus (one female) and R. sanguineus (s.l.) (two larvae, 101 nymphs, 108 males and 100 females). Partial mitochondrial 16S rRNA gene sequences were obtained from 58 R. sanguineus (s.l.) specimens, and all of them were genetically identified as belonging to the so-called temperate lineage of R. sanguineus (s.l.) Conclusions These results strongly suggest that the temperate species of R. sanguineus (s.l.) is the only representative of this tick group found on dogs in Portugal. It also adds weight to the hypothesis that Rhipicephalus turanicus is not present in this country, although further investigations are necessary to confirm this. Electronic supplementary material The online version of this article (doi:10.1186/s13071-017-2072-1) contains supplementary material, which is available to authorized users.

Three species of Hepatozoon, namely, Hepatozoon felis, Hepatozoon canis and Hepatozoon silvestris may affect domestic and/or wild felids. Although hepatozoonosis has been documented in a wide range of mammal species, data on cats are limited. To investigate the occurrence of these pathogens in cats, blood samples were collected from animals living in three provinces of southern Italy (Bari, Lecce, and Matera), and molecularly analysed by PCR amplification and sequencing of segments of the 18S rRNA gene. Out of 196 blood samples collected, Hepatozoon spp. DNA was amplified in ten cats (5.1%, CI: 3%-9%), with the majority of infected animals from Matera (8/34, 23.5%) and one each from the other two provinces. BLAST analysis revealed the highest nucleotide identity with sequences of H. canis, H. felis and H. silvestris deposited in GenBank. Results of this study indicate that these three species of Hepatozoon infect domestic cats in Italy. This is the first report of H. silvestris infection in a domestic cat.

Equids can be infected by a range of skin-dwelling filarial nematodes, including four species of the genus Onchocerca. Current literature on equine onchocercosis is fragmentary and often limited to isolated case reports. The present study aimed to describe a clinical case of equine onchocercosis caused by Onchocerca boehmi (Supperer, 1953) (syn. Elaeophora boehmi) in an 8-year-old gelding Belgian show jumper from northern Italy. The horse was presented with a firm and painless mass on the proximal third of the right metacarpal region. Ultrasound examination showed a peritendinous enlargement around the palmaro-lateral area of the tendons, characterized by an elongated hypoechoic and well-defined structure, embedding a coiled hyperechoic line. The metacarpal nodule was resected and histologically examined. Fragments of a parasitic nematode were detected, isolated and examined. The morphological analysis allowed identifying the nematode as O. boehmi. In addition, total genomic DNA was extracted from individual fragments using a commercial kit for the nematode identification and a comparative sequence analysis of the nematode cytochrome oxidase subunit 1 (cox1) sequence with data available in the GenBankTM database revealed the closest identity (i.e. 91 %) with that of Onchocerca lupi. Thus far, O. boehmi has only been reported in Austria and Iran, and information about its life-cycle and vectors is lacking. The systematic position of this species within the genus Onchocerca, not in Elaeophora where it was originally described, is in concordance with the morphological and molecular analysis. In this article, we describe the first autochthonous case of equine onchocercosis in Italy caused by O. boehmi and discuss novel parasitological, clinical, and pathological data on these pathogens of horses.

Despite their medical and veterinary importance, some tick species are so poorly studied, that their role within pathogen vector transmission cycles is difficult to assess. The tick Ixodes ventalloi is one such species, and its biology and phylogenetic status remain an issue of debate. In the present study, specimens of adult I. ventalloi (n = 65 females; n = 31 males) infesting cats in the Lipari Island (Aeolian archipelago, Sicily, southern Italy) were characterized morphologically and molecularly, the latter based on mitochondrial 16S rRNA and cytochrome c oxidase subunit 1 (cox1) genes. In addition, within the I. ventalloi specimens examined, the genetic data and phylogenetic analyses for both mitochondrial genes suggest the existence of two distinct genogroups. The ecological and epidemiological significance of the genetic structure within the I. ventalloi endemic population remains to be determined. The results highlight the need for further analysis of this tick species, including whole mitochondrial genome sequencing and crossbreeding studies, which will be pivotal to complement features of its status as a vector of disease pathogens.

Anaplasma platys is an obligate intracellular, tick-borne pathogen of dogs, which causes canine infectious cyclic thrombocytopenia (CICT). The vector role of Rhipicephalus sanguineus group ticks has been only suggested, but definitive evidence is lacking. This study aimed to detect and quantify A. platys DNA in infected dogs and in their respective ticks through a quantitative real-time PCR assay. From March to May 2009, blood and tick samples from dogs residing in a CICT-endemic area were collected and molecularly analysed. Differences (p0.05) were detected in the bacterial load between tick nymphs (3.5 × 10(-2)± 2.5 × 10(-2)) and adults (female: 1.2 × 10(-1) ± 1.1 × 10(-1); male: 9.3 × 10(-2)± 1.2 × 10(-2)) and between unengorged (1.1 × 10(-1) ± 2.8 × 10(-2)) and partially or fully engorged ticks (1.9 × 10(-1) ± 2.7 × 10(-2)). No difference was found between ticks collected from positive (1.1 × 10(-1)± 0.6 × 10(-1)) and negative (1.2 × 10(-1) ± 0.3 × 10(-1)) dogs (p0.05). The mean bacterial load detected in positive dogs was lower than that in their respective ticks (p0.05). This study provides circumstantial evidence of the putative role of Rhipicephalus sp. I as a vector of this pathogen.

With the exception of Aelurostrongylus abstrusus, feline lungworms have been poorly studied. Information on their distribution is patchy and mostly limited to case reports. In this study, the occurrence of feline lungworms and co-infecting gastrointestinal parasites has been investigated in 12 European countries (i.e. Austria, Belgium, Bulgaria, France, Greece, Hungary, Italy, Portugal, Romania, Spain, Switzerland and the United Kingdom). An average of 10 domestic cats, with regular outdoor access, was sampled each month for 12 months, and freshly passed faeces were collected. Stools were processed using a McMaster assay and a quantitative Baermann-Wetzel method. Animals positive for lungworms and/or gastrointestinal parasites were treated with a formulation containing fipronil, (S)-methoprene, eprinomectin, and praziquantel (Broadline®, Merial), and re-sampled 28 days post-treatment. The association between lungworm infection and risk factors was analysed using statistical medians/means and the efficacy of the treatment against each lungworm species was assessed. Of 1990 cats sampled, 613 (30.8%) were positive for at least one parasite, while 210 (10.6%) were infected by lungworms. The prevalence of lungworm infection varied between the sampled sites, with the highest recorded in Bulgaria (35.8%) and the lowest in Switzerland (0.8%). None of the cats from Austria or the United Kingdom were infected by lungworms. Aelurostrongylus abstrusus was the species most frequently detected (78.1%), followed by Troglostrongylus brevior (19.5%), Eucoleus aerophilus (14.8%) and Oslerus rostratus (3.8%). The overall efficacy of the treatment was 99% for A. abstrusus and 100% for T. brevior, O. rostratus and E. aerophilus. Data presented provide a comprehensive account of the diagnosis, epidemiology and treatment of feline lungworms in Europe, as well as of the occurrence of co-infections by gastrointestinal parasites.