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Abstract Phlebotomine sand flies (Diptera: Phlebotominae) are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). In Central Europe, Phlebotomus mascittii is the predominant species, but largely understudied. To better understand factors driving its current distribution, we infer patterns of genetic diversity by testing for signals of population expansion based on two mitochondrial genes and model current and past climate and habitat suitability for seven post-glacial maximum periods, taking 19 climatic variables into account. Consequently, we elucidate their connections by environmental-geographical network analysis. Most analyzed populations share a main haplotype tracing back to a single glacial maximum refuge area on the Mediterranean coasts of South France, which is supported by network analysis. The rapid range expansion of Ph. mascittii likely started in the early mid-Holocene epoch until today and its spread possibly followed two routes. The first one was through northern France to Germany and then Belgium, and the second across the Ligurian coast through present-day Slovenia to Austria, toward the northern Balkans. Here we present a combined approach to reveal glacial refugia and post-glacial spread of Ph. mascittii and observed discrepancies between the modelled and the current known distribution might reveal yet overlooked populations and potential further spread.

On Linosa Island, Italy, Dirofilaria immitis infection has been hyperendemic in dogs and seroprevalent among islanders. In 2020, we implemented a heartworm disease elimination program on Linosa Island. Of 54 dogs tested for D. immitis antigen and microfilariae, 28 had positive results and received treatment with oral doxycycline twice daily for 4 weeks plus topical imidacloprid/moxidectin monthly for 12 months. The 26 dogs with negative results received monthly topical imidacloprid/moxidectin as preventive. During month 1, the number of microfilaremic dogs was reduced by 76.5%. From month 2 on, all animals were microfilariae negative, and during months 3 to 9, the number of antigen-positive dogs decreased progressively. Treatment of positive dogs coupled with chemoprophylaxis for noninfected dogs was effective, protecting them from new infections. The elimination program reduced the risk for human infection, representing a One Health paradigm. Monitoring and chemoprophylaxis are advocated to maintain the status of heartworm disease–free area.

Hedgehogs are synanthropic mammals, reservoirs of several vector-borne pathogens and hosts of ectoparasites. Arthropod-borne pathogens (i.e., Rickettsia spp., Borrelia spp., and Anaplasmataceae) were molecularly investigated in ectoparasites collected on hedgehogs (n = 213) from Iran (161 Hemiechinus auritus, 5 Erinaceus concolor) and Italy (47 Erinaceus europaeus). In Iran, most animals examined (n = 153; 92.2%) were infested by ticks (Rhipicephalus turanicus, Hyalomma dromedarii), and 7 (4.2%) by fleas (Archeopsylla erinacei, Ctenocephalides felis). Of the hedgehogs infested by arthropods in Italy (i.e., 44.7%), 18 (38.3%) were infested by fleas (Ar. erinacei), 7 (14.9%) by ticks (Haemaphysalis erinacei, Rh. turanicus, Rhipicephalus sanguineus sensu lato), and 6 (12.8%) by mites (Caparinia tripilis, Acarus nidicolous, Ornithonyssus spp.). Phoretic behavior of C. tripilis on Ar. erinacei was detected in two flea specimens from Italy. At the molecular analysis Rickettsia spp. was detected in 93.3% of the fleas of Italy. In Iran, Rickettsia spp. was detected in 8.0% out of 212 Rh. turanicus ticks, and in 85.7% of the Ar. erinacei fleas examined. The 16S rRNA gene for Ehrlichia/Anaplasma spp. was amplified in 4.2% of the 212 Rh. turanicus ticks. All sequences of Rickettsia spp. from fleas presented 100% nucleotide identity with Rickettsia asembonensis, whereas Rickettsia spp. from Rh. turanicus presented 99.84%–100% nucleotide identity with Rickettsia slovaca, except for one sequence, identical to Rickettsia massiliae. The sequences of the 16S rRNA gene revealed 99.57%–100% nucleotide identity with Anaplasma spp., except for one, identical to Ehrlichia spp. A new phoretic association between C. tripilis mites and Ar. erinacei fleas has been herein reported, which could be an important route for the spreading of this mite through hedgehog populations. Additionally, spotted fever group rickettsiae were herein detected in ticks and fleas, and Anaplasma/Ehrlichia spp. in ticks, suggesting that hedgehogs play a role as reservoirs for these vector-borne pathogens.

Abstract Background Onchocerca lupi and Cercopithifilaria spp. are vector-borne filarioids of dogs, which harbour skin microfilariae (mfs), the former being of zoonotic concern. Proper treatment studies using compounds with microfilaricidal activity have not been performed. Therefore, this study aimed to assess the efficacy of a commercially available spot-on formulation containing moxidectin 2.5%/imidacloprid 10% for the treatment of O. lupi or Cercopithifilaria spp. skin-dwelling mfs in naturally infected dogs. Methods Privately owned dogs (n = 393) from southern Portugal were sampled via skin biopsies to identify and count mfs in 20 µl of skin sediment. A total of 22 mfs-positive dogs were allocated to treatment group (n = 11; G1) or left untreated as a control (n = 11; G2). As a pilot investigation to test the treatment efficacy, five dogs assigned to G1 were treated four times at monthly intervals with moxidectin 2.5%/imidacloprid 10% spot-on formulation on SDs 0, 28 (± 2), 56 (± 2), and 84 (± 2). Based on the negative results for both O. lupi and/or Cercopithifilaria spp. mfs of dogs in the pilot study from SD28 onwards, the remaining six dogs in G1 were treated at SD0 and assessed only at SD28. Results Of the 393 animals sampled, 78 (19.8%) scored positive for skin-dwelling mfs. At the pilot investigation, a mean number of 19.6 mfs for O. lupi was recorded among five infected dogs whereas no mfs were detected at SD28. At SD0, the mean number of Cercopithifilaria spp. larvae was 12.6 for G1 and 8.7 for G2. The mean number of mfs for G2 was 20.09. Conclusions Results herein obtained suggest that a single treatment with moxidectin 2.5%/imidacloprid 10% spot-on formulation is efficacious against skin-dwelling mfs in dogs. The microfilaricidal effect of moxidectin could also be useful in reducing the risk of O. lupi infection for humans.

Species of Ixodiphagus (Hymenoptera: Encyrtidae) are parasitoid wasps whose immature forms develop inside ixodid and argasid ticks (Acari: Ixodida). Following oviposition by adult female wasps into the idiosoma of ticks, larvae hatch and start feeding on their internal contents, eventually emerging as adult wasps from the body of the dead ticks. Species of Ixodiphagus have been reported as parasitoids of 21 species of ticks distributed across 7 genera. There are at least ten species described in the genus, with Ixodiphagus hookeri being the most studied as an agent for biological control of ticks. Although attempts of tick control by means of this parasitoid largely failed, in a small-scale study 150,000 specimens of I. hookeri were released over a 1-year period in a pasture where a small cattle population was kept, resulting in an overall reduction in the number of Amblyomma variegatum ticks per animal. This review discusses current scientific information about Ixodiphagus spp., focusing on the role of this parasitoid in the control of ticks. The interactions between these wasps and the ticks’ population are also discussed, focusing on the many biological and logistical challenges, with limitations of this control method for reducing tick populations under natural conditions.

The proper diagnosis of gastrointestinal parasites of small ruminants requires the development of multivalent techniques characterized by high sensitivity, specificity, precision, reproducibility, and the ability to quickly detect and monitor infections that pose risks to animal health. The aim of this study was to evaluate the performance of Mini-FLOTAC technique and McMaster egg counting (gold standard) in the detection of gastrointestinal parasites of goats and sheep. A total of 789 fecal samples were analyzed (401 of sheep and 388 of goats). From those, nematode eggs were detected in 80.86% (638/789), being 72.57% (291/401) from sheep and 89.43% (347/388) from goats. The Mini-FLOTAC technique presented a better performance to detect helminth eggs in both goats (88.40%; 343/388) and sheep (71.57%; 287/401) (?2 = 10.358; p < 0.0001). Kappa analysis revealed a weak concordance between techniques for goats (k = 0.342; p < 0.001) and sheep (k = 0.281; p < 0.001). Data herein reported suggests that the Mini-FLOTAC is a technique more sensitive than the McMaster egg counting, therefore its use might be adopted for the diagnosis of gastrointestinal infection in small ruminants.

Abstract Volatile organic compounds (VOCs) are chemicals emitted as products of cell metabolism, which reflects the physiological and pathological conditions of any living organisms. These compounds play a key role as olfactory cues for arthropod vectors such as mosquitoes, sand flies, and ticks, which act in the transmission of pathogens to many animal species, including humans. Some VOCs may influence arthropod behaviour, e.g., host preference and oviposition site selection for gravid females. Furthermore, deadly vector-borne pathogens such as Plasmodium falciparum and Leishmania infantum are suggested to manipulate the VOCs profile of the host to make them more attractive to mosquitoes and sand fly vectors, respectively. Under the above circumstances, studies on these compounds have demonstrated their potential usefulness for investigating the behavioural response of mosquitoes, sand flies, and ticks toward their vertebrate hosts, as well as potential tools for diagnosis of vector-borne diseases (VBDs). Herein, we provide an account for scientific data available on VOCs to study the host seeking behaviour of arthropod vectors, and their usefulness as attractants, repellents, or tools for an early diagnosis of VBDs. Graphical Abstract

Abstract ParSCo (Parasitology Summer Course) is an intense, 1-week-long summer course organized by the Parasitology Unit of the Department of Veterinary Medicine, University of Bari, Italy, with the support of the World Association for the Advancement of Veterinary Parasitology (WAAVP), the European Veterinary Parasitology College (EVPC) and Parasites and Vectors. The course, which is conducted in southern Italy, is planned for parasitologists and post-graduate students working in the field of parasitology. The course consists of theoretical and practical lessons, which include the collection, identification and diagnosis of parasites of pets, livestock and wildlife. The participants in ParSCo are afforded the opportunity to be involved in clinical examination and sample collection for the diagnosis of parasitic diseases (e.g. leishmaniosis, thelaziosis and many tick-borne diseases) present in the Mediterranean Basin. The course is conducted at Casa di Caccia, a hunting lodge situated in the Gallipoli Cognato Forest near the Basento River in the Basilicata region in southern Italy. In addition to the training purpose, ParSCo is a great opportunity for sharing knowledge and expertise while becoming part of the parasitology community in a pleasant environment. In this editorial, we share some information and celebrate 10 years of ParSCo, looking forward to forthcoming sessions of this unique parasitology summer course. Graphical Abstract

The eyeworms of the subgenus Thelaziella infect orbital cavities, conjunctival sacs and lachrymal ducts of many wild birds, being able to cause conjunctivitis. In Brazil, at least 10 species of Thelaziella have been described. Here we present a brief review of cases of Thelazia (Thelaziella) aquilina and describe the infection of this parasite in Harpia harpyja, as a new host record. Two nematodes were collected from the surface of the cornea of an adult female H. harpyja in the Amazon region (Brazil) and were morphologically identified as two males of T. (T.) aquilina. This data demonstrated the need for further studies focused on elucidating the transmission, biological cycle, and phylogenetic positioning of Thelaziella as subgenus.

The raccoon Procyon lotor (Carnivora: Procyonidae) is an invasive species of growing importance for the introduction of alien pathogens or as additional hosts for autochthonous pathogens in Europe, including zoonotic parasites. As the population is steadily increasing and outcompeting the red fox (Vulpes vulpes) in Germany, the consumption of raccoon meat raises concerns about pathogens they may transmit. Therefore the presence of Trichinella larvae was here investigated in muscle samples (n = 904) of raccoons from northern Germany. No Trichinella larvae were found, thus confirming the general low occurrence of this parasite in Germany. However, Spirocerca lupi (n = 12) and an unidentified Trichinella-like nematode (n = 1) were accidently detected in the examined samples. The first is not a zoonotic parasite but has a high veterinary relevance as it can cause severe diseases in dogs. It is the first documented autochthonous infection of this nematode in Germany. The larvae of an unidentified Trichinella-like nematode were found in high abundance in all examined muscles of one raccoon, though they could not be identified to species level. Histological investigation revealed intramuscular cystic structures. This is the largest study investigating muscular parasites of raccoons in Europe so far, which suggests that this invasive animal species is infected by S. lupi and by a yet unknown Trichinella -like parasite.

Abstract Filarioids of the genus Cercopithifilaria are little studied, yet widespread parasites, that are relatively unique in being one of the very few nematodes transmitted by hard ticks. These filarioids live in the subcutis while microfilariae are found in the dermis. Definitive hosts include domestic dogs as well as a wide range of vertebrates, such as ruminants, non-human primates, murids, marsupials, porcupines, viverrids, bears and lagomorphs. The genus Cercopithifilaria contains three taxa (i.e. C. bainae, C. grassii and a yet undescribed species, namely Cercopithifilaria sp. II) that are known to infect dogs worldwide, with their occurrence overlapping the distribution of the main tick vector, Rhipicephalus sanguineus sensu lato. In recent decades, more attention has focused on these filarioids since they have been associated with clinical signs of infection, such as dermatitis, chronic polyarthritis and cutaneous cysts, and possibly with facilitating infections caused by other tick-borne pathogens. Nevertheless, these parasites remain largely underdiagnosed in clinical practice due to the lack of awareness of veterinary practitioners and to major obstacles to their diagnosis. In this review, we have assessed currently available data on Cercopithifilaria spp. infecting dogs worldwide and discussed the biological, clinical and epidemiological aspects of these filarioids, with the overall aim to gain a better understanding of their potential role in skin diseases. Graphical abstract

Abstract The zoonotic nematode Thelazia callipaeda infects the eyes of domestic and wild animals and uses canids as primary hosts. It was originally described in Asia, but in the last 20 years it has been reported in many European countries, where it is mainly transmitted by the drosophilid fruit fly Phortica variegata. We report the autochthonous occurrence of T. callipaeda and its vector P. variegata in Austria. Nematodes were collected from clinical cases and fruit flies were caught using traps, netting, and from the conjunctival sac of one dog. Fruit flies and nematodes were morphologically identified and a section of the mitochondrial cytochrome c oxidase subunit I gene (COI) was analysed. A DNA haplotype network was calculated to visualize the relation of the obtained COI sequences to published sequences. Additionally, Phortica spp. were screened for the presence of DNA of T. callipaeda by polymerase chain reaction. Thelazia callipaeda and P. variegata were identified in Burgenland, Lower Austria, and Styria. Thelazia callipaeda was also documented in Vienna and P. variegata in Upper Austria and South Tyrol, Italy. All T. callipaeda corresponded to haplotype 1. Twenty-two different haplotypes of P. variegata were identified in the fruit flies. One sequence was distinctly different from those of Phortica variegata and was more closely related to those of Phortica chi and Phortica okadai. Thelazia callipaeda could not be detected in any of the Phortica specimens. Graphical Abstract

Abstract Background Sergentomyia minuta (Diptera: Phlebotominae) is an abundant sand fly species in the Mediterranean basin and a proven vector of reptile parasite Leishmania (Sauroleishmania) tarentolae. Although it feeds preferentially on reptiles, blood meal analyses and detection of Leishmania (Leishmania) infantum DNA in wild-caught S. minuta suggest that occasional feeding may occur on mammals, including humans. Therefore, it is currently suspected as a potential vector of human pathogens. Methods A recently established S. minuta colony was allowed to feed on three reptile species (i.e. lizard Podarcis siculus and geckos Tarentola mauritanica and Hemidactylus turcicus) and three mammal species (i.e. mouse, rabbit and human). Sand fly mortality and fecundity were studied in blood-fed females, and the results were compared with Phlebotomus papatasi, vector of Leishmania (L.) major. Blood meal volumes were measured by haemoglobinometry. Results Sergentomyia minuta fed readily on three reptile species tested, neglected the mouse and the rabbit but took a blood meal on human. However, the percentage of females engorged on human volunteer was low in cage (3%) and feeding on human blood resulted in extended defecation times, higher post-feeding mortality and lower fecundity. The average volumes of blood ingested by females fed on human and gecko were 0.97 µl and 1.02 µl, respectively. Phlebotomus papatasi females readily fed on mouse, rabbit and human volunteer; a lower percentage of females (23%) took blood meal on the T. mauritanica gecko; reptilian blood increased mortality post-feeding but did not affect P. papatasi fecundity. Conclusions Anthropophilic behaviour of S. minuta was experimentally demonstrated; although sand fly females prefer reptiles as hosts, they were attracted to the human volunteer and took a relatively high volume of blood. Their feeding times were longer than in sand fly species regularly feeding on mammals and their physiological parameters suggest that S. minuta is not adapted well for digestion of mammalian blood. Nevertheless, the ability to bite humans highlights the necessity of further studies on S. minuta vector competence to elucidate its potential role in circulation of Leishmania and phleboviruses pathogenic to humans. Graphical abstract

Abstract Background The Asian elephant (Elephas maximus), which is an endangered species, harbors several parasites. Among the ectoparasites that it harbors, ear mites of the genus Loxanoetus have the potential to cause external otitis, an inflammation that may also be associated with the presence of other microorganisms. We assessed the relationships between ear mites, nematodes, yeast, bacterial rods, and cocci sampled from the ears of captive Asian elephants in Thailand. In addition, we discuss the possibility that dust-bathing behavior may be triggered by ear mite infestation, and that this in turn may lead to contamination of the ears with soil microorganisms. Methods Legally owned captive Asian elephants (n = 64) were sampled. Ear swabs were individually collected from both ears and microscopically examined for the presence of mites, nematodes, yeast, bacterial rods, cocci, and host cells. Mites and nematodes were identified to species level using morphological and molecular methods. Results Loxanoetus lenae mites were present in 43.8% (n = 28/64) of the animals (19 animals with mites in one ear and nine animals with mites in both ears). Nematodes of the genus Panagrolaimus were detected in 23.4% (n = 15/64) of the animals (10 with nematodes in one ear and five with nematodes in both ears). In adult elephants (Fisher’s exact test, P = 0.0278) and female elephants (Fisher’s exact test, P = 0.0107), the presence of nematodes in both ears was significantly associated with the presence of mites. In addition, higher categorical burdens of nematodes were also significantly associated with the presence of mites (Fisher’s exact test, P = 0.0234) and epithelial cells (Fisher’s exact test, P = 0.0108), and marginally significantly associated with bacterial cocci (Fisher’s exact test, P = 0.0499). Conclusions The presence of L. lenae mites in the ear canals of the Asian elephants was significantly associated with the occurrence of other microorganisms, such as soil nematodes, bacteria and yeasts. The presence of mites in their ears may increase the dust-bathing behavior of elephants which, if confirmed, represents a further paradigmatic example of a parasitic infestation affecting animal behavior. Graphical Abstract

Thelazia callipaeda is a zoonotic vector-borne nematode that infects and causes eye disease among a wide range of domestic and wild mammals, including humans. We describe an unusual case of reinfection by this nematode in Serbia and call for a focus on preventive measures in endemic areas.

Abstract Background For a long time known as the oriental eyeworm, Thelazia callipaeda is a zoonotic nematode that infects the eyes of a wide range of vertebrate hosts including dogs, cats, wildlife carnivores, lagomorphs, and humans. The high occurrence of this infection in Europe and the first cases in the United States have increased scientific interest in the parasite, as it also represents a risk for people living in endemic areas. Therefore, treatment and prevention of thelaziosis in canine population are advocated to reduce the risk of human infection as well. Here, we assessed the efficacy of a formulation containing sarolaner/moxidectin/pyrantel (Simparica Trio®) administered orally at monthly intervals, for the prevention of establishment of infection with T. callipaeda in naturally infected dogs. In this formulation, moxidectin is expected to have efficacy against eyeworms, whereas sarolaner and pyrantel are not. Methods The study was conducted in eyeworm endemic areas of Italy and France, where dogs (n = 125) were assigned into two groups consisting of a negative control group (G1; n = 62), in which animals were treated monthly with a control product (sarolaner; Simparica®), and a treatment group (G2; n = 63) in which animals were treated monthly with Simparica Trio (sarolaner/moxidectin/pyrantel) from day 0 to day 150. In total, nine animals were withdrawn from the study (two animals became positive at day 30, and seven for reasons unrelated to eyeworm infection), resulting in 116 animals (n = 58 for G1; n = 58 for G2). Results In G1, 16 out of 58 animals (27.6%) were observed with eyeworms during the study, and none of the animals from G2 were ever observed with eyeworms, resulting in 100% efficacy (P

During 2021, we collected blood and serum samples from 135 persons exposed to tick bites in southern Italy. We serologically and molecularly screened for zoonotic tickborne pathogens and only molecularly screened for Candidatus Midichloria mitochondrii. Overall, 62 (45.9%) persons tested positive for tickborne pathogens. Coxiella burnetii was detected most frequently (27.4%), along with Rickettsia spp. (21.5%) and Borrelia spp. (10.4%). We detected Candidatus M. mitochondrii DNA in 46 (34.1%) participants who had statistically significant associations to tickborne pathogens (p

Abstract Background Some species of drosophilid flies belonging to the genus Phortica feed on ocular secretions of mammals, acting as biological vectors of the zoonotic eyeworm Thelazia callipaeda. This study describes an effective breeding protocol of Phortica variegata and Phortica oldenbergi in insectary conditions. Methods Alive gravid flies of P. oldenbergi, P. variegata and Phortica semivirgo were field collected in wooded areas of Lazio region (Italy) and allowed to oviposit singularly to obtain isofamilies. Flies were maintained in ovipots (200 ml) with a plaster-covered bottom to maintain high humidity level inside. Adult feeding was guaranteed by fresh apples and a liquid dietary supplement containing sodium chloride and mucin proteins, while larval development was obtained by Drosophila-like agar feeding medium. The breeding performances of two media were compared: a standard one based on cornmeal flour and an enriched medium based on chestnut flour. All conditions were kept in a climatic chamber with a photoperiod of 14:10 h light:dark, 26 ± 2 °C and 80 ± 10% RH. Results From a total of 130 field-collected Phortica spp., three generations (i.e. F1 = 783, F2 = 109, F3 = 6) were obtained. Phortica oldenbergi was the species with highest breeding performance, being the only species reaching F3. Chestnut-based feeding medium allowed higher adult production and survival probability in both P. oldenbergi and P. variegata. Adult production/female was promising in both species (P. oldenbergi: 13.5 F1/f; P. variegata: 4.5 F1/f). Conclusions This standardized breeding protocol, based on controlled climatic parameters and fly densities, together with the introduction of an enriched chestnut-based feeding medium, allowed to investigate aspects of life history traits of Phortica spp. involved in the transmission of T. callipaeda. Obtaining F3 generation of these species for the first time paved the road for the establishment of stable colonies, an essential requirement for future studies on these vectors in controlled conditions. Graphical abstract

Thelazia callipaeda is a zoonotic parasite causing ocular disease in domestic dogs, cats, several wild carnivores, hares, and humans. This nematode is widely distributed in Europe, where it is transmitted by the drosophilid fly Phortica variegata. Since the first report of infection in grey wolves (Canis lupus) from southern Italy, other cases of thelaziosis have been recorded in this animal species throughout Europe, raising questions about their role in spreading T. callipaeda. Indeed, for their wandering behavior through long distances and living in woody areas where the vectors thrive, wolves may act as reservoirs and spreaders of thelaziosis. In this study we reviewed the literature about wolves acting as reservoirs of T. callipaeda in Europe. In addition, we report the first detection of T. callipaeda eyeworms in grey wolves in the Italian Alps, discussing its possible implications in the epidemiology of thelaziosis in the Alpine landscape. Animals (n = 3) included in this study were originated from the Italian Alps, one juvenile male wolf was found dead, and the other two were seven-year-old males translocated from Piedmont region to a Zoological Garden, in Tuscany. All animals were infected with eyeworms, which were morphologically and molecularly identified as T. callipaeda. Data herein presented confirm those available in the literature about the circulation of a unique cox1 haplotype in Europe. In addition, the report of T. callipaeda in wolves from the Alps suggests an ecological continuity of habitats which are suitable for the distribution of T. callipaeda from the southern to northern Italy through the Apennine backbone. Retrospectively, it could also explain the spreading of the oriental eyeworm infection in Europe over the last 20 years with many wild carnivores, such as foxes and possibly wolves, playing a pivotal role as reservoirs of the infection for dogs, cats and humans.

Abstract Background Dirofilaria immitis and Dirofilaria repens are vector-borne zoonotic parasites which affect mainly dogs and humans worldwide. In Iran, information about the distribution of those nematodes is scant in several regions. Therefore, we investigated the prevalence of these filarial parasites in stray dogs from five Iranian provinces where no information about these parasites is available. Methods Blood samples were collected from 344 stray dogs in five provinces of Iran (i.e. Mazandaran, Gilan, Esfahan, Qazvin and Loresan). The presence of microfilariae was assessed using direct smear, modified Knott’s test, molecular detection of filarial DNA (cox1 gene) and Wolbachia endosymbiont of parasitic nematodes (ftsZ gene) by conventional PCR (cPCR). All of the PCR products were sequenced and phylogenetic analysis was performed. Results In total, 75 dogs (21.8%) were found to be positive for D. immitis by cPCR. Infection was detected in all provinces, with the highest prevalence in Gilan province (22/28; 78.6%). Acanthocheilonema reconditum was diagnosed in five dogs (1.4%) from three provinces (i.e. Esfahan, Mazandaran, Gilan). Two dogs were infected with both parasites and three were only infected with A. reconditum. Dirofilaria repens infection was not found in the examined population. Representative sequences of the D. immitis cox1 gene from dogs from the northern provinces (Mazandaran, Gilan, Qazvin) were grouped together and distinctly separate from the ones from western and central provinces (Lorestan and Esfahan), suggesting that different nematode populations are present in the country. Conclusion The data reported herein fill existing gaps in knowledge about canine filarial infection in two Iranian provinces and record the highest prevalence of D. immitis ever reported in the country (i.e. 78.6%). A geographical review of the literature about Dirofilaria spp. and A. reconditum infections in dogs and humans has also been summarized, indicating that D. immitis and D. repens are distributed in 22 of 31 provinces in Iran, whereas A. reconditum is present in fewer regions. Effective control strategies are advocated for owned dogs, and a national program for the management of stray dogs is needed to minimize the risk of infection in animals and humans. Graphical Abstract

Abstract Background Due to the complexity of retrieving skin-dwelling microfilariae, filarioids of dogs presenting dermal microfilariae (e.g. Cercopithifilaria spp., Onchocerca lupi) are relatively unknown compared to Dirofilaria spp. and Acanthocheilonema spp. whose microfilariae circulate in the blood. Although Cercopithifilaria spp. and O. lupi filarioids are distributed worldwide, there is a paucity of information on their occurrence in Iran. The aim of this study was to investigate these filarioids in a large population of dogs from different regions of Iran. Methods From October 2018 to September 2020, skin biopsies were obtained from dogs housed in shelters (n = 557) and privately owned dogs (n = 26) in seven provinces of Iran (Hamedan, Kermanshah, Yazd, Mazandaran, Khuzestan, Lorestan, Esfahan), as well as from three road-killed jackals (Canis aureus) and three cats (Felis catus) in Hamedan province. The skin biopsies were first soaked in saline solution at room temperature overnight, and examined for dermal microfilariae under the microscope. Positive skin specimens and sediments were tested by PCR for a 304-bp region of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and amplicons were sequenced. Results Microfilariae of Cercopithifilaria spp. were found in skin biopsies of 32 of the 583 (5.5%) dogs tested, with infection rates of up to 25% in Kermanshah. No microfilariae were recovered from skin biopsy samples collected from dogs in Khorramabad and Ahvaz, nor from the examined jackals and cats. None of the privately owned dogs were found to be infected. Morphologic and morphometric characteristics of the microfilariae were consistent with C. bainae. Eighteen skin samples were positive for the cox1 gene, of which 15 sequences showed a nucleotide identity of 100% and three of 93.4% with the reference sequence of C. bainae available in GenBank (haplotype I; GenBank accession number: JF461457). Conclusions The data from this study broadens current knowledge on the geographical distribution of C. bainae in dogs in Middle Eastern countries. Further studies on different wild canine species in the country (e.g. jackal, fox, wolf) could provide further information on the epidemiology of these filarioids. A particular focus should be put on zoonotic O. lupi given the reports of its presence in human patients from this country. Graphical Abstract

Abstract Background Canine vector-borne diseases (CVBDs) associated to ticks are among the most important health issues affecting dogs. In Italy, Ehrlichia canis, Anaplasma spp., Rickettsia conorii and Borrelia burgdorferi (s.l.) have been studied in both healthy canine populations and those clinically ill with suspected CVBDs. However, little information is currently available on the overall prevalence and distribution of these pathogens in the country. The aim of this study was to assess the prevalence and distribution of tick-borne pathogens (TBPs) in clinically suspect dogs from three Italian macro areas during a 15-year period (2006–2020). Methods A large dataset (n = 21,992) of serological test results for selected TBPs in three macro areas in Italy was analysed using a Chi-square test to evaluate the associations between the categorical factors (i.e. macro area, region, year, sex and age) and a standard logistic regression model (significance set at P = 0.05). Serological data were presented as annual and cumulative prevalence, and distribution maps of cumulative positive cases for TBPs were generated. Results Of the tested serum samples, 86.9% originated from northern (43.9%) and central (43%) Italy. The majority of the tests was requested for the diagnosis of E. canis (47%; n = 10,334), followed by Rickettsia spp. (35.1%; n = 7725), B. burgdorferi (s.l.) (11.6%; n = 2560) and Anaplasma spp. (6.2%; n = 1373). The highest serological exposure was recorded for B. burgdorferi (s.l.) (83.5%), followed by Rickettsia spp. (64.9%), Anaplasma spp. (39.8%) and E. canis (28.7%). The highest number of cumulative cases of Borrelia burgdorferi (s.l.) was recorded in samples from Tuscany, central Italy. Rickettsia spp. was more prevalent in the south and on the islands, particularly in dogs on Sicily older than 6 years, whereas Anaplasma spp. was more prevalent in the north and E. canis more prevalent in the south and on the islands. Conclusions The results of this study highlight the high seroprevalence and wide distribution of the four TBPs in dogs with clinically suspected CVBDs from the studied regions of Italy. The very high seroprevalence of B. burgdorferi (s.l.) exemplifies a limitation of this study, given the use of clinically suspect dogs and the possibility of cross-reactions when using serological tests. The present research provides updated and illustrative information on the seroprevalence and distribution of four key TBPs, and advocates for integrative control strategies for their prevention. Grapic abstract

The trypanosomatid protist Leishmania tarentolae is a saurian-associated parasite vectored by the Sergentomyia minuta sand fly. This study aimed to confirm the circulation of L. infantum and L. tarentolae in sand flies, reptiles and dogs and to isolate new strains of these protists. Reptilian and sheltered dog blood samples were collected, and sand flies were captured. Samples were tested for Leishmania spp. using duplex real-time PCR (dqPCR) and real-time PCR (qPCR); the origin of blood meal was identified in engorged sand flies by conventional PCR. The reptilian blood and intestinal content of sand fly females were cultured. Dog sera were tested by IFAT using both Leishmania species. Four Tarentola mauritanica geckoes were molecularly positive for L. infantum or L. tarentolae, with no co-infections; moreover, amastigote-like forms of L. infantum were observed in the bone marrow. 24/294 sand flies scored positive for Leishmania spp. by dqPCR, 21 S. minuta and two Phlebotomus perniciosus were positive for L. tarentolae, while only a single Ph. perniciosus was positive for L. infantum. Blood meal analysis confirmed reptile and dog in S. minuta, dog and human in Ph. perniciosus and dog in Phlebotomus neglectus. Two axenic strains of L. tarentolae were obtained. Twelve of 19 dogs scored positive for L. infantum and L. tarentolae by IFAT and three of them also for L. infantum by dqPCR, and six by qPCR. These data confirm the sympatric circulation of L. infantum and L. tarentolae in geckoes, sand flies, and dogs, and suggest that geckoes may be infected with L. infantum.

Among the gastrointestinal parasites of dogs that present public health importance, the species of the genus Ancylostoma and Toxocara are the most frequently detected, and have been important cause of soil contamination. The aim of this study was to detect gastrointestinal parasites in dog’s feces collected in households, streets and public squares of the city of Garanhuns, Pernambuco, Brazil. Fecal samples (n = 640) were collected in the environment and evaluated by parasitological methods of Willis-Mollay and Hoffman, Pons and Janer. Out of all samples, 51.09% (327/640) were positive for gastrointestinal parasites. In particular, in 70.94% (232/327) of the samples were detected eggs of Ancylostoma spp., and in 11.01% (36/327) coinfection by Ancylostoma spp. and Toxocara canis. In conclusion, the presence of eggs of zoonotic parasites in feces collected from the environment in the city of Garanhuns represent a risk for the transmission of Cutaneous Larva Migrans and Visceral Larva Migrans for the human population.

Abstract Trypanosoma (Duttonella) vivax is an important cause of economic losses among feedlot cattle. These losses are related to the morbidity, mortality, reproductive issues and decreased production. It is known that the clinical signs observed in infections by this protozoon are similar to other hemoparasitosis, which difficult the diagnosis. Therefore, the aim of this study was to detect and molecularly characterize an outbreak of trypanosomiasis caused by T. (D.) vivax in dairy cattle in the municipality of São Miguel Aleixo, state of Sergipe, Brazil. Blood samples from cattle (n = 15) presenting clinical signs compatible with trypanosomiasis were collected and parasitological and molecular evaluated. Among the samples analyzed, 34% (5/15) were positive from blood smears, 60% (9/15) from the buffy coat method and 80% (12/15) from the molecular method. The DNA sequence obtained (659 bp) showed 99% similarity to T. (D.) vivax sequences that are available in the GenBank database. The presence of this protozoon in cattle herds is a problem for producers. Diagnosing trypanosomiasis is problematic because its evolution is similar to that of other parasitic blood diseases. In addition, this is the first report of infection by T. (D.) vivax in cattle in the state of Sergipe, northeastern Brazil.