Squamata reptiles as a potential source of helminth infections when preyed on by companion animals.

BACKGROUND: Squamate reptiles cohabiting with companion animals may represent a source of helminth infections, especially through predation by dogs and cats with an outdoor lifestyle. METHODS: In order to assess the role of reptiles as intermediate/paratenic hosts of trophically transmitted helminths, synanthropic reptiles (n = 245) captured from different ecological settings (i.e., households, dog shelters, urban, peri-urban and rural areas or natural parks) of southern Italy were examined for endoparasites. Parasitic cysts (i.e., larval forms of acanthocephalans, cestodes and nematodes) and free helminths (i.e., adult nematodes and digeneans) were morphologically and molecularly identified, and statistical analysis was carried out to evaluate the correlations between reptiles, infections, and ecological settings. RESULTS: Overall, 31% of reptiles were positive for at least one helminth, with Podarcis siculus (18.7%) and Tarentola mauritanica (8.1%) being the most frequently infected species. Among the parasites of medical interest, Joyeuxiella echinorhyncoides showed the highest prevalence (19.7%), followed by Diplopylidium acanthotetra (10.5%), Joyeuxiella pasqualei, Mesocestoides lineatus (5.6%) and Physaloptera sp. (3.9%). Macracanthorhynchus hirudinaceus was detected once. Podarcis siculus and T. mauritanica were associated with cestode infections. CONCLUSIONS: The wide range of helminths detected here in reptiles living in sympatry with pets and the fact that many of these helminth species are parasitic and may infect companion animals (e.g., J. pasqualei, J. echinorhyncoides, D. acanthotetra, Physaloptera sp.) and humans (i.e., Macracanthorhynchus hirudinaceus, Mesocestoides lineatus) indicate the potential health risk associated with pets preying on these small vertebrates. Our results indicate the need for complementary investigations of trophically transmitted parasites in dogs and cats living in sympatry with reptiles.

Snakes and Souks: Zoonotic pathogens associated to reptiles in the Marrakech markets, Morocco.

The world-famous markets of Marrakech, also known in Arabic as souks, harbor a vast diversity of reptiles that are sold for medicinal/magic/pet purposes or used for snake charming. This unique epidemiological context has never been studied considering the interactions of humans, reptiles, and zoonotic pathogens. Thus, the aim of this study was to identify the parasites and pathogens present in blood and feces associated with handled reptiles in the markets of Marrakech to assess the risk of zoonotic transmission within the reptile-human interface. Privately owned reptiles (n = 118), coming from vendors or snake charmers, were examined and blood and feces sampled. DNA was extracted and molecular screening (cPCR, nPCR, qPCR, dqPCR) was performed aiming to identify potentially zoonotic pathogens (i.e., Anaplasma/Ehrlichia spp., Rickettsia spp., Borrelia burgdorferi sensu lato, Coxiella burnetii, Babesia/Theileria spp., Cryptosporidium spp., Giardia spp., Leishmania spp., Cestoda). Overall, 28.9% (34/118) of reptiles were positive for at least one pathogen. In blood, Anaplasma spp. were detected in four snakes, with two Montpellier snakes positive for Anaplasma phagocytophilum, while Rickettsia spp. were detected in one Mediterranean chameleon and four puff adders. Leishmania tarentolae was molecularly detected in a Mediterranean chameleon and a Montpellier snake. In feces, the cox1 gene generated a myriad of sequences for nematodes, cestodes, fungi and bacteria. Importantly, Proteus vulgaris was identified from a Mediterranean chameleon. Cryptosporidium spp. nPCR yielded a positive sample (i.e., Cryptosporidium sp. apodemus genotype I) from a Moroccan worm lizard, as well as for bacteria such as Pseudomonas aeruginosa in an Egyptian cobra, and Morganella morganii from a puff adder. Results from this study demonstrated the risk of zoonotic transmission of microorganisms and parasites present in blood and feces from reptiles that are brought to the souks in Marrakech, Morocco, to be sold for medicinal purposes or used for snake charming, being in direct and straight contact with humans.

Description of Joyeuxiella pasqualei (Cestoda: Dipylidiidae) from an Italian domestic dog, with a call for further research on its first intermediate host.

Although Joyeuxiella pasqualei is frequently detected in cats from Mediterranean Europe, information on its biology is still scarce. This cestode is relatively less frequently reported in dogs, possibly because it is often misdiagnosed with the better-known Dipylidium caninum. The occurrence of J. pasqualei proglottids in a dog living in a closed environment triggered us to delve into the biology of this cestode by collecting biological samples from lizards and a road-killed cat. Two reptile species, Podarcis siculus (Lacertidae), and Tarentola mauritanica (Geckonidae) were also collected in the garden and its surroundings. In addition, experimental infections with eggs obtained from gravid proglottids were performed in laboratory mice, and Tenebrio molitor (Coleoptera: Tenebrionidae) beetles. Proglottids from the dog's feces and adult cestodes detected at necroscopy of a cat were morphologically identified as J. pasqualei. Two out of 13 T. mauritanica collected in the garden had natural infections of J. pasqualei cysts in the liver and attached to the intestine. All P. siculus lizards and experimentally infected rodents and beetles were negative. DNA sequences obtained from J. pasqualei showed the highest nucleotide similarities with Versteria sp., Echinococcus sp., Raillietina sonini, Taenia polyacantha and D. caninum. Data herein provided show the inability of rodents to become infected by direct ingestion of gravid proglottids, suggesting a need for an invertebrate first intermediate host in the life cycle. Thus, more research study is advocated to better understand the biology of J. pasqualei such as its first intermediate host and its mechanism of transmission in reptiles and rodents.

Leishmania (Sauroleishmania) tarentolae isolation and sympatric occurrence with Leishmania (Leishmania) infantum in geckoes, dogs and sand flies.

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.

Dermal microfilariae of dogs, jackals and cats in different regions of Iran.

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.

Wild Boar (Sus scrofa) as Reservoir of Zoonotic Yeasts: Bioindicator of Environmental Quality.

Wildlife animals are recognized as reservoirs for zoonotic fungi and their faeces might play an important role in introducing pathogens into the environment. Thought wild boar (Sus scrofa) population has dramatically increased across Europe, information about their possible role in dissemination of zoonotic pathogenic yeasts in the environment is scant. Therefore, fecal samples (n = 124) from wild boars from Campania region (Southern Italy) were collected and yeasts identified biochemically and molecularly by sequencing of the internal transcribed spacer region and their phylogenetical relationship assessed. The antifungal susceptibility profiles of yeasts were also investigated using AFST-EUCAST method. Yeasts were isolated from 50.1% of the samples with the highest occurrence in samples from the province of Salerno (61.1%). A total of 368 Candida strains belonging to nine species were identified, with Candida albicans (45.7%), followed by Candida krusei (15.2%), Kazachstania slooffiae (9.8%) and Candida parapsilosis (7.6%) as the most prevalent identified species. Among C. albicans four sequence types (i.e., ST1-ST4) were identified with an intraspecific nucleotide difference up to 0.21%. The ML tree grouped all representative sequence types as paraphyletic clades with those of the references yeast species, respectively and supported by high bootstrap values. Fluconazole was the less active drug whereas, posaconazole, voriconazole, and isavuconazole the most active one. No resistance phenomena were observed for C. albicans and high MICs values for 5FC, azoles and echinocandines were registered in non-albicans Candida spp. This study showed, for the first time, the important role of wild boars in dissemination of pathogenic fungi in the environment. The absence of resistance phenomena in the Candida spp. might reflect environmental free from residues of azoles antifungals pollution or chemicals and suggests the role of wild boar as bio indicators of environment quality.

Raillietiella hemidactyli (Pentastomida: Raillietiellidae) in Tarentola mauritanica geckoes: A new zoonotic parasite for Europe.

Pentastomida is a subclass of parasitic arthropods, related to crustaceans, which develop in the respiratory tract of vertebrates (i.e., fishes, amphibians, reptiles, birds and mammals). Within this group of parasites, Raillietiella spp. adults develop in the lungs of lizards, snakes and toads, whereas larval stages in insects (e.g., cockroaches), which are intermediate hosts. Lizards were captured under the frame of a study on reptile zoonotic parasites. Feces of the collected animals were examined and pentastomids were diagnosed in Tarentola mauritanica geckoes (1.2%; 3/259) from Linosa island. Adult forms of Railietiella hemidactyli pentastomids were morphologically characterized and molecularly identified through 18S rDNA amplification and sequencing. Positive animals had adult forms of R. hemidactyli pentastomids in the lungs as well as embryonated eggs in feces. Raillietiella was herein identified for the first time in synanthropic geckoes in a confined population of one of the southernmost islands of Italy, representing the first report of this zoonotic pentastomid in synanthropic and invasive reptiles in Europe. Further studies should focus on the prevalence of pentastomids on synanthropic reptiles in other Italian regions to assess the zoonotic risk of infection and to warn veterinarians and physicians about the risk they may represent for several species of hosts, including dogs, cats and humans.

Leishmania spp. in Squamata reptiles from the Mediterranean basin.

Leishmania tarentolae is a nonpathogenic trypanosomatid species isolated from geckoes in the Mediterranean basin. In Italy, L. tarentolae and Leishmania infantum occur in sympatry in areas where canine leishmaniosis (CanL) is endemic. Here, we investigated the prevalence and distribution of L. tarentolae in reptilian hosts and the presence of L. infantum in the same animals in southern Italy. From April 2020 to April 2021, lizards and snakes were captured in urban, peri-urban sites, natural parks, and dog shelters. Blood was individually sampled and assessed for the presence of Leishmania spp. by cytology and/or molecular biology. Captured lizards were euthanized and their tissues/organs were tested by duplex real-time-PCR for the detection of L. infantum and L. tarentolae and the positive ones were amplified by conventional PCR to confirm the species identification by sequencing. Samples were also screened by real-time PCR for L. infantum kDNA minicircle. Of the 259 collected Squamata reptiles, 34 (13.1%) specimens scored positive for Leishmania spp., with a prevalence of 10% for L. tarentolae (n = 26) and of 3.1% for L. infantum (n = 8). Positive lizards were mostly Podarcis siculus (n = 26) from dog shelters, with six specimens positive for L. infantum. In addition, L. tarentolae was detected in one Podarcis filfolensis lizard, and in five T. mauritanica geckos (with one gecko and P. filfolensis lizard showing amastigote-like forms in thrombocytes), whereas L. infantum was detected in one T. mauritanica, and one Hemidactylus turcicus. Data herein reported demostrate that saurian reptiles living in CanL endemic areas of the Mediterranean basin may be exposed to both L. tarentolae and L. infantum indicating a sympatric circulation of these species in different vertebrate hosts. Moreover, this study represents the first molecular evidence of L. infantum infection in European reptiles.

Detection of Leishmania tarentolae in lizards, sand flies and dogs in southern Italy, where Leishmania infantum is endemic: hindrances and opportunities.

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.

Fasciola hepatica in wild boar (Sus scrofa) from Italy.

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.

Role of reptiles and associated arthropods in the epidemiology of rickettsioses: A one health paradigm.

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.

Molecular survey on tick-borne pathogens and Leishmania infantum in red foxes (Vulpes vulpes) from southern Italy.

Red foxes (Vulpes vulpes) have been recognised to harbour and transmit a wide range of tick-borne pathogens (TBPs) including those of zoonotic concern. To investigate the prevalence and the distribution of TBPs and of Leishmania infantum in foxes (n = 244), spleen samples were collected within the frame of a multi-regional wildlife health surveillance program in Italy. A combined PCR/sequencing approach was performed for the detection of Anaplasma spp., Babesia spp., Borrelia spp., Ehrlichia spp., Hepatozoon spp. and L. infantum DNA. Overall, 146 foxes (59.8 %, 95 % CI: 53.6-65.8) tested positive for at least one pathogen with Hepatozoon canis being the most prevalent (i.e., n = 124; 50.8 %, 95 % CI: 44.6-57.0), followed by Babesia vulpes (n = 20; 8.2 %, 95 % CI: 5.4-12.3), different spirochete species from Borrelia burgdorferi sensu lato complex (n = 9; 3.7 %, 95 % CI: 1.9-6.9), Ehrlichia canis and L. infantum (n = 7; 2.9 % each, 95 % CI: 1.4-5.8), Anaplasma platys (n = 4; 1.6 %, 95 % CI: 0.6-4.1), Anaplasma phagocytophilum ecotype I and Candidatus Neoehrlichia sp. (n = 3; 1.2 % each, 95 % CI: 0.4-3.5). All samples scored negative for Babesia canis and Borrelia miyamotoi. This study revealed the presence of spirochetes from B. burgdorferi s.l. complex, Ca. Neoehrlichia sp., A. platys and A. phagocytophilum ecotype I in red fox population from Italy, underling the necessity to monitoring these carnivores, mainly because they live in contact with dogs and humans. Data on the tick fauna circulating on wildlife species will complement information herein obtained, instrumentally to establish preventive strategies for minimizing the risk of infection for animals and humans.

Major antigen and paramyosin proteins as candidate biomarkers for serodiagnosis of canine infection by zoonotic Onchocerca lupi.

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.

Virulence and in vitro antifungal susceptibility of Candida albicans and Candida catenulata from laying hens.

In spite of evidence that domestic and wild birds may act as carriers of human pathogenic fungi, data on the role of laying hens as reservoirs of drug resistant and virulent yeasts is lacking. Here, we assess several virulence factors (phospholipase and haemolysin activity) and the antifungal susceptibility profiles of 84 Candida albicans and 17 Candida catenulata strains isolated from cloacae (group A), faeces (group B) and eggs (group C) of laying hens. Of these strains, 95% C. albicans and 23% C. catenulata strains displayed phospholipase and haemolytic activities. For C. albicans, the highest values of phospholipase (Pz = 0.62) and haemolytic activities (Hz = 0.49) were recorded among the strains from group C whilst for C. catenulata (Pz = 0.54; Hz = 0.49) among those from group A. High minimum inhibitory concentration (MIC) values for azoles and amphotericin B (AmB) were recorded irrespective of their sources in all C. albicans strains. A total of 22 C. albicans strains were multidrug resistant, displaying resistance to fluconazole, itraconazole (ITZ), voriconazole (VOR) and posaconazole (POS). All C. catenulata strains from group C were resistant to ITZ, POS, micafungin and anidulafungin and susceptible to AmB. In this study, C. albicans and C. catenulata isolated from the cloacae, faeces and eggs of laying hens produced phospholipase and haemolysin and might be multidrug resistant. In the environment (faeces) or in eggs, C. albicans and C. catenulata strains might acquire pathogenic virulence traits and/or show multidrug resistance profiles. Based on these results, breeding and handling of laying hens and/or eggs may have implications for human and animal health.

Cercopithifilaria species in dogs and ticks from Greece.

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.

First report of Thelazia callipaeda in a free-ranging Iberian wolf (Canis lupus signatus) from Spain.

Thelazia callipaeda (Spirurida, Thelaziidae) is a vector-borne zoonotic eyeworm able to infect a broad spectrum of carnivores. Here, we describe the first case of bilateral infection by T. callipaeda in the eyes of an adult female Iberian wolf (Canis lupus signatus) in central Spain. Nematodes collected were morphologically identified (n = 42), and two specimens were molecularly characterized. At the sequence analysis of the partial mitochondrial cytochrome c oxidase subunit 1 gene, T. callipaeda haplotype 1 (the only haplotype circulating in Europe) was detected. The role of the Iberian wolf as a natural reservoir for T. callipaeda in the life cycle of this emerging zoonosis and the implications in conservation are discussed.

Zoonotic Vectorborne Pathogens and Ectoparasites of Dogs and Cats in Eastern and Southeast Asia.

To provide data that can be used to inform treatment and prevention strategies for zoonotic pathogens in animal and human populations, we assessed the occurrence of zoonotic pathogens and their vectors on 2,381 client-owned dogs and cats living in metropolitan areas of 8 countries in eastern and Southeast Asia during 2017-2018. Overall exposure to ectoparasites was 42.4% in dogs and 31.3% in cats. Our data cover a wide geographic distribution of several pathogens, including Leishmania infantum and zoonotic species of filariae, and of animals infested with arthropods known to be vectors of zoonotic pathogens. Because dogs and cats share a common environment with humans, they are likely to be key reservoirs of pathogens that infect persons in the same environment. These results will help epidemiologists and policy makers provide tailored recommendations for future surveillance and prevention strategies.

Effect of chlorogenic and gallic acids combined with azoles on antifungal susceptibility and virulence of multidrug-resistant Candida spp. and Malassezia furfur isolates.

Chlorogenic acid (CHA) and gallic acid (GA) are safe natural phenolic compounds that are used as enhancers of some drugs in influencing antioxidant, anticancer, and antibacterial activities. Among fungi, Candida spp. and Malassezia spp. are characterized by an increasing prevalence of multidrug resistance phenomena and by a high morbidity and mortality of their infections. No data are available about the efficacy of CHA and GA combined with azoles on the antifungal susceptibility and on the virulence of both fungi. Therefore, their antifungal and antivirulence effects have been tested in combination with fluconazole (FLZ) or ketoconazole (KTZ) on 23 Candida spp. and 8 M. furfur isolates. Broth microdilution chequerboard, time-kill studies, and extracellular enzymes (phospholipase and hemolytic) activities were evaluated, displaying a synergistic antifungal action between CHA or GA and FLZ or KTZ on C. albicans, C. bovina, and C. parapsilosis, and antagonistic antifungal effects on M. furfur and Pichia kudriavzevii (Candida krusei) isolates. The time-kill studies confirmed the chequerboard findings, showing fungicidal inhibitory effect only when the GA was combined with azoles on Candida strains. However, the combination of phenolics with azoles had no effect on the virulence of the tested isolates. Our study indicates that the combination between natural products and conventional drugs could be an efficient strategy for combating azole resistance and for controlling fungistatic effects of azole drugs.

Phlebotomine sand flies and Leishmania species in a focus of cutaneous leishmaniasis in Algeria.

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.

Efficiency of the Q3 lab-on-chip Real Time-PCR platform for detecting protozoan pathogens in bivalve mollusks.

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., 103, 104 and 105). 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 < 0.01), especially for Toxoplasma in M. galloprovincialis at both 24 h and 72 h after contamination, and in O. edulis at 72 h. The ability of Q3 to detect all the investigated pathogens was similar to that of Real-Time-PCR, and Q3 was efficient in detecting Toxoplasma in both M. galloprovincialis and O. edulis. This is the first study concerning the use of lab-on-chip technology in a food matrix, and in edible marine mollusks in particular.