Mansonella sp. and associated Wolbachia endosymbionts in ring-tailed coatis (Nasua nasua) in periurban areas from Midwestern Brazil.

Coatis (Nasua nasua) are wild carnivorous well adapted to anthropized environments especially important because they act as reservoirs hosts for many arthropod-borne zoonotic pathogens. Information about filarioids from coatis and associated Wolbachia spp. in Brazil is scant. To investigate the diversity of filarial nematodes, blood samples (n = 100 animals) were obtained from two urban areas in midwestern Brazil and analyzed using blood smears and buffy coats and cPCR assays based on the cox1, 12S rRNA, 18S rRNA, hsp70 and myoHC genes for nematodes and 16S rRNA for Wolbachia. When analyzing coati blood smears and buffy coats, 30% and 80% of the samples presented at least one microfilaria, respectively. Twenty-five cox1 sequences were obtained showing 89% nucleotide identity with Mansonella ozzardi. Phylogenetic analyses clustered cox1 sequences herein obtained within the Mansonella spp. clade. Sequences of both myoHC and two hsp70 genes showed 99.8% nucleotide identity with Mansonella sp. and clustered into a clade within Mansonella sp., previously detected in coatis from Brazil. Two blood samples were positive for Wolbachia, with a 99% nucleotide identity with Wolbachia previously found in Mansonella perstans, Mansonella ozzardi and Mansonella atelensis and in ectoparasites of the genus Pseudolynchia, Melophagus and Cimex. The study showed a high prevalence of Mansonella sp. in the coati population examined, suggesting that this animal species play a role as reservoirs of a novel, yet to be described, species within the Onchocercidae family.

Molecular detection of Babesia spp. and Rickettsia spp. in coatis (Nasua nasua) and associated ticks from midwestern Brazil.

Procyonids are reservoirs of many zoonotic infectious diseases, including tick-borne pathogens. The role of coatis (Nasua nasua) in the epidemiology of piroplasmids and Rickettsia has not been fully addressed in Brazil. To molecularly study these agents in coatis and associated ticks, animals were sampled in two urban areas in Midwestern Brazil. Blood (n = 163) and tick (n = 248) DNA samples were screened by PCR assays targeting the 18S rRNA and gltA genes of piroplasmids and Rickettsia spp., respectively. Positive samples were further molecularly tested targeting cox-1, cox-3, ß-tubulin, cytB, and hsp70 (piroplasmid) and ompA, ompB, and htrA 17-kDa (Rickettsia spp.) genes, sequenced and phylogenetically analyzed. All coatis' blood samples were negative for piroplasmids, whereas five pools of ticks (2%) were positive for two different sequences of Babesia spp.. The first from Amblyomma sculptum nymphs was close (i.e., ≥ 99% nucleotide identity) to a Babesia sp. previously found in capybaras (Hydrochoerus hydrochaeris); the second from Amblyomma dubitatum nymphs and Amblyomma spp. larvae was identical (100% nucleotide identity) to a Babesia sp. detected in opossums (Didelphis albiventris) and associated ticks. Four samples (0.8%) were positive by PCR to two different Rickettsia spp. sequences, being the first from Amblyomma sp. larva identical to Rickettsia belli and the second from A. dubitatum nymph identical to Rickettsia species from Spotted Fever Group (SFG). The detection of piroplasmids and SFG Rickettsia sp. highlights the importance of Amblyomma spp. in the maintenance of tick-borne agents in urban parks where humans and wild and domestic animals are living in sympatry.

Molecular detection of zoonotic filarioids in Culex spp. from Portugal.

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.

Seasonal variation in canine anti-Leishmania infantum antibody titres.

Quantitative anti-Leishmania antibody titres are critical in the management of dogs with leishmaniosis, from diagnosis to treatment and follow-up, and there is a paucity of data relating changes in antibody titres to sand fly vector seasonality. This study aimed to evaluate seasonal variations in anti-Leishmania infantum antibody titres in dogs from a hyperendemic area for canine leishmaniosis (CanL). Leishmania infantum-seropositive and clinically healthy dogs (n=65) were sampled in June 2019 (sand fly season) and again in February-March 2020 (non-transmission season) to monitor clinical status and serological titres. There was a reduction in anti-L. infantum antibody titres during the non-transmission season in most dogs (n=36; 55.4%), and 44% of those dogs (n=16/36) became seronegative (i.e. below the cut-off value of 1:80). Given the relevance of serology to epidemiological, preventive and clinical studies related to CanL, seasonal variations in antibody titres are important in areas where phlebotomine vectors have seasonal patterns of activity. Sand fly seasonal period must be considered in the interpretation of annual anti-L. infantum antibody screening test results in asymptomatic dogs, to make clinical decisions about staging, treatment and prevention.

Marked host association and molecular evidence of limited transmission of ticks and fleas between sympatric wild foxes and rural dogs.

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.

Feline leukemia virus in owned cats in Southeast Asia and Taiwan.

Feline leukaemia virus (FeLV) is a retrovirus associated with fatal disease in cats with infection in its progressive form. Although there are numerous reports on the occurrence of FeLV in the feline population worldwide, there is a paucity of data in Asia. In this study, we assessed the circulation of FeLV by ELISA and nested PCR in cats from different countries in Southeast Asia (i.e., Thailand, Malaysia, Singapore, Philippines, Indonesia and Vietnam) and Taiwan during 2017-2018. Forty-seven cats were positive to FeLV by antigen or provirus detection, but 32 samples were considered truly positive on the basis of positive molecular testing. Frequency of occurrence of FeLV proviral DNA ranged from 0% (0/43 positive samples) in Indonesia to 18.5% (22/119 positive samples) in Thailand. A statistically significant association (p < 0.05) was found between country of cats origin, age, lifestyle, abnormal oral mucosa, and FeLV molecular positive results. In-depth studies are needed in other countries in Southeast Asia to elucidate the mosaic of knowledge about FeLV epidemiology.

Beyond taxonomy: species complexes in New World phlebotomine sand flies.

A species complex (= species group, species series) is an assemblage of species, which are related morphologically and phylogenetically. Recent research has revealed several arthropod vector species that were believed to be a single nominal species actually representing a group of closely related species, which are sometimes morphologically indistinguishable at one or more developmental stages. In some instances, differences in terms of vector competence, capacity, or both have been recorded. It highlights the importance of detecting and studying species complexes to improve our understanding of pathogen transmission patterns, which may be vectored more or less efficiently by different species within the complex. Considering more than 540 species, about one-third of the phlebotomine sand flies in the New World present males and/or females morphologically indistinguishable to one or more species. Remarkably, several of these species may act in transmission of pathogenic agents. In this article, we review recent research on species complexes in phlebotomine sand flies from the Americas. Possible practical implications of recently acquired knowledge and future research needs are also discussed.

Troglostrongylus brevior is the dominant lungworm infecting feral cats in Jerusalem.

Feline lungworms infect the respiratory tract of wild and domestic cats, causing infection often associated with clinical disease. Until recently, Aelurostrongylus abstrusus has been considered the most relevant species of lungworm, while Troglostrongylus brevior was considered of less significance. Fecal samples of feral cats from Jerusalem, Israel, collected over a year, were examined for first stage lungworm larvae (L1) using the Baermann method. Positive samples were morphologically identified, and their species identity was molecularly confirmed. Forty of 400 (10.0%) cats were lungworm-positive, of which 38/40 (95.0%) shed Troglostrongylus brevior and 6/40 (15.0%) shed Aelurostrongylus abstrusus. Four cats (10.0%) had mixed infections with both lungworm species. L1 shedding was associated with clinical respiratory signs in 11 (19.0%) T. brevior shedding cats of a total of 58 cats manifesting respiratory signs, while 23/342 (6.7%) cats without respiratory signs were L1-positive (p = 0.006). Non-respiratory clinical signs were also found to be more prevalent in L1 shedders (p = 0.012). A young kitten ≤ 4 weeks of age shed T. brevior L1 larvae. DNA sequences of both lungworm species using the ribosomal internal transcribed spacer 2 (ITS2) locus were > 99% similar to other sequences deposited in GenBank, suggesting that T. brevior and A. abstrusus ITS2 sequences are both highly conserved. In conclusion, L1 shedding in feral cats from Jerusalem were mostly caused by T. brevior with only a small proportion involving A. abstrusus, different from many studies from other geographical regions.

Molecular detection of Leishmania (Sauroleishmania) tarentolae in human blood and Leishmania (Leishmania) infantum in Sergentomyia minuta: unexpected host-parasite contacts.

The detection of atypical Kinetoplastida in vertebrate hosts and vectors might suggest unexpected host-parasite contacts. Aside to major vectors of Leishmania (Leishmania) infantum in Italy (e.g. Phlebotomus perniciosus and Phlebotomus perfiliewi), the sand fly fauna also includes Sergentomyia minuta, herpetophilic and proven vector of Leishmania (Sauroleishmania) tarentolae, in which records of blood meal on mammals and detection of L. infantum DNA are increasing. This study was conducted in Central Italy aiming to molecularly detect potential atypical Leishmania host-vector contacts. Detection of Leishmania spp. DNA was performed by polymerase chain reaction (SSU rRNA, ITS1 targets) on field-collected sand fly females (N = 344), blood samples from humans (N = 185) and dogs (N = 125). Blood meal identification was also performed on engorged sand flies. Leishmania spp. DNA was found in 13.1% sand flies, 3.7% humans and 14.4% dogs. Sequence analysis identified L. infantum in S. minuta (4.4%), P. perniciosus (9.1%), humans (2.2%) and dogs (14.4%). Leishmania tarentolae was detected in S. minuta (12.6%), P. perfiliewi (6.6%) and human (1.6%) samples. Of 28 S. minuta examined for blood meal, 3.6 and 21.4% scored positive for human and lizard DNA, respectively. These results indicate the importance of one-health approach to explore new potential routes of transmission of leishmaniasis involving S. minuta.

Species diversity and molecular insights into phlebotomine sand flies in Sardinia (Italy)-an endemic region for leishmaniasis.

This study provides updated information on the distribution of the phlebotomine sand fly species and their genetic characterization in Sardinia, a Mediterranean island where leishmaniasis is endemic. From April to November 2017, sand flies were trapped in five different capture sites using sticky traps and light traps as collection methods, operated nearby sheep, poultry, cat, and dog shelters. Phlebotomine specimens (n = 513) collected were morphologically and molecularly identified as Phlebotomus perniciosus (249/513, 48.5%), Phlebotomus perfiliewi (236/513; 46%), and Sergentomyia minuta (28/513, 5.5%). Sand flies were collected from the second half of May to October confirming the well-defined seasonal activity, which peaks in August in Sardinia. Overall, correlation analyses indicated a significant positive association between the monthly number of sand flies collected and the mean temperature (r = 0.88, rho = 0.87, and tau = 0.69, P < 0.05), while there was non-significant, moderately negative correlation between the monthly number of sand flies collected and the monthly mean relative humidity and wind (r = - 0.22, rho = 0.02, and tau = 0.04, P > 0.05). This study provides the first data on the molecular characterization of phlebotomine sand flies in this region and confirms the presence of three sand fly species. Molecular results suggest that the morphological features used for analysis represent synapomorphic-derived characters which are shared among descendant taxa and the common ancestor.

Survival of Phortica variegata experimentally and naturally infected with Thelazia callipaeda.

Knowledge of the effects that Thelazia callipaeda (Spirurida, Thelaziidae) infection has on the survival of its vector Phortica variegata (Drosophilidae, Steganinae) is scarce. The present study aimed to: (a) assess the rate of infection between experimentally infected (EI) and not experimentally infected (NEI) flies and (b) determine how T. callipaeda infection may affect the survival of P. variegata. In addition, fat composition was evaluated in flies that died during overwintering. Molecular analysis showed that T. callipaeda prevalence in flies that died before experimental infection, plus those from the NEI group, is 0.75% (i.e. 11 out of 1462 individuals). The EI group showed a significantly higher positivity to T. callipaeda (i.e. 51 out of 682 individuals; 7.48%) compared with the NEI group (i.e. 9 out of 750 individuals; 1.2%). Thelazia callipaeda DNA was detected until 147 days after experimental infection. This demonstrates that larvae of this eyeworm may survive in the fly for a significant period of the winter. Fat composition analysis showed that flies produced more unsaturated than saturated fatty acids during diapause, probably because unsaturated fatty acids remain in a liquid state at lower temperatures, providing anti-freeze properties to survive winter.

Rapid diagnosis of parasitic diseases: current scenario and future needs.

BACKGROUND: Parasitic diseases are one of the world's most devastating and prevalent infections, causing millions of morbidities and mortalities annually. In the past, many of these infections have been linked predominantly to tropical or subtropical areas. Nowadays, however, climatic and vector ecology changes, a significant increase in international travel, armed conflicts, and migration of humans and animals have influenced the transmission of some parasitic diseases from 'book pages' to reality in developed countries. It has also been noted that many patients who have never travelled to endemic areas suffer from blood-borne infections caused by protozoa. In the light of existing knowledge, this new trend can be explained by the fact that in the process of migration a large number of asymptomatic carriers become a part of the blood bank donor and transplant donor populations. Accurate and rapid diagnosis represents the crucial weapon in the fight against parasitic infections. AIMS: To review old and new approaches for rapid diagnosis of parasitic infections. SOURCES: Data for this review were obtained through searches of PubMed using combinations of the following terms: parasitological diagnostics, microscopy, lateral flow assays, immunochromatographic assays, multiplex-PCR, and transplantation. CONTENT: In this review, we provide a brief account of the advantages and limitations of rapid methods for diagnosis of parasitic diseases and focus our attention on current and future research in this area. The approximate costs associated with the use of different techniques and their applicability in endemic and non-endemic areas are also discussed. IMPLICATIONS: Microscopy remains the cornerstone of parasitological diagnostics, especially in the field and low-resource settings, and provides epidemiological assessment of parasite burden. However, increased use and availability of point-of-care tests and molecular assays in modern era allow more rapid and accurate diagnoses and increased sensitivity in the identification of parasitic infections.

Level of agreement between two commercially available rapid serological tests and the official screening test used to detect Leishmania seropositive dogs in Brazil.

This study compared the level of agreement between two commercially available rapid serological tests and the official screening test used to detect Leishmania seropositive dogs in Brazil. Ninety-five canine sera from a visceral leishmaniasis endemic area were tested by using the official immunochromatographic test (T1; rK28 antigen) based on dual path platform technology, a rapid ELISA (T2; purified Leishmania antigens) and an immunochromatographic test (T3; rK28 antigen) based on lateral flow. There was substantial agreement (Kappa 0.77; 95% confidence interval, CI, 0.62-0.91; P<0.001; observed agreement 90.5%) between T1 and T2, and a fair agreement (Kappa 0.26; 95% CI 0.08-0.43; P<0.001; observed agreement 74.7%) between T1 and T3. Sixteen dogs positive at T1 and T2 were negative at T3. T2 may be a reliable alternative to T1, while T3 could lead to an underestimation of the actual number of seropositive dogs.

International dog travelling and risk for zoonotic Onchocerca lupi.

Onchocerca lupi is a recently recognized threat for the health of animals and humans in European, American, African and Middle Eastern countries. We describe a case of imported O. lupi infection in Italy and report the lifespan of this parasite in a non-endemic area, to advocate increased awareness of the veterinary community for this zoonotic parasitosis.

Spreading of Thelazia callipaeda in Greece.

Thelazia callipaeda (Spirurida, Thelaziidae), the so-called oriental eyeworm, has been increasingly reported as an agent of infection in animals and humans from many European countries. Clinical signs range from subclinical to moderate or severe ocular disorders (e.g., epiphora, photophobia, conjunctivitis, keratitis, ulcers). The disease has been also diagnosed in animals from countries of the Balkan area (e.g., Croatia, Bosnia and Herzegovina and Serbia), but only a single case of canine thelaziosis, presumably autochthonous, was reported in Northern Greece. In this study, we provide robust information of the occurrence of thelaziosis in Greece by reporting autochthonous cases of thelaziosis in dogs (n = 46), cats (n = 3) and in one rabbit (Oryctolagus cuniculus) living in Northern and Central regions of Greece. The occurrence of a single haplotype of the cytochrome oxidase subunit 1 gene confirms that the same zoonotic haplotype of the parasite circulating in Europe is also spreading in Greece. The increased awareness of this parasitosis is crucial to limit the risk of further infections in both humans and animals in European countries.

Filaroidosis Infection in an Immunocompetent Adult Dog from France.

A dog from Paris (France) was referred with a 2-week history of dry cough, intermittent acute onset of dyspnoea, and acute abdominal pain. A generalised bronchoalveolar infiltrate with a patchy distribution was observed at chest x-rays and computed tomography (CT) scans. Negative results were obtained through several faecal examinations for cardiorespiratory nematodes by using the Baermann technique and at two blood analysis with a commercially available test for the detection of A. vasorum antigen (the first one at the first visit and second one at the control visit, one month later). PCR methods for the identification of A. vasorum and C. vulpis were also accomplished. At the control visit, nematode L1s were found during direct microscopic examination of bronchoalveolar lavage fluid (BALF). Thus, a different antigen-based assay for the detection of A. vasorum was performed with a positive result. Moreover, based on morphology, isolated larvae were identified as Filaroides hirthi. The dog was treated with fenbendazole (50 mg/kg per os once daily) for two consecutive weeks. After five months, the dog was referred again for the intermittent acute onset of dyspnoea and was found to be still positive for F. hirthi larvae at BALF examination. A 15-day treatment regimen with fenbendazole in combination with three subcutaneous injections of ivermectin (0.4 mg/kg, once every two weeks), was then performed. No larvae were detected at two BALF microscopical examinations performed one month apart. Results from this case report underline the importance of including F. hirthi infections in the differential diagnosis of dog bronchopneumonia.

Larval development of Angiostrongylus chabaudi, the causative agent of feline angiostrongylosis, in the snail Cornu aspersum.

Nematodes of the Angiostrongylidae family, such as Angiostrongylus vasorum and Angiostrongylus cantonensis, may cause potentially life-threatening diseases in several mammal species. Alongside these well-known species, Angiostrongylus chabaudi has been recently found affecting the cardiopulmonary system of domestic and wild cats from Italy, Germany, Greece, Romania and Bulgaria. Nonetheless, significant gaps in the understanding of A. chabaudi epidemiology include the lack of information of species acting as intermediate host and of the morphological description of larval stages. Cornu aspersum (n = 30) land snails were infected with 100 first-stage larvae of A. chabaudi collected from a naturally infected wildcat in Romania. Larvae at different developmental stages were found in 29 out of 30 (96·7%) infected snails and a total of 282 (mean 9·8 ± 3·02 larvae per each specimen) were collected from the gastropods. Here we demonstrate that A. chabaudi develops in snails and report C. aspersum as potential intermediate host for this parasitic nematode. Findings of this study are central to understand the ecological features of feline angiostrongylosis and its epidemiology within paratenic and intermediate hosts.

A comparative test of ixodid tick identification by a network of European researchers.

This study reports the results of a comparative test of identification of ticks occurring in Western Europe and Northern Africa. A total of 14 laboratories were voluntarily enrolled in the test. Each participant received between 22 and 25 specimens of adult and nymphal ticks of 11 species: Dermacentor marginatus, D. reticulatus, Haemaphysalis punctata, Hyalomma lusitanicum, Hy. marginatum, Ixodes ricinus, I. hexagonus, Rhipicephalus annulatus, R. bursa, R. rossicus, and/or R. sanguineus s.l. Ticks were morphologically identified by three of the co-authors and the identification confirmed by a fourth co-author who used molecular methods based on several genes. Then ticks were randomly selected and blindly distributed among participants, together with a questionnaire. Only specimens collected while questing and, if possible, in the same survey, were circulated. Because of the random nature of the test, a participant could receive several specimens of the same species. Species in the different genera had variable misidentification rates (MR) of 7% (Dermacentor), 14% (Ixodes), 19% (Haemaphysalis), 36% (Hyalomma), and 54% (Rhipicephalus). Within genera, the MR was also variable ranging from 5.4% for I. ricinus or 7.4% for D. marginatus or D. reticulatus to 100% for R. rossicus. The test provided a total misidentification rate of 29.6% of the species of ticks. There are no significant differences in MR according to the sex of the tick. Participants were requested to perform a second round of identifications on the same set of ticks, using only purposely prepared keys (without illustrations), circulated to the enrolled participants, including 2 species of the genus Dermacentor, 8 of Haemaphysalis, 10 of Hyalomma, 23 of Ixodes, and 6 of Rhipicephalus. The average MR in the second round was 28%: 0% (Dermacentor), 33% (Haemaphysalis), 30% (Hyalomma) 18% (Ixodes), and 50% (Rhipicephalus). Species which are not reported in the countries of a participating laboratory had always highest MR, i.e. purely Mediterranean species had highest MR by laboratories in Central and Northern Europe. Participants expressed their concerns about a correct identification for almost 50% of the ticks of the genera Hyalomma and Rhipicephalus. The results revealed less than total confidence in identifying the most prominent species of ticks in the Western Palearctic, and underpin the need for reference libraries for specialists involved in this task. Results also showed that a combination of certain genes may adequately identify the target species of ticks.