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.

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.

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.

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.

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.

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.

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.

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.

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.

Potential role of ATP-binding cassette transporters against acaricides in the brown dog tick Rhipicephalus sanguineus sensu lato.

ATP-binding cassette (ABC) transporters have been shown to be involved in pesticide detoxification in arthropod vectors and are thought to contribute to the development of drug resistance. Little is currently known about the role they play in ticks, which are among the more important vectors of human and animal pathogens. Here, the role of ABC transporters in the transport of fipronil and ivermectin acaricides in the tick Rhipicephalus sanguineus (Ixodida: Ixodidae) was investigated. Larvae were treated with acaricide alone and acaricide in combination with a sub-lethal dose of the ABC transporter inhibitor cyclosporine A. The LC50 doses and 95% confidence intervals (CIs) estimated by mortality data using probit analysis were 67.930 p.p.m. (95% CI 53.780-90.861) for fipronil and 3741 p.p.m. (95% CI 2857-4647) for ivermectin. The pre-exposure of larvae to a sub-lethal dose of cyclosporine A reduced the LC50 dose of fipronil to 4.808 p.p.m. (95% CI 0.715-9.527) and that of ivermectin to 167 p.p.m. (95% CI 15-449), which increased toxicity by about 14- and 22-fold, respectively. The comparison of mortality data for each separate acaricide concentration showed the synergic effect of cyclosporine A to be reduced at higher concentrations of acaricide. These results show for the first time a strong association between ABC transporters and acaricide detoxification in R.sanguineus s.l.

Simultaneous infection by four feline lungworm species and implications for the diagnosis.

Besides Aelurostrongylus abstrusus, other parasites belonging to the superfamily Metastrongyloidea, namely Oslerus rostratus, Troglostrongylus brevior and to the family Trichuridae, i.e. Eucoleus aerophilus (syn. Capillaria aerophila), have also been reported as agents of respiratory infection in domestic cats. A case of simultaneous infection by four feline lungworm species in Sardinia is herein described. An adult female cat (Felis silvestris catus), road-killed in the southeast part of Sardinia (municipality of Villacidro, province of Cagliari), Italy, was referred to the Laboratory of Parasitology of the Veterinary Teaching Hospital in Sassari. At necropsy, the lungs were examined and dissected under a stereomicroscope for the presence of parasites, and first-stage larvae (L1) of broncho-pulmonary nematodes were searched for in a faecal sample using the Baermann method. Parasites collected in the lungs were morphologically identified as A. abstrusus, E. aerophilus, and O. rostratus. In addition to the above species, L1s of Troglostrongylus spp. were detected at coproscopy but no adult specimen was found in the lungs. The morphological identification was confirmed by the molecular amplification and sequencing of cox1 mitochondrial gene, 18S and ITS2 ribosomal DNA. This finding stands as the first simultaneous infection by four feline lungworm species in the same animal, and as the first report of O. rostratus and E. aerophilus in Sardinia.

A look into the Medical and Veterinary Entomology crystal ball.

Medical and Veterinary Entomology (MVE) represents a leading periodical in its field and covers many aspects of the biology and control of insects, ticks, mites and other arthropods of medical and veterinary importance. Since the first issue of the journal, researchers working in both developed and developing countries have published in MVE, with direct impact on current knowledge in the field. An increasing number of articles dealing with the epidemiology and transmission of vector-borne pathogens have been published in MVE, reflecting rapid changes in vector distribution, pathogen transmission and host-arthropod interactions. This article represents a gaze into the crystal ball in which we identify areas of increasing interest, discuss the main changes that have occurred in the epidemiology of parasitic arthropods since the first issue of MVE, and predict the principal scientific topics that might arise in the next 25 years for scientists working in medical and veterinary entomology.

Cercopithifilaria spp. in dogs in Sardinia Island (Italy).

A survey on Cercopithifilaria spp. was carried out on owned and kennelled dogs in Sardinia, Italy. A total of 180 dogs were sampled and tested by microscopic detection or PCR of dermal microfilariae in skin snip sediments. The overall prevalence for Cercopithifilaria spp. at both microscopy and molecular tests was 9.4 % (17/180), while 8.3 % (15/180) of dogs scored positive at microscopic detection of sediments only. Of the 225 microfilariae measured, 212 were identified as Cercopithifilaria bainae and the remaining as Cercopithifilaria sp. II. All samples were molecularly processed for specific amplification of cytochrome oxidase subunit 1 (cox1) and ribosomal 12S gene fragments. The Basic Local Alignment Search Tool analysis of the cox1 and 12S sequences here obtained showed a high nucleotide similarity (99 and 100 %, respectively) with those of C. bainae available in GenBank. In particular, cox1 haplotype I (HI; n=14), haplotype HXVIII (n=2), and a new haplotype, named HXIX (n=1), differing for a single polymorphism from HI, were detected. This study reports data on the occurrence, distribution, and genetic makeup of C. bainae and Cercopithifilaria sp. II infesting dogs in Sardinia, suggesting that these filarioids are spread in areas where Rhipicephalus sanguineus sensu lato ticks occur.

Evidences of increasing risk of dirofilarioses in southern Italy.

Given the spread of Aedes albopictus from northern to southern Italy, and the lack of updated data on Dirofilaria infections, this study was carried out to assess the infection risk for dogs and cats in Apulia region. During a 2-year study, 175 A. albopictus female specimens and samples of blood from 427 dogs (309 privately owned dogs and 118 shelter dogs) and 12 cats were collected. All blood samples were subjected to a modified Knott method, to a test for the detection of circulating Dirofilaria immitis antigen, and to a Dirofilaria species-specific real-time PCR for the simultaneous detection of D. immitis and Dirofilaria repens, targeting on partial cytochrome oxidase subunit 1 and internal transcribed spacer-2, respectively. Two abdomen and one thorax pools from A. albopictus were positive for D. immitis, with minimum infection rates of 1.14 and 0.51, respectively, and a probability of a single positive specimen to be infected of P = 0.6 % (95 % confidence interval (CI) = 0.12-1.73). Out of 439 examined subjects, 22 (5.0 %) tested positive for Dirofilaria spp. in at least one diagnostic test. A specific D. immitis infestation rate of 3.5 % was found among the privately owned dogs, while shelter dogs tested positive only for D. repens with a prevalence of 3.4 %; one cat tested molecularly positive for D. immitis. There was a significantly higher rate of positivity among guard dogs for D. immitis (odds ratio, 6.24, 95 % CI, 1.26-25.28; P < 0.05). The increasing risk of D. immitis infection in southern Italy is supported by the noteworthy positivity of A. albopictus populations and the cat. Our data highlight the usefulness to include filarioid infestation in routine diagnosis.

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.

Defining the concept of 'tick repellency' in veterinary medicine.

Although widely used, the term repellency needs to be employed with care when applied to ticks and other periodic or permanent ectoparasites. Repellency has classically been used to describe the effects of a substance that causes a flying arthropod to make oriented movements away from its source. However, for crawling arthropods such as ticks, the term commonly subsumes a range of effects that include arthropod irritation and consequent avoiding or leaving the host, failing to attach, to bite, or to feed. The objective of the present article is to highlight the need for clarity, to propose consensus descriptions and methods for the evaluation of various effects on ticks caused by chemical substances.

Physiological and molecular characterization of atypical lipid-dependent Malassezia yeasts from a dog with skin lesions: adaptation to a new host?

Three lipid-dependent Malassezia isolates (here named 114A, 114B and 114C) recovered from a dog with skin lesions were phenotypically and genotypically characterized. All presented ovoid cells and buds formed on a narrow base. Most of the results from physiological tests were consistent with those of Malassezia furfur. The phylogenetic analysis of ITS-1 and LSU nucleotide sequences was concordant in placing all three clinical Malassezia isolates close to M. furfur. However, the phylogenetic data on the chs-2 sequence revealed that clinical isolate 114A is distinct from M. furfur and was closely affiliated to the sequence of M. pachydermatis with high nodal support. In particular, lipid-dependent isolates 114A displayed chs-2 sequences similar (100%) to that of the non-lipid dependent species Malassezia pachydermatis. The presence of the genetic and physiological polymorphisms detected in these three isolates of M. furfur could have resulted from a process of adaptation of this anthropophilic species to a new host.

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.