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Background Metastrongyloid parasites Angiostrongylus vasorum and Crenosoma vulpis infect wild and domestic canids and are important pathogens in dogs. Recent studies indicate that gastropod intermediate hosts infected with various metastrongyloids spontaneously shed infective third-stage larvae (L3) into the environment via feces and mucus under laboratory conditions. Shed L3 retain motility up to 120 days, but whether they retain infectivity was unknown. Methods To assess the infectivity of shed L3, the heart/lungs of six red foxes ( Vulpes vulpes ) were obtained from trappers in Newfoundland, Canada. Lungs were examined for first-stage larvae (L1) by the Baermann technique. A high number of viable A. vasorum L1 and a low number of C. vulpis L1 were recovered from one fox; these were used to infect naïve laboratory-raised Limax maximus . L3 recovered from slugs by artificial digestion were fed to two naïve purpose-bred research beagles (100 L3/dog). L1 shed by these two dogs was used to infect 546 L. maximus (2000–10,000 L1/slug). L3 shedding was induced by anesthetizing slugs in soda water and transferring them into warm (45 °C) tap water for at least 8 h. Shed L3 recovered from slugs were aliquoted on romaine lettuce in six-well tissue culture plates (80–500 L3/well) and stored at 16 °C/75% relative humidity. Four naïve research beagles were then exposed to 100 L3/dog from larvae stored for 0, 2, 4, or 8 weeks, respectively, after shedding. Results All four dogs began shedding C. vulpis L1 by 26–36 days post-infection (PI). All four dogs began shedding A. vasorum L1 by 50 days PI. Conclusions L3 infectivity for the definitive host was retained in both metastrongyloids, indicating the potential for natural infection in dogs through exposure from environmental contamination. As an additional exposure route, eating or licking plant or other material(s) contaminated with metastrongyloid L3 could dramatically increase the number of dogs at risk of infection from these parasites. Graphical Abstract

Background Canine vector-borne diseases (CVBDs) represent a wide group of diseases of major significance for canine health. In addition to their veterinary importance, many of these diseases are of great zoonotic concern, posing a risk of potential transmission to humans. To date, there has been scant knowledge regarding the prevalence, distribution and risk factors of CVBDs in Greece. Therefore, the objectives of the present study were to update the current knowledge on the seroprevalence of Dirofilaria immitis , Anaplasma spp., Ehrlichia spp. and Borrelia burgdorferi ( sensu lato ) in dogs in Greece and, furthermore, to assess possible environmental and any other risk factors associated with these infections. A total of 1000 apparently healthy and randomly selected dogs, presented in veterinary clinics, were involved at the national level ( n  = 66 municipalities). Serum samples were obtained from each individual dog and were tested using the SNAP ® 4Dx ® Plus kit from IDEXX Laboratories. Possible risk factors were assessed using binary regression models, including dogs’ lifestyle, climatological parameters and the altitude of the region. Results Overall, 21.8% (95% CI: 19.4–24.5%) of the sampled dogs were found to be seropositive to at least one of the four pathogens examined. The most prevalent pathogen was Ehrlichia spp. (12.5%, 95% CI: 10.6–14.7) followed by D. immitis (9.0%, 95% CI: 7.8–11.5) and Anaplasma spp. (6.2%, 95% CI: 4.9–7.9). The lowest prevalence (0.1%) was recorded for B. burgdorferi ( s.l. ) where only one dog was found to be positive. Among the examined risk factors, low mean temperature was found to increase the prevalence of Ehrlichia spp. ( P  ≤ 0.001) and Anaplasma spp. ( P  ≤ 0.001), while low minimum temperature increased the prevalence of D. immitis ( P  ≤ 0.001). In addition, low total annual rainfall had an effect of the prevalence of Ehrlichia spp. ( P  ≤ 0.01). Altitude also had a significant effect on the prevalence of D. immitis ( P  ≤ 0.05) and Anaplasma spp. ( P  ≤ 0.01). Conclusions To our knowledge, this is the first large-scale seroepidemiological study of CVBDs in Greece. It has been evidenced that environmental factors such as temperature, rainfall and altitude can influence the prevalence and distribution of CVBDs.

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.

Rodents are important intermediate and paratenic hosts for carnivore parasites, including the important zoonotic agents Toxoplasma, Echinococcus and Toxocara. Monitoring of such parasites in rodents can be used to detect increasing risks for human and veterinary public health. Rodents were trapped at four sites in Berlin, two near the city center, two at the periphery. PCRs were conducted to detect Coccidia (target ITS-1) and specifically Toxoplasma gondii (repetitive element) in brain and ascarids (ITS-2) in muscle or brain tissue. During necropsies, metacestodes were collected and identified using ITS-2 and 12S rRNA PCRs. An ELISA to detect antibodies against Toxocara canis ES antigens was performed. Within the 257 examined rodents, the most frequently observed parasite was Frenkelia glareoli predominantly found in Myodes glareolus. T. gondii was only detected in 12 rodents and Microtus spp. (although strongly underrepresented) had a significantly increased chance of being positive. Neither Echinococcus nor typical Taenia parasites of dogs and cats were found but Mesocestoides litteratus and Taenia martis metacestodes were identified which can cause severe peritoneal or ocular cysticercosis in dogs, primates and humans. Using PCR, the ascarids T. canis (n = 8), Toxocara cati (4) and Parascaris sp. (1) were detected predominantly in muscles. Seroprevalence of T. canis was 14.2% and ELISA was thus more sensitive than PCR to detect infection with this parasite. Non-parametric multidimensional scaling and cluster analysis revealed that parasite communities could be grouped into an urban and a peri-urban cluster with high frequency of ascarid-positive rodents in urban and high frequency of F. glareoli in peri-urban sites. Prevalence rates of parasites in rodents with potential impact for human or veterinary public health are considerable and the monitoring of transmission cycles of carnivore parasites in intermediate rodent hosts is recommended to estimate the health risks arising from wild and domesticated carnivores.

Background Angiostrongylus vasorum , Crenosoma vulpis and Eucoleus aerophilus are a source of increasing concern, potentially causing significant pulmonary and severe cardiac/systemic diseases in domestic dogs and wild canids, especially red foxes ( Vulpes vulpes ). To investigate the prevalence and geographical distribution of these parasites in central Germany, a total of 569 foxes were examined by dissection. Methods Pluck (heart and lung) and faecal samples of red foxes were collected from three regions of Germany. Lungs, hearts and adjacent vessels were processed for adult nematode detection. Parasitological diagnoses of faecal samples were performed by SAF technique, Giardia - and Cryptosporidium -Coproantigen-ELISAs and by a duplex copro-PCR for the detection of A. vasorum and C. vulpis DNA. Results Foxes originated from three Federal States of central Germany: Thuringia ( n  = 359); Rhineland-Palatinate ( n  = 121) and Hesse ( n  = 89). High prevalences for all three nematodes were detected, with E. aerophilus (69.4%; 395/569), followed by C. vulpis (32.3%; 184/569) and A. vasorum (14.1%; 80/569). In case of A. vasorum , prevalences varied significantly between Federal States, with the highest prevalence of 27.3% in Rhineland-Palatinate, followed by 19.1% and 8.4% in Hesse and Thuringia, respectively. The presence of A. vasorum in fox populations showed a rather patchy distribution, increasing from north-eastern to south-western regions. Analyses on C. vulpis revealed prevalences of 35.1%, 30.3% and 25.6% (Thuringia, Hesse and Rhineland-Palatinate, respectively). The most prevalent lungworm nematode was E. aerophilus , with a prevalence of 75.2%, 71.9% and 66.9% (Rhineland-Palatinate, Hesse and Thuringia, respectively) and an almost area-wide equal distribution. Significant differences for single parasite prevalences within geographical regions of the Federal States could be detected whilst no correlation between age or gender and parasite occurrence was estimated. Weak seasonality for the winter months for A. vasorum , stronger correlation to spring and late summer for C. vulpis and no correlation to any season for E. aerophilus were detected. The method of dissection revealed a significantly higher sensitivity for C. vulpis when compared with the results of the duplex copro-PCR. Conclusions A sylvatic cycle was confirmed for all three lungworm nematodes in the examined area. The prevalences for all three lungworm nematodes are some of the highest recorded so far in German foxes. The data suggest that A. vasorum might be spreading from south-western to north-eastern parts of Germany.

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. Graphical Abstract

BACKGROUND: Giardia is now considered the most common enteric parasite in well cared for dogs and cats in developed countries. The ecology, epidemiology and clinical impact of infections with this parasite in such animals is still not fully understood due to variable results across different studies. METHODS: Faecal samples were collected between 2009 and 2012 from privately owned cats and dogs in Germany presented to local veterinarians for a variety of reasons. Giardia positive samples were identified by microscopy and coproantigen methods. Total faecal DNA was extracted from Giardia positive samples and multilocus genotyping methods (18S rDNA, β-giardin, GDH) were applied. Relationships between host age, sex, and breed, season of presentation and the different species of Giardia detected were assessed. RESULTS: A total of 60 cat and 130 dog samples were identified as Giardia positive. Potentially zoonotic Giardia was identified in both animal species. Cats had a similarly high rate of infection with the G. duodenalis and G. cati. Cats less than 1 year were more likely to have G. duodenalis than cats older than 1 year. Pure breed cats demonstrated a greater proportion of zoonotic species than mixed breed cats. In samples from dogs, G. canis (C and D genotypes) were identified most commonly. Male dogs were more likely to have G. canis (genotype D) than female dogs. The 18S rDNA PCR protocol was the most successful followed by the β-giardin and GDH (amplifying from 92%, 42% and 13% of samples respectively). CONCLUSIONS: The potentially zoonotic species G. duodenalis and G. enterica were found in cat and dog samples, with G. duodenalis found in greater numbers; however, this may be due to the detection techniques utilised. Cats appeared to show a relationship between G. duodenalis and G. cati with age and breed, which may be explained by different housing habitats for pure and mixed breed cats. The different success rates for the three loci utilised highlights the usefulness of the 18S locus as a screening tool, as well as the importance of using multiple loci for genotyping to fully determine the level of multiple infection of Giardia present.

Background Phagocyte-derived extracellular traps (ETs) were recently demonstrated mainly in vertebrate hosts as an important effector mechanism against invading parasites. In the present study we aimed to characterize gastropod-derived invertebrate extracellular phagocyte trap (InEPT) formation in response to larval stages of important canine and feline metastrongyloid lungworms. Gastropod haemocytes were isolated from the slug species Arion lusitanicus and Limax maximus , and the snail Achatina fulica , and exposed to larval stages of Angiostrongylus vasorum , Aelurostrongylus abstrusus and Troglostrongylus brevior and investigated for gastropod-derived InEPT formation. Results Phase contrast as well as scanning electron microscopy (SEM) analyses of lungworm larvae-exposed haemocytes revealed ET-like structures to be extruded by haemocytes thereby contacting and ensnaring the parasites. Co-localization studies of haemocyte-derived extracellular DNA with histones and myeloperoxidase in larvae-entrapping structures confirmed classical characteristics of ETs. In vivo exposure of slugs to A. vasorum larvae resulted in InEPTs being extruded from haemocytes in the slug mucous extrapallial space emphasizing the pivotal role of this effector mechanism against invasive larvae. Functional larval entrapment assays demonstrated that almost half of the haemocyte-exposed larvae were contacted or even immobilized by released InEPTs. Overall, as reported for mammalian-derived ETs, different types of InEPTs were here observed, i.e. aggregated, spread and diffused InEPTs. Conclusions To our knowledge, this study represents the first report on metastrongyloid lungworm-triggered ETosis in gastropods thereby providing evidence of early mollusc host innate immune reactions against invading larvae. These findings will contribute to the better understanding on complex parasite-intermediate host interactions since different gastropod species bear different transmitting capacities for metastrongyloid infections.

Cutaneous dirofilariosis is a canine mosquito-borne zoonosis that can cause larva migrans disease in humans. Dirofilaria repens is considered an emerging pathogen occurring with high prevalence in Mediterranean areas and many parts of tropical Asia. In Hong Kong, a second species, Candidatus Dirofilaria hongkongensis, has been reported. The present study aimed to compare mitochondrial genomes from these parasites and to obtain population genetic information. Complete mitochondrial genomes were obtained by PCR and Sanger sequencing or ILLUMINA sequencing for four worms. Cytochrome oxidase subunit 1 sequences identified three as D. repens (all from Europe) and one as C. D. hongkongensis (from India). Mitochondrial genomes have the same organization as in other spirurid nematodes but a higher preference for thymine in the coding strand. Phylogenetic analysis was in contradiction to current taxonomy of the Onchocercidae but in agreement with a recent multi-locus phylogenetic analysis using both mitochondrial and nuclear markers. D. repens and C. D. hongkongensis sequences clustered together and were the common sister group to Dirofilaria immitis. Analysis of a 2.5 kb mitochondrial genome fragment from macrofilaria or canine blood samples from Europe (42), Thailand (2), India (1) and Vietnam (1) revealed only small genetic differences in the D. repens samples including all European and the Vietnam sample. The Indian C. D. hongkongensis and the two Thai samples formed separate clusters and differences were comparatively large. Genetic differences between Dirofilaria spp. causing cutaneous disease can be considerable whereas D. repens itself was genetically quite homogenous. C. D. hongkongensis was identified for the first time from the Indian subcontinent. The full mitochondrial genome sequence strengthens the hypothesis that it represents an independent species and the Thai samples might represent another cryptic species, Candidatus Dirofilaria sp. 'Thailand II', or a quite divergent population of C. D. hongkongensis.

Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus and Troglostrongylus brevior can cause severe cardiovascular and pulmonary symptoms in companion animals and wildlife. Recently, these nematodes were reported to spread within Europe and South America. The reasons behind this are still unknown, but obligate gastropod intermediate host populations might play a role. Therefore, lungworm infections in terrestrial slug populations in selected geographic areas of the Federal States of Bavaria and of Baden-Wuerttemberg, Germany, were studied. In total, 517 slugs (462 Arion spp., 51 Deroceras reticulatum, one Limax maximus, and three unknown slug species) were collected in the summer and autumn seasons, artificially digested and microscopically and molecularly analyzed for the presence of metastrongyloid lungworm larvae. Overall, gastropods showed a prevalence of 11.61% (60/517) for A. vasorum, 1.74% (9/517) for A. abstrusus, 0.77% (4/517) for C. vulpis and 0.97% (5/517) for T. brevior infections, respectively. In Obrigheim (Baden-Wuerttemberg), a hyperendemic focus of canine angiostrongylosis was identified. Here, gastropod infection rates rose from 13.60% (17/125) to 62.96% (34/54) within a few months. In total, 25.61% (84/328) of analysed terrestrial gastropods from Baden-Wuerttemberg were positive for metastrongyloids. In contrast, Bavarian gastropods showed a much lower prevalence of 4.76% (9/189). For the first time, the presence of T. brevior was confirmed for Arion spp. in Baden-Wuerttemberg via molecular analyses. Overall, the current data confirm that canine angiostrongylosis occurs in hyperendemic foci in certain geographic areas with high infection rates in intermediate host populations. As a result, the prevalence for a specific region can rise remarkably within a short period of time. Thus, for a better understanding of lungworm epidemiology in Germany and to protect dogs from angiostrongylosis in hyperendemic foci, it seems mandatory to enhance current efforts on Metastrongyloidea-targeted monitoring on a geographical and time span-related level.