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The diagnosis of eimeriosis in calves mainly relies on the presence of diarrhoea and the excretion of Eimeria oocysts in the faeces. Restraining the animals to collect rectal samples for diagnostic purposes is stressful and time-consuming. The aim of this study was to evaluate a method for the quantification of oocysts in environmental barn straw samples. To investigate the recovery rate of the method, straw and Eimeria negative faeces were spiked with Eimeria oocysts in plastic bags and mixed with water and 0.05% Tween 20 (v/v); the liquids were filtered twice through sieves (mesh size 300 and 52 μm), centrifuged and the number of oocysts in the sediment determined using a McMaster counting chamber. A recovery rate of 52.4% (95% confidence interval: 48.2–56.5%) was obtained. In the following, field straw (n = 156) and individual faecal samples (n = 195, also analysed by McMaster counting chambers) were collected on four different farms. Eimeria oocysts were present on all farms in faecal (84/195, 43.1%) and straw samples (119/156, 76.3%). In 37 (23.7%) straw samples, sporulated oocysts were observed, with a sporulation rate ranging from 0 to 40%. Despite high variability between farms and examination days, mean numbers of oocysts in the straw positively correlated with mean numbers of oocysts excreted in the faeces (ρSpearman = 0.60). The examination of environmental straw samples may represent an easy-to-perform, non-invasive, inexpensive preliminary diagnostic approach for surveillance of eimeriosis at group level, having the potential to assess the infection pressure.

Background The lungworm Aelurostrongylus abstrusus infects wild and domestic feline species worldwide and is considered a primary respiratory parasite of cats. Definitive diagnosis is based on identification of first stage larvae (L1s) released in faeces approximately 5 to 6 weeks after infection. More recently, serology has been shown to be a diagnostic alternative for A. abstrusus infection in cats. The present study was aimed at evaluating the diagnostic performance of serological antibody detection compared to faecal examination for A. abstrusus infection in a population of cats with known infection status from endemic areas in Italy and to identify factors (larval scores, age, co-infections with other helminths) that may influence test sensitivity and specificity. Methods All cats resulting positive in the Baermann technique (n=78) were tested with the A. abstrusus ELISA An additional 90 serum samples from cats living in three geographical areas with infection prevalence greater than 10%, but that resulted negative in Baermann, were also tested. Results Among 78 cats copromicroscopically positive for L1s of A. abstrusus (Group 1), 29 (37.2%) were seropositive in ELISA. Of the 90 cats from Group 2 (cats living in three geographical areas in Italy with A. abstrusus prevalence greater than 10%, but negative in Baermann examination), 11 (12.2%) were positive in ELISA. The overall seroprevalence was 23.8%. There was no statistical difference either between average optical density (OD) values of cats excreting >100 L1s vs. cats excreting P value = 0.3247) or comparing the OD values with age of infected cats. Few Baermann-negative cats positive for Toxocara cati or hookworms were seropositive, supporting lack of cross reactivity to these nematodes. Conclusions Results from the present study suggest that relying solely on faecal examination may underestimate prevalence of A. abstrusus infection in cats and that field surveys based on antibody detection is useful for establishing true prevalence of infected and/or exposed animals.

The skin microbiota plays an essential role in the protection against pathogens. It is our skin microbiota that makes us smell different from each other, rendering us more or less attractive to mosquitoes. Mosquitoes exploit skin bacterial odours to locate their hosts and are vectors of pathogens that can cause severe diseases such as malaria and dengue fever. A novel solution for long-lasting protection against insect vectors of disease could be attained by manipulating the bacterial commensals on human skin. The current options for protection against biting insects usually require topical application of repellents that evaporate within hours. We discuss possible routes for the use of commensal bacteria to create a microbial-based repellent.

Anthelmintic resistance (AR) has thus far only rarely been reported for intestinal helminths of dogs and cats, in contrast to parasites of livestock and horses. We highlight possible reasons for this striking and important discrepancy, including ecological, biological and genetic factors and/or intervention regimens of key intestinal helminths concerning both host groups. In view of the current knowledge related to the genetics, mechanisms and principles of AR development, we point at issues which in our view contribute to a comparatively lower risk of AR development in intestinal helminths of dogs and cats. Finally, we specify research needs and provide recommendations by which, based on the available information about AR in ruminant and equine helminths, the development of AR in dog and cat helminths may best be documented, prevented or at least postponed., Graphical abstract Image 1, Highlights • Identifying causes of differences in anthelmintic resistance between livestock and dogs & cats. • Discussion of roles of AR mechanisms, genetics and husbandry in delayed AR evolution in dogs & cats. • Research needs for improved tools to monitor AR development in dogs & cats.

The larval stage of the helminthic cestode Echinococcus multilocularis can inflict tumor-like hepatic lesions that cause the parasitic disease alveolar echinococcosis in humans, with high mortality in untreated patients. Opportunistic properties of the disease have been established based on the increased incidence in immunocompromised patients and mouse models, indicating that an appropriate adaptive immune response is required for the control of the disease. However, cellular interactions and the kinetics of the local hepatic immune responses during the different stages of infection with E. multilocularis remain unknown. In a mouse model of oral infection that mimics the normal infection route in human patients, the networks of the hepatic immune response were assessed using single-cell RNA sequencing (scRNA-seq) of isolated hepatic CD3+ T cells at different infection stages. We observed an early and sustained significant increase in natural killer T (NKT) cells and regulatory T cells (Tregs). Early tumor necrosis factor (TNF)- and integrin-dependent interactions between these two cell types promote the formation of hepatic lesions. At late time points, downregulation of programmed cell death protein 1 (PD-1) and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1)-dependent signaling suppress the resolution of parasite-induced pathology. The obtained data provide fresh insight into the adaptive immune responses and local regulatory pathways at different infection stages of E. multilocularis in mice.

With the current expansion of vector-based research and an increasing number of facilities rearing arthropod vectors and infecting them with pathogens, common measures for containment of arthropods as well as manipulation of pathogens are becoming essential for the design and running of such research facilities to ensure safe work and reproducibility, without compromising experimental feasibility. These guidelines and comments were written by experts of the Infravec2 consortium, a Horizon 2020-funded consortium integrating the most sophisticated European infrastructures for research on arthropod vectors of human and animal diseases. They reflect current good practice across European laboratories with experience of safely handling different mosquito species and the pathogens they transmit. As such, they provide experience-based advice to assess and manage the risks to work safely with mosquitoes and the pathogens they transmit. This document can also form the basis for research with other arthropods, for example, midges, ticks or sandflies, with some modification to reflect specific requirements. info:eu-repo/semantics/publishedVersion

The endangered Bengal tiger (Panthera tigris tigris) is a keystone species playing an essential role in ecology as well as in the social and spiritual lives of the Himalayan people. The latest estimate of the Bengal tiger population in Bhutan accounts for 103 individuals. Infectious organisms, including zoonotic parasites causing high burden in human health, have received little attention as a cause of mortality in tigers. Taeniosis/cysticercosis, caused by the cestode Taenia solium, is considered one of the major neglected tropical diseases in Southeast Asia. We present here a case of neurocysticercosis in a Bengal tiger showing advanced neurological disease outside Thimphu, the capital city of Bhutan. After palliative care, the animal died, and necropsy revealed multiple small cysts in the brain. Here we show the presence of two genetic variants of T. solium in the parasite material collected based on PCR and sequencing of the complete cox1 and cytB genes. The sequences form a discrete branch within the Asia plus Madagascar cluster of the parasite. On other hand, tests for feline morbillivirus, feline calicivirus, canine distemper virus, Nipah, rabies, Japanese encephalitis, feline leukaemia and feline immunodeficiency virus were negative. In contrast, PCR for feline herpesvirus was positive and a latex agglutination test revealed an elevated antibody titer against Toxoplasma gondii (titer 1:256). The molecular examination of taeniid eggs isolated from the tiger faeces produced sequences for which the highest homology in GenBank is between 92% and 94% with T. regis and T. hydatigena. This fatal case of T. solium neurocysticercosis, a disease previously unrecorded in tigers or other non-domestic felids, demonstrates an anthropogenically driven transmission of a deadly pathogen which could become a serious threat to the tiger population., Graphical abstract Image 1, Highlights • This is the first report of a fatal infection with Taenia solium in a Bengal tiger. • There is a need for research into infectious disease threats to tigers in Bhutan. • The importance of a ‘One Health’ approach is demonstrated and recommended. • Non-domestic felids.

Metastrongylus sp. is a lungworm with worldwide distribution in wild boars and other suids. In Switzerland, two wild boar populations are geographically divided by the Alps. We investigated 84 wild boars, 52 from north and 32 from south of the Alps, different in sex and age (juveniles, subadults, adults), shot between September and December 2018. The lungs were macroscopically examined and dissected for the detection of Metastrongylus specimens. Additionally, faecal samples were obtained from 55 animals and analysed by sedimentation/flotation and the mini-FLOTAC® method. Overall, 12′774 Metastrongylus sp. specimens were isolated: prevalence was 77.4% and mean burden 196.5 (range: 1–2589), with no significant differences between north (80.8%, 218.0) and south (71.9%, 157.4) nor between sexes and age groups. Macroscopically, dense nodular lesions associated with Metastrongylus sp. were present in 19 out of 65 (33.9%) positive lungs. Five Metastrongylus sp. were detected: M. pudendotectus (67.9%), M. salmi (63.1%), M. confusus (56%), M. apri (44%) and M. asymmetricus (17.9%), with a significant difference (p = 0.012) between north (32.7%) and south (62.5%) for M. apri. The lungworm population was female biased. The number of Metastrongylus sp. eggs in faecal samples did not correlate with worm burdens. Furthermore, the following endoparasites were detected: Isospora suis/Eimeria sp. (74.5%), Strongyloides suis (27.3%%), Trichuris suis (20.0%), Hyostrongylus rubidus/Oesophagostomum sp. (18.2%), Globocephalus sp. (9.1%), Capillaria sp. (7.3%), Ascaris suum (3.6%), Giardia sp. (3.6%) and Balantidium coli (1.8%), with significant differences for S. suis (north 36.1% > south 10.5%) and Globocephalus sp. (only south, 26.3%). Although geographically separated, both Swiss wild boar populations share similar parasite spectra, while also showing some regional idiosyncrasies partially explained by ecological and climatic factors. Despite their clinical relevance being unknown, accurate knowledge concerning the distribution of endoparasites in the wildlife reservoir is relevant to better understand risk factors for the domestic pig population., Graphical abstract Image 1, Highlights • 12′774 Metastrongylus sp. were isolated from wild boars north and south of the Alps. • Metastrongylus prevalence was 77.4%, mean burden 196.5, five species were detected. • No significant differences between wild boar sexes and age groups. • Overall, similar parasite spectra with regional idiosyncrasies were identified. • Known parasite occurrence is relevant to evaluate risk factors for domestic pigs.

Red deer (Cervus elaphus) carcasses showing grey-greenish discolouration have been increasingly observed in the canton of Grisons, Switzerland. We investigated whether Sarcocystis infections were associated with this pathology, and whether wild and domestic canids were involved in their transmission. Meat from affected red deer (n = 26), faeces and intestines from red foxes (Vulpesvulpes) (n = 126), and faeces from hunting dogs (n = 12) from the region, were analysed. Eosinophilic myositis and/or fasciitis were diagnosed in 69% of the deer, and sarcocysts were observed in 89% of the animals. Molecular typing targeting a ~700bp variable region of the 18S rRNA gene revealed Sarcocystis hjorti in 73%, S. venatoria/S. iberica in 54%, S. linearis/S. taeniata in 12%, S. pilosa in 8% and S. ovalis in 4% of the deer samples. No inflammatory changes were observed in red deer carcasses with normal appearance (n = 8); however, sarcocysts were observed in one sample, and S. hjorti, S. venatoria/S. iberica or S. silva DNA was detected in five samples. Sarcocystis oocysts/sporocysts were observed in 11/106 faecal and 6/20 intestinal fox samples, and in 2/12 canine samples. Sarcocystis tenella (n = 8), S. hjorti (n = 2), S. gracilis (n = 2), and S. miescheriana (n = 1) were identified in foxes, and S. gracilis (n = 2), S. capreolicanis (n = 1) and S. linearis/S. taeniata (n = 1) in dogs. This study provides first molecular evidence of S. pilosa and S. silva infection in red deer and S. linearis/S. taeniata in dogs and represents the first record of S. ovalis transmitted by corvids in Central Europe. Although Sarcocystis species infecting red deer are not regarded as zoonotic, the affected carcasses can be declared as unfit for human consumption due to the extensive pathological changes., Graphical abstract Image 1, Highlights • Red deer with eosinophilic myositis/fasciitis were infected with Sarcocystis. • Red foxes and hunting dogs in Grisons transmit Sarcocystis spp. to cervids, ruminants and swine. • Sarcocystis ovalis, transmitted by corvid birds, infect red deer in Central Europe. • Sarcocystis silva and S. pilosa were first detected in red deer by molecular methods. • Red foxes are natural definitive hosts of Sarcocystis hjorti.

The stable fly Stomoxys calcitrans (Diptera: Muscidae) is considered as the main mechanical vector of the lumpy skin disease virus (LSDV). In addition, the mosquito species Aedes aegypti (Diptera: Culicidae) was shown to transmit the virus from donor to receptor animals. Retention of the virus for several days was shown for two additional tropical mosquito species and the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae). In the present study, viral retention for 10- or 7-days post feeding on virus-spiked blood through a membrane was shown for field-collected Aedes japonicus and laboratory-reared Culex pipiens, two widely distributed mosquito species in temperate regions. Viral DNA could be detected from honey-coated Flinders Technology Associates (FTA) cards and shedded faeces for 1 or 4 days after an infectious blood meal was given to Ae. aegypti. Virus increase over time and virus dissemination was observed in laboratory-reared C. nubeculosus, but the virus could be isolated from field-collected biting midges only from the day of exposure to the blood meal. Thus, mosquitoes might serve as mechanical vectors of LSDV in case of interrupted feeding. A putative biological virus transmission by Culicoides biting midges, as suspected from field observations, deserves further investigations.

Background Infection with the cardiopulmonary nematode Angiostrongylus vasorum may cause severe disease in dogs, therefore prophylactic treatments are necessary to prevent infection in dogs at risk. A clinical field study was conducted to demonstrate the efficacy and safety of an oral combination of sarolaner, moxidectin and pyrantel (Simparica Trio®) for the prevention of A. vasorum infection in dogs (prevention study). A survey study was conducted concurrently to determine the infection pressure in the same areas. Methods Prevention and survey studies were both conducted at the same veterinary clinics in endemic hot spots for A. vasorum in Denmark and Italy. The prevention study was a randomized, placebo controlled, double masked study where 622 client-owned dogs were treated and tested at 30 days intervals for 10 months. In the survey study 1628 dogs that were at risk of infection and/or were suspected to be infected were tested by fecal and/or serological methods, and the percent of dogs positive for A. vasorum was calculated. Results In the prevention study, there were no adverse events related to treatment with Simparica Trio®. Two placebo-treated animals became infected with A. vasorum during the 10-month study period, while none of the dogs in the combination product-treated group became infected. In the survey study, 12.2% of the study dogs were found positive to A. vasorum, indicating high exposure to the parasite during the period of the prevention study. Conclusions Monthly oral treatment with the combination of sarolaner, moxidectin and pyrantel (Simparica Trio®) was 100% effective in the prevention of natural infection with A. vasorum in dogs in highly endemic areas. In endemic areas, A. vasorum occurrence in dogs at risk is considerable.

Canine angiostrongylosis is a potentially lethal parasitic disease that can manifest itself with a broad spectrum of clinical signs, including respiratory distress, neurological and bleeding disorders, or non-specific signs. The occurrence of Angiostrongylus vasorum is widely reported in Europe, but very little is known about its presence in Austria. In this first large-scale survey, 1279 sera were collected from Austrian dogs and tested by an ELISA for the detection of circulating antigen of A. vasorum (sensitivity: 95.7%, specificity 94.0%) and by a separate ELISA detecting specific antibodies (sensitivity 81.0%, specificity 98.8%). Furthermore, 1040 faecal samples were tested for the presence of lungworm first stage larvae (L1). One dog (0.1%, 95% confidence intervals [CI]: 0.0–0.4%) was positive in both ELISAs, while 1.2% (n = 15, CI: 0.7–1.9%) of the tested dogs were antigen-positive and 1.5% (n = 19, CI: 0.9–2.3%) were positive for specific antibodies. Overall, 13 dogs (1.3%; CI: 0.7–2.1%) were positive for A. vasorum L1 while 31 dogs were positive for Crenosoma vulpis L1 (3.0%; CI: 2.0–4.2%). One dog shed L1 from both A. vasorum and C. vulpis (0.1%, CI: 0.0–0.5%). Dogs positive for A. vasorum originated from northeast, southeast and south Austria (antigen and/or antibody detection), but also from north, west and southwest Austria (antibody detection) and from northeast and west Austria (L1 detection). One of 88 blood samples (1.1%, CI: 0.0–6.2%) submitted from the eastern part of Austria was positive by a rapid assay for A. vasorum antigen detection (Angio Detect™). Crenosoma vulpis positive samples originated from northwest, north, northeast, south and west Austria. These results confirm the very sporadic occurrence of A. vasorum in the investigated areas of the country. However, due to the substantial infectious pressure from the surrounding countries and the free circulation of dogs and foxes acting as wildlife reservoirs and due to clinical relevance for infected dogs, it is crucial to maintain disease awareness also in areas where the parasite has not yet been detected.

The occurrence of the nematode Angiostrongylus vasorum, also known as the French heartworm, is increasingly being reported from various European countries. The adults of this parasite species live in the pulmonary arteries and right cardiac ventricle of wild canids and domestic dogs. Larval stages and eggs in the lungs induce inflammatory verminous pneumonia, causing severe respiratory disease in dogs. Furthermore, haematological and neurological signs and even death may occur. In Hungary, A. vasorum has been identified in red foxes, golden jackals and in two dogs and some slugs. In this first large-scale survey, 1247 sera from pet dogs were collected and tested by an ELISA for the detection of circulating antigen of A. vasorum and by a separate ELISA to detect specific antibodies against the parasite. A total of 1.36% (n = 17, 95 % confidence intervals, CI: 0.80 - 2.17 %) of the animals were positive in both ELISAs, while 1.76 % (n = 22, CI: 1.11 - 2.66 %) of the tested dogs were antigen-positive only and 2.73 % (n = 34, CI: 1.90 - 3.79 %) were positive for specific antibodies only. Regions with antigen- and antibody-positive animals overlapped and were distributed over nearly the whole sampled areas of the country. A considerable number of cases was observed in Budapest and also in the southern part of the country bordering Croatia, while in the most eastern part bordering Ukraine no positive samples were detected. These results confirm the endemic occurrence of A. vasorum in dogs originating from different parts of Hungary and the significant advantages of A. vasorum serology in epidemiological studies.

Cystic echinococcosis (CE) is widespread and locally frequent in southern Africa where it affects humans, livestock, and wild mammals. However, most data from the region are old and do not provide information on the causative Echinococcus species. For Namibian livestock only anecdotal records were available prior to this preliminary survey. Our retrospective analysis of slaughterhouse records of CE in cattle from the commercial farming area in central and southern Namibia resulted in 1.65% CE prevalence among 35,143 slaughtered cattle in the period 2015-2016. For comparison, carcasses of ruminant livestock were prospectively examined in the communal farming areas of northern Namibia, resulting in three CE cases among only 12 cattle, and no cases among nine goats. To determine the Echinococcus species affecting Namibian livestock, a total of 53 cysts were collected from all parts of the country and analysed for species and genotype by amplification and sequencing of the nad1 gene. All 50 cattle cysts (isolated from 40 cattle), both from the commercial and communal farming areas, were Echinococcus ortleppi (all fertile, and 42/50 from the lungs), while three opportunistically collected cysts from three sheep in southern Namibia were E. canadensis G7. Our data suggest that E. ortleppi is the only CE agent that is relevant for cattle infection in Namibia, and that low prevalence in the commercial farming areas contrasts with high CE burden in the northern traditional husbandry systems. The present data provide baseline information to stimulate epidemiological studies on the transmission pathways of various CE agents in livestock, wildlife, and humans in Namibia and neighbouring countries.

The diagnosis of human taeniosis can be achieved through coproscopy, ELISA or PCR. An important limitation of these methods is the high turnaround time for stool sample collection and preparation, indicating the need for a straightforward sampling strategy. Due to the high metabolic activity and reproductive potential of Taenia spp., we hypothesise that parasite DNA (cells and eggs) present in the peri-anal region of the host can be exploited as a target for molecular diagnosis. We evaluated the feasibility of recovering parasite DNA from the peri-anal area of foxes naturally infected with Taenia spp. Before necropsy, cotton swabs were rubbed at the peri-anal region of foxes. DNA was extracted using alkaline lysis coupled with a commercial DNA isolation kit (method A) or alkaline lysis alone (method B). DNA was used in the multiplex-PCR assay (previously described and called here swab-PCR) and a novel LAMP assay detecting Taenia spp. commonly found in foxes (swab-LAMP). The results of these assays from 105 foxes were compared with the presence of intestinal helminths determined at necropsy and by the sedimentation and counting technique (SCT). The sensitivity of swab-PCR for detecting Taenia (n = 68) was 89.8% (95% CI, 77.7–96.6) and 89.5% (66.9–98.7) using methods A and B, respectively. The sensitivity of the swab-LAMP assay was 83.7% (70.3–92.7) using method A and 89.5% (66.9–98.7) with method B. We postulate that peri-anal swab sampling followed by simplified DNA extraction and LAMP might be a suitable strategy for surveillance of human taeniosis in resource-limited settings in the future.

The Coccidia is the largest group of parasites within the Apicomplexa, a phylum of unicellular, obligate parasites characterized by the possession of an apical complex of organelles and structures in the asexual stages of their life cycles, as well as by a sexual reproductive phase that occurs enterically in host animals. Coccidian sexual reproduction involves morphologically distinct microgametes and macrogametes that combine to form a diploid zygote and, ultimately, following meiosis and mitosis, haploid, infectious sporozoites, inside sporocysts within an oocyst. Recent transcriptomic analyses have identified genes involved in coccidian sexual stage development and reproduction, including genes encoding for microgamete- and macrogamete-specific proteins with roles in gamete motility, fusion and fertilization, and in the formation of the resilient oocyst wall that allows coccidians to persist for long periods in the environment. Transcriptomics has also provided important clues about the regulation of gene expression in the transformation of parasites from one developmental stage to the next, a complex sequence of events that may involve transcription factors such as the apicomplexan Apetala2 (ApiAP2) family, alternative splicing, regulatory RNAs and MORC (a microrchida homologue and regulator of sexual stage development in Toxoplasma gondii). The molecular dissection of coccidian sexual development and reproduction by transcriptomic analyses may lead to the development of novel transmission-blocking strategies.

For a long time, wildlife carnivores have been disregarded for their potential in transmitting zoonotic nematodes. However, human activities and politics (e.g., fragmentation of the environment, land use, recycling in urban settings) have consistently favoured the encroachment of urban areas upon wild environments, ultimately causing alteration of many ecosystems with changes in the composition of the wild fauna and destruction of boundaries between domestic and wild environments. Therefore, the exchange of parasites from wild to domestic carnivores and vice versa have enhanced the public health relevance of wild carnivores and their potential impact in the epidemiology of many zoonotic parasitic diseases. The risk of transmission of zoonotic nematodes from wild carnivores to humans via food, water and soil (e.g., genera Ancylostoma, Baylisascaris, Capillaria, Uncinaria, Strongyloides, Toxocara, Trichinella) or arthropod vectors (e.g., genera Dirofilaria spp., Onchocerca spp., Thelazia spp.) and the emergence, re-emergence or the decreasing trend of selected infections is herein discussed. In addition, the reasons for limited scientific information about some parasites of zoonotic concern have been examined. A correct compromise between conservation of wild carnivores and risk of introduction and spreading of parasites of public health concern is discussed in order to adequately manage the risk of zoonotic nematodes of wild carnivores in line with the ‘One Health’ approach., Graphical abstract Image 1, Highlights • A large variety of wild carnivore nematodes have zoonotic potential. • Limited epidemiological information is available for nematode zoonoses of wild carnivores. • Some nematode zoonosis of wild carnivores may display a considerable morbidity. • A compromise between animal conservation and zoonotic infection is advocated. • Strict control of exotic carnivore species invading wildlife are urgently needed.

Wild mustelids and canids are definitive hosts of Taenia and Versteria spp. while rodents act as natural intermediate hosts. Rarely, larval stages of these parasites can cause serious zoonoses. In Europe, four cases of Taenia martis cysticercosis have been diagnosed in immunocompetent women, and two cases in zoo primates since 2013. In North America, a zoonotic genotype related but distinct from Versteria mustelae has been identified in 2014, which had caused a fatal infection in an orangutan and liver- and disseminated cysticercoses in two severely immune deficient human patients in 2018, respectively. Additionally, we could attribute a historic human case from the USA to this Versteria sp. by reanalysing a published nucleotide sequence. In the last decades, sporadic zoonotic infections by cysticerci of the canid tapeworm Taenia crassiceps have been described (4 in North America, 8 in Europe). Besides, 3 ocular cases from North America and one neural infection from Europe, all in immunocompetent patients, 6 cutaneous infections were described in severely immunocompromised European patients. Correspondingly, besides oral infections with taeniid eggs, accidental subcutaneous oncosphere establishment after egg-contamination of open wounds was suggested, especially in cases with a history of cutaneous injuries at the infection site. Taenia multiceps is mainly transmitted in a domestic cycle. Only five human coenurosis cases are published since 2000. In contrast, T. serialis coenurosis (1 human case since 2000) is primarily transmitted by wild canids. The etiological diagnosis of exotic cysticercoses is challenging. Usually, clinical material does not allow for a morphological identification, and serological tests are not available. These limitations have partly been overcome by molecular tools. Without claiming any dramatic emergence of cysticercoses and coenuroses transmitted by wild carnivores, further sporadic cases of such ‘exotic’ infections have to be expected., Graphical abstract Taenia crassiceps cysticerci from the abdomen of an infected rodent.Image 1, Highlights • Wild canids and mustelids transmit rare but potentially fatal cysticercoses and coenuroses. • Martens and weasels can rarely transmit dangerous parasitic infections. • Tapeworm eggs may contaminate wounds and develop locally. • In North America, the mustelid tapeworm Versteria causes severe human infections. • Molecular analyses from minute clinical material allows for a specific diagnosis.

Aelurostrongylus abstrusus (Nematoda, Metastrongyloidea) is a worldwide occurring lungworm causing verminous pneumonia in cats. To date the Baermann method is the most used procedure to diagnose A. abstrusus infection by isolating first stage larvae from faeces, though its sensitivity and specificity can be impaired by several factors. An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against A. abstrusus has been recently developed as a diagnostic alternative. The present study evaluated the seroprevalence for A. abstrusus infection in cats from two endemic areas of Italy. Overall, 250 sera were sampled and tested for the presence of antibodies against A. abstrusus. Based on the results obtained from 20 cats proven to be infected by A. abstrusus using Baermann technique and molecular methods, and from 20 negative cats (Subset A), a cut off value of 0.347 optical density (OD) was determined, leading to a sensitivity of 95% and a specificity of 100%. Two-hundred and ten cats (142 and 68 from Abruzzo and Umbria regions, respectively) were included in Subset B (i.e. 202 negative by Baermann examination and 8 positive for Troglostrongylus brevior). Antibodies against A. abstrusus were detected in forty-five (21.4%, 95% CI: 16.1-27.6%) samples. This study confirms the occurrence of A. abstrusus in endemic areas of Italy and indicates that one-fifth of randomly selected cats have or had a lungworm infection with production of antibodies.

Feline lungworms affect the respiratory tract of domestic cats causing respiratory conditions of various degrees. In this study, we investigated the exposure of cats to feline lungworm infections by detecting antibodies in a large population of animals from several regions of Italy. Sera of 1087 domestic cats living in regions of the north (n = 700), the centre (n = 227) and the south (n = 160) of Italy were examined by a newly developed indirect ELISA conceived for detection of antibodies against the most frequently occurring feline lungworm Aelurostrongylus abstrusus. Individual cat data (i.e., age, sex, neutering status and provenience) were analysed as potential risk factors for exposure to lungworm infections. Samples were additionally screened for feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) proviral DNAs. Overall, 9% (98/1087; 95% confidence interval (CI) 7.4-10.9%) of the animals tested seropositive to lungworm antibodies. Positive cats were identified in the north (7.1%; CI 5.5-9.3%), in the centre (5.3%; CI 3.0-9.0%) and in the South (22.5%; CI 16.7-29.6%), with more seropositive animals in the latter area (p

Background Dogs are the main reservoir hosts of Leishmania infantum; nevertheless, recent investigations indicate a likely role for cats in the epidemiology of Leishmania infection. Feline leishmaniosis (FeL) remains poorly characterised, partly due to the lack of suitable diagnostic tools. This study aimed to compare serum amyloid A (SAA) levels and serum protein electrophoresis (SPE) profiles (specifically, alpha 2 and gamma globulins) in cats naturally exposed to or infected by L. infantum from southern Italy versus those of healthy controls and versus cats with neoplastic or inflammatory conditions from non-endemic areas. Methods Serum or plasma samples from four cohorts of cats were analysed for SAA levels and by SPE: (i) G1: healthy controls from Leishmania-non-endemic regions of Switzerland; (ii) G2: cats pre-diagnosed with neoplastic or inflammatory conditions available from the University of Cambridge sample archive; (iii) G3: L. infantum-seropositive, quantitative (q)PCR-negative cats from southern Italy; (iv) G4: L. infantum-seropositive and qPCR-positive cats from southern Italy. SAA data were assessed for normality and homoscedasticity using the Shapiro–Wilk and Levene’s tests, respectively; the Kruskall–Wallis test, followed by Dunn’s test with Bonferroni correction were subsequently used to compare SAA serum levels between groups. A weighted generalised linear model with a binomial distribution was used to assess statistically significant differences in the numbers of animals displaying elevated gamma globulins and increased alpha 2 globulins between groups. Results Overall, 68 samples were analysed (G1: n = 16, G2: n = 20, G3: n = 20, G4: n = 12). Cats suffering from neoplastic and inflammatory conditions (G2 ) showed significantly higher SAA levels than healthy controls (G1) (median values [interquartile range]: G1: 0.00 [0.00–0.00] mg/l versus G2: 0.85 [0.00–49.55] mg/l). G2, G3 and G4 cats showed higher percentages of individuals with increased alpha 2 globulins (percentages ± standard error: G1 = 20.0% ± 10.3, G2 = 80.0% ± 8.9, G3 = 70.0% ± 10.2, G4 = 75.0% ± 12.5) and gamma globulins (G1 = 0.0% ± 0, G2 = 65.0% ± 10.7, G3 = 50.0% ± 11.2, G4 = 58.3% ± 14.2) than healthy control cats (G1). For all three markers, no significant difference between cats within G2, G3 and G4 was recorded. Conclusions This study indicates that the proportions of animals with elevated levels of alpha 2 and gamma globulins are significantly higher in cats exposed to and infected with L. infantum. Levels of SAA and alpha 2 and gamma globulins may not be used to differentiate between L. infantum infection or exposure, and neoplastic and/or inflammatory conditions. Graphical Abstract

In the past 50 years, enormous progress has been made in the diagnosis, treatment and control of taeniid cestode infections/diseases and in the scientific understanding thereof. Most interest in this group of parasites stems from the serious diseases that they cause in humans. It is through this lens that we summarize here the most important breakthroughs that have made a difference to the treatment of human diseases caused by these parasites, reduction in transmission of the taeniid species associated with human disease, or understanding of the parasites’ biology likely to impact diagnosis or treatment in the foreseeable future. Key topics discussed are the introduction of anti-cestode drugs, including benzimidazoles and praziquantel, and the development of new imaging modalities that have transformed the diagnosis and post-treatment monitoring of human echinococcoses and neurocysticercosis. The availability of new anti-cestode drugs for use in dogs and a detailed understanding of the transmission dynamics of Echinococcus granulosus sensu lato have underpinned successful programs that have eliminated cystic echinococcosis in some areas of the world and greatly reduced the incidence of infection in others. Despite these successes, cystic and alveolar echinococcosis and neurocysticercosis continue to be prevalent in many parts of the world, requiring new or renewed efforts to prevent the associated taeniid infections. Major advances made in the development of practical vaccines against E. granulosus and Taenia solium will hopefully assist in this endeavour, as might the understanding of the parasites’ biology that have come from an elucidation of the nuclear genomes of each of the most important taeniid species causing human diseases.

Background Since the huge epidemic of Zika virus (ZIKV) in Brazil in 2015, questions were raised to understand which mosquito species could transmit the virus. Aedes aegypti has been described as the main vector. However, other Aedes species (e.g. Ae. albopictus and Ae. japonicus) proven to be competent for other flaviviruses (e.g. West Nile, dengue and yellow fever), have been described as potential vectors for ZIKV under laboratory conditions. One of these, the Asian bush mosquito, Ae. japonicus, is widely distributed with high abundances in central-western Europe. In the present study, infection, dissemination and transmission rates of ZIKV (Dak84 strain) in two populations of Ae. japonicus from Switzerland (Zürich) and France (Steinbach, Haut-Rhin) were investigated under constant (27 °C) and fluctuating (14–27 °C, mean 23 °C) temperature regimes. Results The two populations were each able to transmit ZIKV under both temperature regimes. Infectious virus particles were detected in the saliva of females from both populations, regardless of the incubation temperature regime, from 7 days post-exposure to infectious rabbit blood. The highest amount of plaque forming units (PFU) (400/ml) were recorded 14 days post-oral infection in the Swiss population incubated at a constant temperature. No difference in terms of infection, dissemination and transmission rate were found between mosquito populations. Temperature had no effect on infection rate but the fluctuating temperature regime resulted in higher dissemination rates compared to constant temperature, regardless of the population. Finally, transmission efficiency ranged between 7–23% and 7–10% for the constant temperature and 0–10% and 3–27% under fluctuating temperatures for the Swiss and the French populations, respectively. Conclusions To the best of our knowledge, this is the first study confirming vector competence for ZIKV of Ae. japonicus originating from Switzerland and France at realistic summer temperatures under laboratory conditions. Considering the continuous spread of this species in the northern part of Europe and its adaptation at cooler temperatures, preventative control measures should be adopted to prevent possible ZIKV epidemics.

Helminth parasites have a remarkable ability to persist within their mammalian hosts, which is largely due to their secretion of molecules with immunomodulatory properties. Although the soluble components of helminth secretions have been extensively studied, the discovery that helminths release extracellular vesicles (EVs) has added further complexity to the host-parasite interaction. Whilst several studies have begun to characterise the molecules carried by helminth EVs, work aimed at investigating their biological functions has been hindered by a lack of helminth-specific EV markers. To begin to address this, we summarised helminth EV literature to date. With a focus on the protein and microRNA (miRNA) cargo, we aimed to detect similarities and differences across those major groups of helminths for which data are available; namely nematodes, trematodes and cestodes. Pfam analysis revealed that although there is no universal EV marker for all helminth species, the EF-hand protein family was present in all EV datasets from cestodes and trematodes, and could serve as a platyhelminth EV biomarker. In contrast, M13 metallopeptidases and actin may have potential as markers for nematode EVs. As with proteins, many miRNA families appeared to be species-, stage-, or dataset-specific. Two miRNA families were common to nematode EVs (mir-10 and let-7); the miRNA cargo of EVs secreted by clade I species appeared somewhat different from species from other clades. Five miRNA families (mir-71, mir-10, mir-190, let-7 and mir-2) were shared by all trematode species examined. Our analysis has identified novel markers that may be used in studies aimed at characterising helminth EVs and interrogating their function at the host-parasite interface. In addition, we discuss the heterogeneity of methods used for helminth EV isolation and emphasise the need for a standardised approach in reporting on helminth EV data. Fil: Sotillo, Javier. Instituto de Salud Carlos Iii (isciii); España Fil: Robinson, Mark W.. The Queens University of Belfast; Irlanda Fil: Kimber, Michael J.. Queen’s University ; Reino Unido Fil: Cucher, Marcela Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Ancarola, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones en Microbiología y Parasitología Médica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones en Microbiología y Parasitología Médica; Argentina Fil: Nejsum, Peter. Aarhus University. Aarhus Institute Of Advanced Studies.; Dinamarca Fil: Marcilla, Antonio. Universidad de Valencia; España Fil: Eichenberger, Ramon M.. Universitat Zurich; Suiza Fil: Tritten, Lucienne. Universitat Zurich; Suiza

Control of cystic echinococcosis (CE) relies on interrupting Echinococcus granulosus sensu lato transmission through interventions in dogs and livestock. However, primary prevention measures aimed at avoiding ingestion of Echinococcus eggs may help reduce the burden of human CE. CE is generally considered, to variable extents, to be foodborne, but there is little evidence on the actual contamination of matrices and sociocultural factors involved in parasite transmission. An overall appraisal of published literature suggests that environmental contamination, possibly through hand-to-mouth transmission, may be of primary importance. While in most endemic areas sufficient epidemiological information is available to start CE control programs, identifying the main sources of infection to humans would allow optimization of site-specific interventions while avoiding irrelevant health education messages.

Toxoplasma gondii-infected pigs play a major role as a source of infection for humans and detection of high-risk herds is essential to implement control measures at the farm level. The aim of this study was to determine whether oral fluid (OF) could be used as a matrix to detect antibodies against T. gondii in infected pigs by immunoblot (IB). For this, OF from experimentally inoculated sows (n = 8) (serial samples) and naturally exposed group-housed fatteners (n = 42 groups, one sample/group) were analysed for IgG and IgA against T. gondii-SAG1 antigen by IB. Simultaneously, each animal was serologically tested for anti-T. gondii IgG by ELISA. Specific IgG was detected in the sera of all inoculated sows from 2 to 3 weeks post inoculation (pi) and in 3.4 to 92% of the pigs in 13 out of 42 groups. Experimentally inoculated sows showed positive OF-IB results for IgA (100%) and IgG (87.5%) at 1.5 weeks pi and continued yielding positive results for IgA (87.5-75%) and IgG (50%) until 4 weeks pi; however, from 8 weeks pi the frequency of detection of both isotypes was lower, despite constantly positive IgG values in serum-ELISA. Interestingly, consecutive daily samplings for 4 days at 13 and 30 weeks pi showed inconsistent results for some sows, showing that the antibody concentration in OF is prone to timely variations. Pooled OF from groups with 91 and 92% of seropositive pigs yielded positive IB results for IgG and IgA. Fattener groups with ≤13% of seropositive pigs gave negative IB results to both isotypes. Our results showed that antibodies to T. gondii can be detected in OF from infected pigs, and that IgA seems to be a more adequate target than IgG. Although OF does not seem to be a robust matrix to assess the serological status for T. gondii in individual animals, this diagnostic approach represents an interesting non-invasive, low-cost and animal welfare friendly option as a screening method at the farm level to determine high exposure to T. gondii in the herd.

Phosphorylated derivatives of phosphatidylinositol (PIPs) are key membrane lipid residues involved in clathrin-mediated endocytosis (CME). CME relies on PIP species PI(4,5)P2 to mark endocytic sites at the plasma membrane (PM) associated to clathrin-coated vesicle (CCV) formation. The highly diverged parasitic protist Giardia lamblia presents disordered and static clathrin assemblies at PM invaginations, contacting specialized endocytic organelles called peripheral vacuoles (PVs). The role for clathrin assemblies in fluid phase uptake and their link to internal membranes via PIP-binding adaptors is unknown. Here we provide evidence for a robust link between clathrin assemblies and fluid-phase uptake in G. lamblia mediated by proteins carrying predicted PX, FYVE and NECAP1 PIP-binding modules. We show that chemical and genetic perturbation of PIP-residue binding and turnover elicits novel uptake and organelle-morphology phenotypes. A combination of co-immunoprecipitation and in silico analysis techniques expands the initial PIP-binding network with addition of new members. Our data indicate that, despite the partial conservation of lipid markers and protein cohorts known to play important roles in dynamic endocytic events in well-characterized model systems, the Giardia lineage presents a strikingly divergent clathrin-centered network. This includes several PIP-binding modules, often associated to domains of currently unknown function that shape and modulate fluid-phase uptake at PVs., Author summary In well-characterized model eukaryotes, clathrin-mediated endocytosis is a key process for uptake of extracellular material and is regulated by more than 50 known proteins. A large number of these carry phosphoinositide (PIP)-binding domains and play a central role in the regulation of endocytosis. Here, we report on the detailed functional characterization of PIP-binding proteins in the intestinal parasitic protist Giardia lamblia. We show evidence that proteins carrying specific PIP-binding domains are directly involved in fluid-phase uptake. Furthermore, using co-immunoprecipitation assays, we confirm these proteins’ association to G. lamblia’s clathrin assemblies. In addition, using state-of-the-art imaging strategies, we demonstrate a previously unappreciated level of complexity involving PIPs and their partner proteins in marking and shaping G. lamblia’s unique endocytic compartments. Our data contribute substantially to an updated working model for G. lamblia’s host-parasite interface, demonstrating how uptake in this parasite is directly regulated by a variety of PIP residues and PIP-binding modules, which have been re-routed from conserved pathways, likely as a result of host-parasite co-evolution.

Background In three randomized, controlled laboratory efficacy studies, the efficacy in the prevention of patent infections of a topical combination of imidacloprid 10%/moxidectin 1% (Advocate® spot-on formulation for cats, Bayer Animal Health GmbH) against larval stages and immature adults of Aelurostrongylus abstrusus, as well as the treatment efficacy of a single or three monthly treatments against adult A. abstrusus, were evaluated. Methods Cats were experimentally inoculated with 300–800 third-stage larvae (L3). Each group comprised 8 animals and the treatment dose was 10 mg/kg bodyweight (bw) imidacloprid and 1 mg/kg bw moxidectin in each study. Prevention of the establishment of patent infections was evaluated by two treatments at a monthly interval at three different time points before and after challenge infection. Curative efficacy was tested by one or three treatments after the onset of patency. Worm counts at necropsy were used for efficacy calculations. Results In Study 1, the control group had a geometric mean (GM) of 28.8 adult nematodes and the single treatment group had a GM of 3.4 (efficacy 88.3%). In Study 2, the control group had a GM of 14.3, the prevention group had a GM of 0 (efficacy 100%), while the treatment group had a GM of 0.1 (efficacy 99.4%). In Study 3, the GM worm burden in the control group was 32.6 compared to 0 in all three prevention groups (efficacy 100% for all of those groups). Conclusions The monthly administration of Advocate® reliably eliminated early larval stages and thereby prevented lung damage from and patent infections with A. abstrusus in cats. Regarding treatment, a single application of Advocate® reduced the worm burden, but it did not sufficiently clear the infection. In contrast, three monthly treatments were safe and highly efficacious against A. abstrusus.

In 2016, a field study was implemented in all Armenian provinces in order to update knowledge on the presence and distribution of both native and invasive mosquito species. Larvae and adult mosquitoes were sampled and identified on the basis of their morphology. Supplementary field surveys were performed in 2017-2018.Between June 20 and July 12, 2016, 117 localities were visited. A total number of 197 sampling units were checked, of which 143 (73%) were positive for mosquitoes (with 1-6 species per sampling unit). A total number of 4157 mosquito specimens were identified to species or species complex level. Ten species represent first records for Armenia: Aedes albopictus, Ae. annulipes, Ae. cataphylla, Ae. cinereus/geminus (probably Ae. cinereus), Ae. flavescens, Anopheles plumbeus, Coquillettidia richiardii, Culex martinii, Cx. torrentium and Culiseta subochrea. The invasive species Ae. albopictus was recorded in a single locality (Bagratashen) at the border point with Georgia, along the main road Tbilisi-Yerevan. This species was further recorded in 2017 and 2018, demonstrating its establishment and spread in north Armenia. These surveys confirm the presence of vectors of malaria parasites (in particular An. sacharovi) and West Nile virus (Cx. pipiens).The knowledge of the Armenian mosquito fauna is extended to a list of 28 species. The record of Aedes albopictus, an important potential vector of many arboviruses, has important implications for public health.Les moustiques d’Arménie : synthèse des connaissances et résultats d’une étude de terrain avec une première mention pour Aedes albopictus.Contexte : En 2016, nous avons réalisé une étude sur le terrain dans toutes les provinces du pays dans le but d’actualiser la présence et la distribution des espèces de moustiques aussi bien natives qu’invasives. Les moustiques récoltés aux stades larvaires et adultes ont été identifiés sur des critères morphologiques. Des suivis additionnels ont été réalisés en 2017 et 2018. Résultats : Entre le 20 juin et le 12 juillet 2016, 117 localités ont été visitées. Au total, 197 unités de collecte ont été prospectées dont 143 (73%) se sont révélées positives pour les moustiques (avec de 1 à 6 espèces par unité de collecte). Au total, 4157 spécimens ont été identifiés au niveau de l’espèce ou du complexe d’espèces. Dix espèces ont été observées pour la première fois en Arménie : Aedes albopictus, Ae. annulipes, Ae. cataphylla, Ae. cinereus/geminus (probablement Ae. cinereus), Ae. flavescens, Anopheles plumbeus, Coquillettidia richiardii, Culex martinii, Cx. torrentium et Culiseta subochrea. L’espèce invasive Ae. albopictus a été observée dans une seule localité (Bagratashen) à la frontière avec la Géorgie, sur l’axe routier principal Tbilisi-Yerevan. Cette espèce a également été observée en 2017 et en 2018, faisant la preuve de son installation et de sa diffusion dans le nord de l’Arménie. Ces suivis confirment la présence des vecteurs de Plasmodium humains (en particulier An. sacharovi) et du virus West Nile (Cx. pipiens). Conclusion : La connaissance de la faune culicidienne d’Arménie progresse, avec une liste actualisée à 28 espèces. L’observation d’Aedes albopictus, un important vecteur potentiel de nombreux arbovirus, a d’importantes implications en termes de santé publique.

The metacestode of Echinococcus multilocularis is the etiological agent of alveolar echinococcosis. The metacestode stage used for research is maintained in rodents by serial passages. In order to determine whether cryopreservation of E. multilocularis metacestodes would be suitable for long-term maintenance and replace serial passages, isolates of different geographic origin were cryopreserved in 1984-1986. The aim of the current study was to test the viability of cryopreserved isolates following long-term cryopreservation (up to 35 years) and to determine the phylogenetic clades these isolates belonged to. Cryopreserved isolates were tested for viability in vitro and in vivo in gerbils. In vitro results of 5 isolates indicated protoscolex survival in 13 of 17 experiments (76%) and metacestode survival in 5 of 12 (42%) in vivo experiments. In vivo results showed 'abortive lesions' in 13 of the 36 animals, 15 were negative and 8 harboured proliferating metacestode tissue containing protoscoleces. Genetic analysis confirmed the isolates belonged to European, Asian and North-American clades. In conclusion, the results of the current study indicate that metacestodes of E. multilocularis are able to survive long-term cryopreservation. Therefore, cryopreservation is a suitable method for long-term storage of E. multilocularis metacestode isolates and reduces the number of experimental animals.

Background Emerging arboviral diseases like Zika, dengue and chikungunya that are transmitted by Aedes aegypti mosquitoes, are increasingly threatening human health. Blends of human-like synthetic chemical attractants can be used to attract host-seeking mosquitoes. The aim of this study was to test new combinations of traps and odour baits in the laboratory, followed by testing the best candidates in the field to improve Ae. aegypti monitoring and surveillance. Methods First, the BG-Suna trap was evaluated for capturing laboratory-reared Ae. aegypti by testing normal and inverted positions in screen cage tests. Secondly, the attractiveness of the MB5 blend, CO2, and their combination was tested. Thirdly, we tested the attractiveness of different trap types (BG-Suna, BG-Sentinel, MM-X and CDC light trap). Finally, we confirmed laboratory results in the field in Paramaribo, Suriname, using the MB5 and BG-Lure odour blends, CO2 and the BG-Sentinel and BG-Bowl trap using a Latin Square design. Results The MB5 blend in combination with CO2 outperformed traps baited only with CO2 or MB5 in screen cage tests (P P = 0.729). In the field, we captured Ae. aegypti, Cx. quinquefasciatus and Cx. nigripalpus. We confirmed the laboratory results and found that the combination of the MB5 blend and CO2 almost doubled Ae. aegypti female captures (P = 0.004) and more than doubled Culex spp. female captures (P = 0.005) compared to using only CO2. Interestingly, the MB5 blend outperformed the commercially available BG-Lure, in the BG-Sentinel (P Ae. aegypti when baited with the MB5 blend in similar numbers as the BG-Sentinel baited with the MB5 (P = 0.362). Conclusions Our study demonstrated that the BG-Sentinel trap baited with the MB5 blend and CO2 outperforms the current golden standard (BG-Sentinel trap with BG-Lure) for monitoring Ae. aegypti females and males, in both laboratory and field experiments. The BG-Bowl baited with the MB5 blend is a good candidate for home use. Finally, the results show that CO2 is an indispensable component of the attractive blend.

Vector‐borne diseases often originate from wildlife and can spill over into the human population. One of the most important determinants of vector‐borne disease transmission is the host preference of mosquitoes. Mosquitoes with a specialised host preference are guided by body odours to find their hosts in addition to carbon dioxide. Little is known about the role of mosquito host preference in the spillover of pathogenic agents from humans towards animals and vice versa. In the Republic of Congo, the attraction of mosquitoes to primate host odours was determined, as well as their possible role as malaria vectors, using odour‐baited traps mimicking the potential hosts of mosquitoes. Most of the mosquito species caught showed a generalistic host preference. Anopheles obscurus was the most abundant Anopheles mosquito, with a generalistic host preference observed from the olfactory response and the detection of various Plasmodium parasites. Interestingly, Culex decens showed a much higher attraction towards chimpanzee odours than to human or cow odours. Human Plasmodium parasites were observed in both human and chimpanzee blood, although not in the Anopheles mosquitoes that were collected. Understanding the role of mosquito host preference for cross‐species parasite transmission provides information that will help to determine the risk of spillover of vector‐borne diseases., The majority of the mosquito species caught showed a generalistic host preference. Anopheles obscurus was the most abundant Anopheles mosquito, with a generalistic host preference, whereas Culex decens showed a much higher attraction towards chimpanzee odours than to human or cow odoursHuman Plasmodium parasites were observed in both human and chimpanzee blood, although not in the Anopheles mosquitoes that were collected.

Background The yellow fever mosquito Aedes aegypti transmits viral diseases that have plagued humans for centuries. Its ancestral home are forests of Africa and ~400–600 years ago it invaded the New World and later Europe and Asia, causing some of the largest epidemics in human history. The species was rarely detected in countries surrounding the Mediterranean Sea after the 1950s, but during the last 16 years it re-appeared in Madeira, Russia and in the eastern coast of the Black Sea. We genotyped Ae. aegypti populations from the Black Sea region to investigate whether this is a recent invasion (and if so, where it came from) or a remnant of pre-eradication populations that extended across the Mediterranean. We also use the Black Sea populations together with a world reference panel of populations to shed more light into the phylogeographical history of this species. Results Microsatellites and ~19,000 genome-wide single nucleotide polymorphisms (SNPs) support the monophyletic origin of all populations outside Africa, with the New World as the site of first colonization. Considering the phylogenetic relationships, the Black Sea populations are basal to all Asian populations sampled. Bayesian analyses combined with multivariate analyses on both types of markers suggest that the Black Sea population is a remnant of an older population. Approximate Bayesian Computation Analysis indicates with equal probability, that the origin of Black Sea populations was Asia or New World and assignment tests favor the New World. Conclusions Our results confirmed that Ae. aegypti left Africa and arrived in New World ~500 years ago. The lineage that returned to the Old World and gave rise to present day Asia and the Black Sea populations split from the New World approximately 100–150 years ago. Globally, the Black Sea population is genetically closer to Asia, but still highly differentiated from both New World and Asian populations. This evidence, combined with bottleneck signatures and divergence time estimates, support the hypothesis of present day Black Sea populations being remnants of older populations, likely the now extinct Mediterranean populations that, consistent with the historic epidemiological record, likely represent the original return of Ae. aegypti to the Old World. Electronic supplementary material The online version of this article (10.1186/s13071-018-2933-2) contains supplementary material, which is available to authorized users.

Over the past years, the subcellular organization of the Excavata member Giardia lamblia (syn. duodenalis, intestinalis) has been investigated in considerable detail. There are several reasons for this endeavour which go beyond this parasite's medical importance and are mostly concerned with its reduced subcellular complexity and debated evolutionary status. One may say that simplification has emerged as a paradigm for the evolution of Giardia's subcellular architecture. However, a complete appreciation of the evolutionary and ecological significance of this phenomenon is far from complete. In this chapter, we present and discuss the most recent data on the main trafficking pathways in G. lamblia which include endo- and exo-cytosis, organellar import and function. We provide perspectives on open questions concerning organelle replication and inheritance and include a technical outlook on methods and approaches to genetic manipulations in G. lamblia. A better understanding of G. lamblia subcellular organization at the morphological and molecular level complements any effort aimed at elucidating this parasitic species' evolutionary status and could provide us with the basis for novel strategies to interfere with parasite transmission and/or pathogenesis.

Angiostronglyus vasorum is a cardiopulmonary nematode infecting mainly canids such as dogs (Canis familiaris) and foxes (Vulpes vulpes). Natural infections have also been reported in mustelids and red pandas (Ailurus fulgens fulgens). We report the occurrence of natural A. vasorum infections in a group of captive meerkats (Suricata suricatta), housed at a university facility in Switzerland. A. vasorum first-stage larvae (L1) were initially identified in a pooled faecal sample. Individual samples, investigated with the Baermann-Wetzel technique, revealed that 41% (7/17) of the meerkats were infected, with ranges of 2–125 L1/g faeces. PCR and sequencing of part of the ITS-2 region resulted in 100% identity with A. vasorum. Infected animals did not show clinical signs. One meerkat died two days after diagnosis. Upon necropsy one adult specimen was recovered; histological examination of the lung revealed granulomatous pneumonia caused by A. vasorum larvae and eggs as well as intima and media hyperplasia and isolated arteriosclerosis of larger lung vessels. However, the cause of death was a spleen rupture with associated blood loss. All meerkats were topically treated with 10 mg imidacloprid/2.5 mg moxidectin per animal, after which they became negative in all follow up faecal examinations. Potential intermediate (gastropods) and paratenic hosts (birds) were collected from within or outside the meerkats enclosure. Gastropods were examined by PCR and bird samples by digestion. Four out of 193 (2.1%) gastropod samples were positive for A. vasorum, whereas none of the bird samples were positive. Meerkats, belonging to the Herpestidae, therefore are suitable definitive hosts for A. vasorum, with production and excretion of live L1. Meerkats kept in captivity in areas where A. vasorum is endemic and with potential contact to intermediate hosts are at risk of infection. Regular faecal examinations including Baermann-Wetzel technique should be considered., Graphical abstract Image 1, Highlights • Meerkats are suitable definitive hosts for Angiostrongylus vasorum. • This is the first case of A. vasorum in the Herpestidae family. • A. vasorum larvae and eggs caused granulomatous pneumonia in an infected meerkat. • 2% of gastropods around the meerkats enclosure were positive for A. vasorum L3. • Captive animal species in areas where A. vasorum is endemic are at risk of infection.

The French heartworm Angiostongylus vasorum is found in European red fox (Vulpes vulpes) and dog populations, where it appears to be spreading geographically. Once introduced into new areas, it establishes in local fox populations, typically to over 50% prevalence in a few years. High susceptibility and constant excretion of first stage larvae (L1) by the definitive hosts are prerequisites for sustaining high parasite biomass in a particular habitat. The present study explores the hypothesis that repeated ingestion of gastropods in nature will result in accumulation of adult worms and elevated excretion of L1 in feces. Experimentally infected foxes were subsequently inoculated via stomach tube once (9 weeks post initial inoculation) or twice (9 and 13 weeks post inoculation (wpi)) with 100 third stage A. vasorum larvae (L3) previously isolated from aquatic snails infected with L1 from a naturally infected dog. Despite large variation in fecal larval excretion for the individual animals within the groups, excretion of L1 was significantly higher in foxes twice inoculated as compared to foxes inoculated only once. With an outlier in the once inoculated group removed, excretion became significantly higher in the three times inoculated group. Establishment of adult worms varied and only a trend to higher worm burdens was found in the group of foxes inoculated three times. However, this became significant with the same single outlier removed. Overall, it appears that protective immunity to A. vasorum does not appear to occur in V. vulpes with animals exhibiting high infection intensities without obvious clinical signs. The increasing larval excretion in foxes being repeatedly exposed to A. vasorum L3 support the hypothesis that foxes under natural conditions may repeatedly ingest infected gastropods and remain a source of environmental contamination for several months, potentially contributing to the establishment of endemic foci through increasing L1 excretion., Graphical abstract Image 1, Highlights • Foxes were experimentally infected with 100 Angiostrongylus vasorum larvae (L3). • Two groups were later challenged once or twice. • Increasing larval counts linked to time post infection and repeated inoculation. • Data suggests role of foxes in rapid establishment of the parasite in new areas.

Background Aedes albopictus, a known worldwide vector of several mosquito-borne disease pathogens including dengue, chikungunya and Zika viruses, was introduced into Europe in the late 1970s through global trade. First recorded in northern Italy in 1990, this mosquito species has rapidly spread throughout the country, where it was responsible for an outbreak of chikungunya in 2007 that affected more than 200 people. As part of the VectorNet project, which is aimed at improving preparedness and responsiveness for animal and human vector-borne diseases in Europe, a mosquito targeted study was carried out on the three southernmost Italian islands. The objective was to verify the current European southern distribution limits of Ae. albopictus and the potential occurrence of other invasive mosquito species, in the light of the introduction of high risk for vector-borne disease pathogens into Europe via migration flows. Results In the summer 2015, six surveys for container-breeding mosquitoes were carried out by setting up a network of oviposition traps and BG Sentinel traps in selected areas on the islands of Pantelleria, Lampedusa and Linosa. Aedes albopictus was found on all three islands under investigation. The consequences on public health with regard to the presence of this mosquito vector and the migrant people entering the country from Africa and the Middle East are also discussed here. Conclusions The detection of the Asian tiger mosquito on these islands, which represent the last European strip of land facing Africa, has important implications for public health policy and should prompt the national authorities to implement tailored surveillance activities and reinforce plans for preparedness strategies in such contexts.

Background: West Nile virus (WNV) is currently the most important mosquito-borne pathogen spreading in Europe. Data on overwintering of WNV in mosquitoes are crucial for understanding WNV circulation in Europe; nonetheless, such data were not available so far.Results: A total of 28,287 hibernating mosquitoes [27,872 Culex pipiens, 73 Anopheles maculipennis (sensu lato), and 342 Culiseta annulata], caught in February or March between 2011 and 2017 in a WNV-endemic region of South Moravia, Czech Republic, were screened for the presence of WNV RNA. No WNV positive pools were found from 2011 to 2016, while lineage 2 WNV RNA was detected in three pools of Culex pipens mosquitoes collected in 2017 at two study sites.Conclusions: To the best of our knowledge, this is the first record of WNV RNA in overwintering mosquitoes in Europe. The data support the hypothesis of WNV persistence in mosquitoes throughout the winter season in Europe.

Besnoitia besnoiti is an apicomplexan parasite responsible for bovine besnoitiosis, a chronic and debilitating disease that causes systemic and skin manifestations and sterility in bulls. Neither treatments nor vaccines are currently available. In the search for therapeutic candidates, calcium-dependent protein kinases have arisen as promising drug targets in other apicomplexans (e.g. Neospora caninum, Toxoplasma gondii, Plasmodium spp. and Eimeria spp.) and are effectively targeted by bumped kinase inhibitors. In this study, we identified and cloned the gene coding for BbCDPK1. The impact of a library of nine bumped kinase inhibitor analogues on the activity of recombinant BbCDPK1 was assessed by luciferase assay. Afterwards, those were further screened for efficacy against Besnoitiabesnoiti tachyzoites grown in Marc-145 cells. Primary tests at 5µM revealed that eight compounds exhibited more than 90% inhibition of invasion and proliferation. The compounds BKI 1294, 1517, 1553 and 1571 were further characterised, and EC99 (1294: 2.38µM; 1517: 2.20µM; 1553: 3.34µM; 1571: 2.78µM) were determined by quantitative real-time polymerase chain reaction in 3-day proliferation assays. Exposure of infected cultures with EC99 concentrations of these drugs for up to 48h was not parasiticidal. The lack of parasiticidal action was confirmed by transmission electron microscopy, which showed that bumped kinase inhibitor treatment interfered with cell cycle regulation and non-disjunction of tachyzoites, resulting in the formation of large multi-nucleated complexes which co-existed with viable parasites within the parasitophorous vacuole. However, it is possible that, in the face of an active immune response, parasite clearance may occur. In summary, bumped kinase inhibitors may be effective drug candidates to control Besnoitiabesnoiti infection. Further in vivo experiments should be planned, as attainment and maintenance of therapeutic blood plasma levels in calves, without toxicity, has been demonstrated for BKIs 1294, 1517 and 1553.

Background In the last decade serological tests for detection of circulating Angiostrongylus vasorum antigen and specific antibodies have been developed and adopted for individual diagnosis and epidemiological studies in dogs. Although confirmed positive at necropsy, antigen detection was not possible in single experimentally, as well as naturally infected dogs, possibly due to immune complex formation. The aim of this study was to evaluate the effect of heat treatment on detection of A. vasorum antigen in sera of experimentally (n = 21, 119 follow-up sera) and naturally (n = 18) infected animals. In addition, sera of dogs showing clinical signs consistent with angiostrongylosis (n = 10), of randomly selected dogs (n = 58) and of dogs with other parasitic infections (n = 15) were evaluated. Sera were subjected to heat treatment at 100 °C after addition of 0.5 M EDTA (dilution 1:5) and tested with ELISAs for detection of circulating A. vasorum antigen before and after treatment. Results Between 5 and 11 weeks post-inoculation (wpi) the percentage of positive untreated samples (experimentally infected dogs) increased over time from 33.3 to 90%. Single samples were still negative between 12 and 15 wpi. Overall, between 5 and 15 wpi, 50.6% (45/89) of the available samples were seropositive. From 3 to 6 wpi EDTA/heat treatment caused a change in 8/34 (23.5%) of the samples, with most (n = 6, 17.6%) converting from positive to negative. In contrast, from 7 to 10 wpi, treatment induced a change in 19/52 (36.5%) samples, with all but one converting from negative to positive. Thirteen of 18 naturally infected dogs were antigen positive before and 15 after EDTA/heat treatment, respectively. Untreated samples of 3 dogs with suspected angiostrongylosis were antigen positive, of which only one remained positive after EDTA/heat treatment. One of 58 untreated random samples was antigen positive; this sample became negative after treatment, while another turned positive. One of 15 dogs infected with other parasites than A. vasorum was positive before but negative after treatment. Conclusion Although heat treatment improves A. vasorum antigen detection between 7 and 10 wpi by immune complex disruption, we do not recommend systematic pretreating sera because of reduced antigen detection between 3 and 6 wpi and impairment of antibody detection, if performed contemporaneously.

Babesia canis is the predominant Babesia species in dogs in Europe and is responsible for a severe and fatal disease. An increase in global pet tourism and a widening of the geographic distribution of the tick vector has led to the emergence of infections in areas where previously only imported cases have been reported. Due to the potential for rapid and serious disease progression, direct parasite detection by stained blood smears and light microscopy or DNA-based methods have traditionally been used for the diagnosis of acute infections. This study describes the production of a murine monoclonal antibody ('mAb BcFIII 7/1/2') that reacts to a 65kDa corpuscular epitope present in B. canis-infected erythrocytes and can be used in an ELISA to detect circulating Babesia antigen during acute infections. The sensitivity of the ELISA was 100% (95%CI: 84.5-100) as determined using blood lysate samples from 27 dogs with acute B. canis infections. Sensitivity was reduced to 53.8% in 13 patent Babesia vogeli infections (95%CI: 26.1-79.6) based on the current test design using convalescent serum from a B. canis-infected dog. The specificity was determined to be 86.4% (95%CI: 64-96.4) using 22 samples from healthy canine blood donors. In the course of acute B. canis infections, the ELISA showed a positive result at the same time as a positive PCR result was recorded. This was 24-48h before parasites could be detected by light microscopy. Convalescent samples collected from 6 B. canis-infected dogs at least 14days post treatment resulted in negative ELISA reactions. The hyper-acute to acute phase of a B. canis infection represents an emergency situation with high mortality. To increase the chances of survival, a fast and accurate diagnosis and immediate treatment is required. The current study demonstrates the opportunity of an early and specific detection of acute infections by an AgELISA that is potentially translatable to a rapid diagnostic test design.