Fifth European Dirofilaria and Angiostrongylus Days (FiEDAD) 2016

Peer reviewe

Host-specific serological response to Angiostrongylus vasorum infection in red foxes (Vulpes vulpes) and impact of heat treatment on antigen detection in dog sera

Angiostrongylus vasorum is a cardiopulmonary nematode increasingly found in dogs and foxes in many European countries. ELISAs for the detection of circulating A. vasorum antigen or specific antibodies in dogs were evaluated for foxes. Blood from 75 farmed Danish foxes experimentally infected with A. vasorum and from 215 dissected wild Swiss foxes were evaluated for detection of circulating A. vasorum antigen and specific antibodies. Antigen detection had 91.2% sensitivity and 89.4% specificity, and antibody detection 42.2% and 92.0%, respectively. The experimentally infected foxes became antigen positive 5 to 10 weeks post inoculation (wpi) and remained positive up to 22 wpi. The antibody responses in the same foxes were highly variable, irrespectively of further challenge inoculations, reaching seropositivity 5 to 7 wpi and followed by a decrease in over half of the animals despite persistent infections. Infected foxes may develop a variable and non-protective immunity which contributes to long term survival of A. vasorum that could explain its efficient spread within the fox population. Furthermore, the effect of EDTA/heat treatment on A. vasorum antigen detection in dog sera was assessed with 205 sera before and after treatment. An improvement of 34.6% was observed in treated samples between 7 and 10 wpi, while detection was impaired between 3 and 5 wpi. The same procedures applied to 74 samples tested with a rapid assay did not substantially improve antigen detection

Impact of heat treatment on antigen detection in sera of Angiostrongylus vasorum infected dogs

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

Genetic diversity of the cardiopulmonary canid nematode Angiostrongylus vasorum within and between rural and urban fox populations

Angiostrongylus vasorum is an emerging parasitic cardiopulmonary nematode of dogs, foxes, and other canids. In dogs, the infection causes respiratory and bleeding disorders along with other clinical signs collectively known as canine angiostrongylosis, while foxes represent an important wildlife reservoir. Despite the spread of A. vasorum across various countries in Europe and the Americas, little is known about the genetic diversity of A. vasorum populations at a local level in a highly endemic area. Thus, in the present study, we investigated the genetic diversity of 323 adult A. vasorum nematodes from 64 foxes living in the canton of Zurich, Switzerland. Among those, 279 worms isolated from 20 foxes were analyzed separately to investigate the genetic diversity of multiple worms within individual foxes. Part of the mitochondrial cytochrome c oxidase subunit I (mtCOI) gene was amplified and sequenced. Overall, 16 mitochondrial haplotypes were identified. The analysis of multiple worms per host revealed 12 haplotypes, with up to 5 different haplotypes in single individuals. Higher haplotype diversity (n = 10) of nematodes from foxes of urban areas than in rural areas (n = 7) was observed, with 5 shared haplotypes. Comparing our data with published GenBank sequences, five haplotypes were found to be unique within the Zurich nematode population. Interestingly, A. vasorum nematodes obtained from foxes in London and Zurich shared the same dominating haplotype. Further studies are needed to clarify if this haplotype has a different pathogenicity that may contribute to its dominance. Our findings show the importance of foxes as a reservoir for genetic parasite recombination and indicate that high fox population densities in urban areas with small and overlapping home ranges allow multiple infection events that lead to high genetic variability of A. vasorum

Capillaria boehmi (syn. Eucoleus boehmi): challenging treatment of a rarely diagnosed nasal nematode in dogs and high prevalence in Swiss foxes

Despite morphological differences of eggs and adults, Capillaria boehmi infections have been occasionally misdiagnosed as C. aerophila infections in the past. Capillaria boehmi is found in the nasal and paranasal sinuses of wild canids and dogs, which may suffer from nasal discharge, sneezing, epistaxis and, importantly, their scent can be impaired. In this study we present three challenging cases of nasal capillariosis in dogs, report and review the variable success of anthelmintic treatments and investigate C. boehmi prevalence in Swiss red foxes, considered as potential wild life reservoir. Out of two females and one male dog (all scent hounds, aged 3-9 years and weighing 19-31 kg), two dogs were previously coproscopically misdiagnosed with Trichuris infections. Two dogs showed clinical signs such as sneezing, coughing and impaired scent. From one dog adult living C. boehmi were obtained by nasal lavage. The identity of worms and eggs of all three dogs were genetically confirmed (18S rRNA, 100 % identity in 578 base pairs). Dogs 1-3 were followed-up for overall 54, 8, and 67 months, respectively. All dogs repeatedly excreted C. boehmi eggs in faecal samples despite treatments with the following compounds, in various dosage and retreatment protocols: fenbendazole, milbemycin oxime (orally), moxidectin/imidacloprid/ (spot-on) and levamisole (intramuscularly). The different anthelmintic compounds showed variable success regarding their effect on clinical outcome and on stopping egg excretion. Reinfections due to a contaminated environment could not be fully excluded. In winter 2016 and 2017, 218 foxes from the canton of Zurich, Switzerland, were examined. Tissues of nasal and paranasal sinuses were investigated for adult Capillaria specimens and eggs. We describe for the first time C. boehmi infections in Switzerland, observing a high prevalence (190/218, 87.2 %). Overall, 107 of 126 adults (84.9 %, 95 % Confidence Interval, CI: 77.5-90.7 %) and 83 of 92 youngsters (90.2 %, CI: 82.2-95.4 %) were infected. The presence of C. boehmi did not correlate with age (P = 0.209), but correlated significantly with sex: male foxes (102 of 107, 95.3 %, CI: 89.4-98.5 %) were significantly (P = 0.001) more often infected than females (88 of 111, 79.3 %, CI: 70.5-86.4 %). Worm burden ranged from 1 to 72 adult specimens (geometric mean: 5.7). In conclusion, C. boehmi infections can be mis- and/or underdiagnosed in dogs. Appropriate anthelmintic treatments, preventing coprophagia and egg contamination of the surroundings and performing coproscopic controls after treatments are fundamental aspects. Potentially, nasal washing may represent an auxiliary alternative. However, the successful elimination of C. boehmi infections in dogs remains challenging

Genome sequence of the cardiopulmonary canid nematode Angiostrongylus vasorum reveals species-specific genes with potential involvement in coagulopathy

Angiostrongylus vasorum is an emerging parasitic nematode of canids and causes respiratory distress, bleeding, and other signs in dogs. Despite its clinical importance, the molecular toolbox allowing the study of the parasite is incomplete. To address this gap, we have sequenced its nuclear genome using Oxford nanopore sequencing, polished with Illumina reads. The size of the final genome is 280 Mb comprising 468 contigs, with an N50 value of 1.68 Mb and a BUSCO score of 93.5%. Ninety-three percent of 13,766 predicted genes were assigned to putative functions. Three folate carriers were found exclusively in A. vasorum, with potential involvement in host coagulopathy. A screen for previously identified vaccine candidates, the aminopeptidase H11 and the somatic protein rHc23, revealed homologs in A. vasorum. The genome sequence will provide a foundation for the development of new tools against canine angiostrongylosis, supporting the identification of potential drug and vaccine targets

Quantitative proteomics analysis of Angiostrongylus vasorum-induced alterations in dog serum sheds light on the pathogenesis of canine angiostrongylosis

Blood contains hundreds of proteins, reflecting ongoing cellular processes and immune reactions. Infections with the blood-dwelling cardiopulmonary nematode Angiostrongylus vasorum in dogs manifest with a broad spectrum of clinical signs including respiratory distress, bleeding diathesis and neurological signs, and are associated with a perturbed blood protein profile in dogs. However, current knowledge does not completely explain the observed pathologies induced by A. vasorum infections, including bleeding disorders. Using sera from experimentally infected dogs, dog serum proteome was analysed by quantitative mass spectrometry methods over several time points before and after inoculation. Following computational analysis, we identified 139 up- and downregulated proteins after infection (log2 ratio cut-off ≥ 1.0; q-value ≤ 0.05). Among upregulated proteins were chitinase 3-like 1 and pulmonary surfactant-associated protein B (log2 fold-changes ≥ 5). Pathway enrichment revealed the complement (especially the lectin pathway) and coagulation cascades as significantly affected upon analysis of downregulated proteins. Among them were mannan-binding lectin serine peptidases, ficolin, and coagulation factor XIII-B. These results bring new elements towards understanding the underlying pathomechanisms of bleeding diatheses observed in some A. vasorum-infected dogs

Quantitative proteomics analysis of Angiostrongylus vasorum-induced alterations in dog serum sheds light on the pathogenesis of canine angiostrongylosis

Blood contains hundreds of proteins, reflecting ongoing cellular processes and immune reactions. Infections with the blood-dwelling cardiopulmonary nematode Angiostrongylus vasorum in dogs manifest with a broad spectrum of clinical signs including respiratory distress, bleeding diathesis and neurological signs, and are associated with a perturbed blood protein profile in dogs. However, current knowledge does not completely explain the observed pathologies induced by A. vasorum infections, including bleeding disorders. Using sera from experimentally infected dogs, dog serum proteome was analysed by quantitative mass spectrometry methods over several time points before and after inoculation. Following computational analysis, we identified 139 up- and downregulated proteins after infection (log2 ratio cut-off ≥ 1.0; q-value ≤ 0.05). Among upregulated proteins were chitinase 3-like 1 and pulmonary surfactant-associated protein B (log2 fold-changes ≥ 5). Pathway enrichment revealed the complement (especially the lectin pathway) and coagulation cascades as significantly affected upon analysis of downregulated proteins. Among them were mannan-binding lectin serine peptidases, ficolin, and coagulation factor XIII-B. These results bring new elements towards understanding the underlying pathomechanisms of bleeding diatheses observed in some A. vasorum-infected dogs.ISSN:2045-232