Fox Serum Proteomics Analysis Suggests Host-Specific Responses to Angiostrongylus vasorum Infection in Canids.

Dogs infected with the cardiopulmonary nematode Angiostrongylus vasorum may suffer from respiratory distress and/or bleeding disorders. Descriptions of clinical signs in foxes are rare, despite high prevalence. To evaluate the impact of infection on coagulation and immune response, serum proteins from eight experimentally infected foxes before and after inoculation (day 0, 35, 84, 154) were subjected to differential proteomic analyses based on quantitative data and compared to available data from dogs. The number of proteins with differential abundance compared to the uninfected baseline increased with chronicity of infection. Bone marrow proteoglycan, chitinase 3-like protein 1 and pulmonary surfactant-associated protein B were among the most prominently increased proteins. The abundance of several proteins involved in coagulation was decreased. Enriched pathways obtained from both increased and decreased proteins included, among others, "platelet degranulation" and "haemostasis", and indicated both activation and suppression of coagulation. Qualitative comparison to dog data suggests some parallel serum proteomic alterations. The comparison, however, also indicates that foxes have a more adequate immunopathological response to A. vasorum infection compared to dogs, facilitating persistent infections in foxes. Our findings imply that foxes may be more tolerant to A. vasorum infection, as compared to dogs, reflecting a longer evolutionary host-parasite adaptation in foxes, which constitute a key wildlife reservoir.

The Angiostrongylus vasorum Excretory/Secretory and Surface Proteome Contains Putative Modulators of the Host Coagulation.

Angiostrongylus vasorum is a cardiopulmonary nematode of canids and is, among others, associated with bleeding disorders in dogs. The pathogenesis of such coagulopathies remains unclear. A deep proteomic characterization of sex specific A. vasorum excretory/secretory proteins (ESP) and of cuticular surface proteins was performed, and the effect of ESP on host coagulation and fibrinolysis was evaluated in vitro. Proteins were quantified by liquid chromatography coupled to mass spectrometry and functionally characterized through gene ontology and pathway enrichment analysis. In total, 1069 ESP (944 from female and 959 from male specimens) and 1195 surface proteins (705 and 1135, respectively) were identified. Among these were putative modulators of host coagulation, e.g., von Willebrand factor type D domain protein orthologues as well as several proteases, including serine type proteases, protease inhibitors and proteasome subunits. The effect of ESP on dog coagulation and fibrinolysis was evaluated on canine endothelial cells and by rotational thromboelastometry (ROTEM). After stimulation with ESP, tissue factor and serpin E1 transcript expression increased. ROTEM revealed minimal interaction of ESP with dog blood and ESP did not influence the onset of fibrinolysis, leading to the conclusion that Angiostrongylus vasorum ESP and surface proteins are not solely responsible for bleeding in dogs and that the interaction with the host's vascular hemostasis is limited. It is likely that coagulopathies in A. vasorum infected dogs are the result of a multifactorial response of the host to this parasitic infection.

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.

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.

Conquering Switzerland: the emergence of Angiostrongylus vasorum in foxes over three decades and its rapid regional increase in prevalence contrast with the stable occurrence of lungworms.

Angiostrongylus vasorum, Crenosoma vulpis and Capillaria aerophila are the most common lungworms of domestic and wild canids. We investigated the short- and long-term lungworm prevalence changes in the Swiss fox population with a focus on A. vasorum. Between 2012 and 2017, lungs and hearts of 533 foxes from north-eastern Switzerland were necropsied and blood samples tested for circulating A. vasorum antigen. Angiostrongylus vasorum prevalence increased steadily from 21.5% in 2012 to 81.8% in 2017. In contrast, C. aerophila and C. vulpis prevalences fluctuated between 41.8 and 74.7%, and 3.6 and 14.9%, respectively. Based on 3955 blood samples collected between 1986 and 2017 from three geographic areas and during four time periods, antigen seropositivity increased from 2.4 to 62.0%. In north-eastern Switzerland, seropositivity was initially low (1.9 and 1.7% in the first two time periods) but increased in the following two decades to 22.2 and 62.0%, respectively. Our findings depict the spectacular expansion of A. vasorum in the past three decades. Regionally, the prevalence in foxes increased 4-fold within 6 years in some regions. This underpins the important role of foxes as reservoir hosts, likely explaining the increasing number of cases of canine angiostrongylosis in Switzerland. Our findings are representative of central Europe and may help anticipating future developments in areas where A. vasorum is present but (still) infrequent.

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.

The first seroepidemiological survey for Angiostrongylus vasorum in domestic dogs from Romania.

BACKGROUND: Angiostrongylus vasorum is a metastrongyloid nematode localized in the right heart and the pulmonary arteries of domestic dogs. The number of reports in Europe has recently increased, presumably as a consequence of a growing awareness among clinicians, animal owners and researchers, but also due to a growing incidence and territorial spread. So far, no studies have been conducted to assess the prevalence and distribution of A. vasorum in domestic dogs in Romania, and the awareness among veterinarians is limited or absent. The aim of the present study was to evaluate the countrywide seroprevalence of circulating antigens of A. vasorum and specific antibodies against A. vasorum in domestic dogs from Romania. METHODS: Between November 2016 and July 2017, blood was sampled from a total of 1545 domestic dogs from 23 counties of Romania. Details about their gender, age, breed, housing, use and origin were collected. All serum samples were tested for the presence of A. vasorum circulating antigens (AG) using monoclonal and polyclonal antibodies in a sandwich ELISA. Additionally, a sandwich ELISA using A. vasorum adult somatic antigen purified by monoclonal antibodies was used for specific antibody (AB) detection. RESULTS: A total of 33 dogs (2.14%, 95% CI: 1.82-3.56%) were seropositive for A. vasorum antigen or antibodies against the parasite. Three dogs were positive for antigen only (0.19%, 95% CI: 0.07-0.57%) and 30 dogs (1.94%, 95% CI: 1.36-2.76%) were positive for antibodies only. No dog was positive for both tests. The overall prevalence (AB or AG) and the AB prevalence were significantly higher in pure breed dogs compared to mixed breeds and mongrel dogs (P < 0.05) and in shepherd dogs compared to other groups (P < 0.05). There was no significant difference between males and females, between urban and rural dogs, between dogs with unrestricted access and with restricted access to the environment, and between dogs living outdoors and indoors. CONCLUSIONS: Our data suggest that the disease is present in Romania in dogs, as it was previously demonstrated in foxes. However, so far, no clinical case has been reported in the country and this may be related to a low awareness among vets.

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.

Intrinsic factors, adrenal gland morphology, and disease burden in captive cheetahs (Acinonyx jubatus) in South Africa.

Adrenal gland weight (AW) and corticomedullary ratio (ACMR) are used as indicators of stress in animals. Captive cheetahs (Acinonyx jubatus) have higher ACMRs than free-ranging ones and stress has been linked to gastritis, amyloidosis, glomerulosclerosis, and myocardial fibrosis. We reviewed age, sex, body weight (BW), kidney weight (KW), and left AW and ACMR with necropsy findings in 51 South African captive cheetahs. Eleven common histopathologic lesions were counted for each animal as measure of its disease burden. Adrenal corticomedullary hyperplasia was significantly correlated with left AW and ACMR. Males had significantly higher AWs than females; other parameters showed no difference between the sexes. Disease burden, gastritis, and myocardial fibrosis were moderately correlated with adrenal morphology supporting prior evidence that gastritis and myocardial fibrosis are linked to stress. Glomerulosclerosis was not correlated with adrenal morphology and neither kidney nor liver amyloidosis contributed significantly to variation in AW or ACMR on multivariate analyses. Interstitial nephritis showed much stronger correlations with kidney and liver amyloidosis than gastritis. All three adrenal parameters were correlated with age; age was the only significant variable affecting ACMR on the multivariate analyses; and disease burden as well as systemic amyloidosis and kidney disease (except for fibrosis) showed moderate correlations with age. Age may, therefore, be important in the pathogenesis of disease in captive cheetahs, particularly of amyloidosis and kidney disease. None of the intrinsic measurements or adrenal parameters were sufficiently closely linked to disease to be used as ante-mortem proxies for disease burden or specific diseases. Zoo Biol. 36:40-49, 2017. © 2016 Wiley Periodicals, Inc.

Meerkats (Suricata suricatta), a new definitive host of the canid nematode Angiostrongylus vasorum.

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.