Detection of specific antibodies in dogs infected with Angiostrongylus vasorum

Abstract

Canine angiostrongylosis, caused by the nematode Angiostrongylus vasorum, is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We determined the diagnostic value of the specific detection of antibodies against A. vasorum adult somatic antigen, adult excretory/secretory (E/S) antigen and first stage larvae (L1) somatic antigen in ELISAs. Also, A. vasorum adult somatic antigen purified by monoclonal antibodies (mAb) was evaluated in a sandwich-ELISA. Among the crude antigens, the best sensitivities when testing 21 naturally infected dogs were obtained using adult E/S and somatic antigen (85.7% and 76.2%, respectively), which were comparable with the results of the sandwich-ELISA based on mAb-purified antigens (81%). The ELISA performed with L1 antigen had the lowest sensitivity (42.9%). In experimentally inoculated dogs, the sensitivities ranged from 97.7% to 100% with all test settings. The specificity was 98.8% (92.5-99.9%, 95% CI) with all ELISAs using sera of 82 randomly selected dogs. Cross-reactions using adult somatic, adult E/S and L1 somatic antigen were observed in sera of dogs infected with Crenosoma vulpis, Dirofilaria immitis, Dirofilaria repens, and Eucoleus aerophilus. In contrast, using the mAb-purified antigens, the cross-reactions were minimal. Depending on the antigens used, specific antibodies were detected starting between 13 and 21 days post experimental inoculation (dpi), and at latest between 35 and 48 dpi, thus before or around the onset of patency. The serological follow-up of four A. vasorum-infected dogs after anthelmintic treatment at 88 dpi showed a decrease of antibody levels after drug administration, and the animals became seronegative 2-9 weeks later. Two untreated dogs remained seropositive. In four dogs treated 4 dpi, virtually no antibody-reaction was detectable, with the exception of the ELISA performed with L1 antigen. The early detection of specific antibodies against A. vasorum by ELISA represents a valid alternative for a reliable diagnosis and for follow-up investigations after anthelmintic treatment.

Combined Serological Detection of Circulating Angiostrongylus vasorum Antigen and Parasite-specific Antibodies in Dogs from Hungary

“…Subsamples were sent to the Institute of Parasitology, Vetsuisse Faculty, University of Zurich, Switzerland and were analysed for the presence of circulating A. vasorum antigens using monoclonal and polyclonal antibodies in a sandwich ELISA with a sensitivity of 95.7 % and a specificity of 94.0 %, as previously described . Additionally, a sandwich ELISA (sensitivity 81.0 %, specificity 98.8 %) using A. vasorum adult somatic antigen purified by monoclonal antibodies (mAb Av 5/5) was used for specific antibody detection (Schucan et al 2012). Test thresholds were regionally determined with 300 randomly selected samples based on the mean value of optical density (A 405 nm ) plus 3 (antigen detection) or 4 (antibody detection) standard deviations.…”

Specific Antibody Detection in Dogs with Filarial Infections

“…Thirty-four sera from healthy blood donor dogs originated from a known Dirofilaria non-endemic area in Switzerland were used to determine the cut-off value (mean value plus 3 standard deviations). Specificity was calculated with randomly selected sera of 69 dogs presented at the Clinic for Small Animals and tested at the Clinical Laboratory of the Vetsuisse Faculty, University of Zurich, Switzerland, for various reasons excluding suspected parasitological infections (Schucan et al 2012). Potential cross-reactions were tested with sera from dogs, experimentally infected with Toxo cara canis (n = 5), Ancylostoma caninum (n = 4) and Trichuris vulpis (n = 2) (last two mentioned were kindly provided by the Institute of Parasitology, University of Veterinary Medicine, Hannover); from experimentally (n = 10) and naturally (n = 7) infected dogs with Angiostrongylus vasorum (Schucan et al 2012); from naturally infected dogs with Crenosoma vulpis (n = 6) diagnosed by the presence of L1 in faeces (Barutzki and Schaper 2009); from naturally infected Italian dogs tested positive for the presence of Capillaria aerophila eggs (n = 7) by PCR-coupled sequencing (Traversa et al 2011); and from naturally infected dogs with Acanthocheilone ma reconditum (n = 10) and Dipetalonema dracun culoides (n = 9) (Magnis et al 2013).…”

“…repens adult somatic antigen was prepared as described by Schucan et al (2012). Protein concentrations were assessed by absorbance assay at 280 nm using a spectrometer (NanoDrop ® ND-1000; Witec AG, Littau, Switzerland).…”

“…ELISAs have been performed in principle as described by Schucan et al (2012). Briefly, plates were coated with D. immitis E/S (1:200 dilution in coating buffer: 0.1 M carbonate/bicarbonate, pH 9.6) and D. immitis or D. repens adult somatic antigen (10 µg/ml coating buffer) at 4 °C overnight in a humid chamber.…”

Detection of Angiostrongylus vasorum in Red Foxes (Vulpes vulpes) from Brandenburg, Germany

“…The transient presence of larvae and adults in the circulation that has been suggested, challenges all diagnostic methods ). More recently other ELISA-based assays detecting A. vasorum antigen and antibodies have been utilised for large-scale regional and country-wide investigations based on larger numbers of samples (Schnyder et al 2011;Schucan et al 2012;Schnyder et al 2013). A test for the serological detection of circulating antigen (Angio Detect TM Test, IDEXX Laboratories, Westbrook, Maine, USA) for rapid diagnosis is now also commercially available for use in veterinary clinics (Schnyder et al 2014).…”