Originally published at: Schnyder, M; Tanner, I; Webster, P; Barutzki, D; Deplazes, P (2011). An ELISA for sensitive and specific detection of circulating antigen of Angiostrongylus vasorum in serum samples of naturally and experimentally infected dogs. Veterinary Parasitology, 179(1-3):152-158.An ELISA for sensitive and specific detection of circulating antigen of Angiostrongylus vasorum in serum samples of naturally and experimentally infected dogs Abstract Canine angiostrongylosis is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We developed a sandwich-ELISA based on a monoclonal antibody (mAb Av 56/1/2) and on polyclonal rabbit antibodies directed against Angiostrongylus vasorum adult excretory/secretoryantigen for the detection of circulating serum antigen of A. vasorum. The sensitivity of the test was 95.7% (78.1-99.9, 95% CI) as determined with sera of 23 dogs naturally infected with A. vasorum. The specificity was 94.0% (83.5-98.7, 95% CI) using 50 dog sera (control group) submitted for reasons other than parasitic infections. Potential cross-reactions were investigated with sera of a group of totally 61 dogs with proven infections with Dirofilaria immitis (n = 23), Crenosoma vulpis (n = 14), Ancylostoma caninum (n=4) or Toxocara canis (n = 20). No significant difference was observed concerning the proportion of positive reactions between the control group and the group with proven helminth infections other than A. vasorum. In experimentally inoculated dogs with proven worm burdens of A. vasorum, the proportion of seropositive dogs increased over the first 3 months of infection, starting from 35 days post inoculation (dpi) which was before the onset of larval excretion. Ten weeks post inoculation, 98.6% of the dogs were seropositive, and circulating antigen persisted in two dogs with long-term follow-up over 286 and 356 days, respectively. In contrast, in dogs with a single treatment with imidacloprid/ moxidectin at four or 32 dpi, no circulating antigen was observed, while in dogs treated at 88-92 dpi, OD values decreased within 13-34 days. The specific detection of circulating A. vasorum antigen by ELISA represents a valid alternative for reliable diagnosis and for follow-up investigations after anthelmintic treatment. Moreover, the test can be used for mass screening in large epidemiological investigations.1 In experimentally inoculated dogs with proven worm burdens of A. vasorum, the proportion of seropositive 26 dogs increased over the first 3 months of infection, starting from 35 days post inoculation (dpi) which was 27 before the onset of larval excretion. Ten weeks post inoculation, 98.6% of the dogs were seropositive, and 28 circulating antigen persisted in two dogs with long-term follow-up over 286 and 356 days, respectively. In 29 contrast, in dogs with a single treatment with imidacloprid/moxidectin at four or 32 dpi, no circulating 30 antigen was observed, while in dogs treated at 88-92 dpi, OD values decreased within 13-34 days. The 31 specific detectio...

A sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Echinococcus multilocularis coproantigens (EM-ELISA) was developed with polyclonal rabbit (solid phase) and chicken egg (catching) antibodies that were directed against E. multilocularis coproantigens and somatic worm antigens, respectively. In experimentally infected dogs and cats, coproantigens were first detectable 6-17 days postinfection (PI) in samples of 8 dogs (worm burdens at necropsy: 6,330-43,200) and from 11 days PI onward in samples of 5 cats infected with 20-6,833 worms. After anthelmintic treatment of 4 dogs and 5 cats at day 20 PI, coproantigen excretion disappeared within 3-5 days. The sensitivity of the ELISA was 83.6% in 55 foxes infected with 4-60,000 E. multilocularis, but reached 93.3% in the 45 foxes harboring more than 20 worms. The EM-ELISA was used in surveys of "normal" dog and cat populations in Switzerland. Among 660 dogs and 263 cats, 5 dogs and 2 cats exhibited a positive reaction. In 2 of these dogs (0.30%) and 1 cat (0.38%), intestinal E. multilocularis infections were confirmed by necropsy, polymerase chain reaction PCR, or both. The specificities of the ELISA in these groups were found to be 99.5% and 99.6%, respectively, if positive ELISA results that could not be confirmed by other methods were classified as "false positive" reactions. An accurate determination of the prevalence of E. multilocularis in populations of final hosts is an essential requirement for establishing epidemiological baseline data and for estimating the potential infection risk for humans (Eckert, 1998). Currently, the most reliable technique for the diagnosis of E. multilocularis infection in foxes and other definitive hosts is parasitological examination of the small intestine at necropsy. Until recently, methods for an accurate and sensitive identification or exclusion of the infection in living animals were not available. The standard purgation technique with arecoline hydrobromide routinely used for screening dog populations for Echinosoccus granulosus is not applicable to foxes and cats. In dogs, this technique is hampered by its relatively low sensitivity (65.2% after 1 dose, 78.3% after 2 doses), and it is inefficient in up to 32% of the dogs that do not purge (Schantz, 1997). Moreover, the technique is biohazardous, labor intensive, and costly.Recent developments in serum antibody, fecal antigen, and DNA detection for the diagnosis of intestinal infections with E. granulosus or E. multilocularis have provided alternatives to current techniques (reviewed by Craig et al., 1996; Deplazes and Eckert, 1996). In particular, the detection of parasite coproantigens by sandwich enzyme-linked immunsorbent assay (ELISA) has become a general focus of interest in the diagnosis In a previous publication, Deplazes et al. (1992) reported for the first time the detection of E. multilocularis coproantigens in fecal samples of foxes and dogs by a sandwich ELISA. However, the sensitivity of this test system designed for the detection...

S U M M A R YRodents are shared intermediate or paratenic hosts for Echinococcus multilocularis, Toxocara spp. and Toxoplasma gondii, and may serve as valuable indicators for assessing the occurrence and the level of environmental contamination and infection pressure with free-living stages of these zoonotic parasites. We investigated 658 non-commensal rodents for parasite infections in the canton of Geneva, Switzerland. The prevalence of infection with E. multilocularis was highest in Arvicola terrestris captured in the north-western area (16 . 5%, CI: 10 . 1%-24 . 8%), possibly reflecting a higher red fox density due to the low incidence of sarcoptic mange in this part of the canton. The exposure rate to Toxocara spp. was highest in the urban area (13 . 2%, CI: 7 . 9%-20 . 3%), and may account for higher densities of domestic carnivore and red fox definitive hosts within the city. Exposure to T. gondii was widespread (5 . 0%, CI: 3 . 2-7 . 4 %), indicating a ubiquitous distribution of infected cat definitive hosts. Interestingly, a widespread distribution of Taenia taeniaeformis, a parasite mainly transmitted by cats, was similarly evidenced in A. terrestris. Distinct spatial patterns for the different zoonotic parasites likely reflected differences in distribution, abundance, and habitat use of the respective definitive hosts. These results highlight the potential value of rodents as shared indicators for these pathogens.

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.

To assess the zoonotic potential of Encephalitozoon-like microsporidia, we isolated and cultivated spores from specimens of urine, respiratory secretions, and stool from six patients infected with human immunodeficiency virus and from nine rabbits. Because spores of Encephalitozoon-like species are indistinguishable by microscopy, we characterized the isolates by western blot analysis and by restriction enzyme analysis of the small subunit (SSU) rDNA after amplification by the polymerase chain reaction. We identified Septata intestinalis in one patient and Encephalitozoon hellem in two symptomatic patients. Encephalitozoon cuniculi was found in all rabbits and in three patients. One of these patients had clinical manifestations of infection with this parasite (severe interstitial pneumonitis). We observed abatement of symptoms and cessation of parasite excretion when these patients were treated with albendazole. Our findings suggest that E. cuniculi may be pathogenic in humans and that it is a zoonotic parasite.