Comparison of microscopic, molecular, and serological methods for the diagnosis and post-treatment monitoring of Echinococcus granulosus in dogs

Cystic echinococcosis, caused by Echinococcus granulosus sensu lato, is a zoonotic disease that results in significant economic losses in infected animals and poses serious public health concerns in humans. Dogs play a critical role in the transmission cycle as the definitive host of the parasite. This thesis aimed to compare microscopic, molecular, and serological diagnostic methods in experimentally infected dogs to determine the most reliable approach for diagnosing and monitoring E. granulosus sensu stricto following treatment. For experimental infection, 20.000 protoscoleces obtained from the liver and lungs of a sheep with hydatid cysts were orally administered to two dogs (ED-2 and ED-3), each housed in separate kennels. A control dog (ED-1) received 10 mL of distilled water. To obtain antigens for serological analysis, an additional dog—separate from the experimental group—was also infected with 20.000 protoscoleces, from which adult parasites were later recovered. Blood and fecal samples were collected daily from the experimental dogs for 50 days post-infection and for an additional 30 days following treatment. Fecal samples were examined microscopically using the Fülleborn and sieving flotation techniques. For molecular analyses, genomic DNA was extracted from feces and analyzed using conventional PCR, real-time quantitative PCR (qPCR), and digital PCR (dPCR). To prepare antigens for serological analysis, arecoline hydrobromide was administered to the experimentally infected dog on day 50 post-infection, and adult parasites were subsequently collected. E. granulosus soluble crude antigen (EgSCA) and recombinant E. granulosus fibronectin antigen (rEgFN162) prepared from these parasites were used to evaluate IgG and IgM responses in the dogs' serum samples via ELISA and Western blot. Microscopic examination revealed parasite eggs in feces between days 44 and 46 post-infection, whereas parasite DNA was detected by conventional PCR as early as day 20. Both real-time qPCR and dPCR yielded consistent results; however, dPCR demonstrated higher sensitivity, successfully detecting parasite DNA even in samples with low egg counts. The administered anthelmintic treatment (praziquantel) ceased egg shedding within 2 to 4 days. Nevertheless, parasite DNA continued to be detected intermittently for up to 30 days post-treatment through molecular methods. Serological evaluations showed that assays based on somatic antigens exhibited high sensitivity; however, their specificity was limited due to cross-reactions. Although the recombinant antigen improved specificity, it did not achieve adequate sensitivity for early diagnosis. IgG responses were found to be more reliable for serological diagnosis, while IgM responses were inconsistent. In conclusion, molecular methods provided accurate results in the early stages of infection, while serological tests were more effective in the later stages. Although microscopic examination offered supportive diagnostic information, due to the limited sensitivity and the risk of cross-reactions associated with all methods, it is recommended that these techniques be used collectively and supported by antigen optimization.