Cytauxzoonosis: Novel diagnostics and therapeutics

Abstract

Cytauxzoonosis is a tick-borne disease in the domestic cats with high mortality and a narrow therapeutic window for initiation of treatment. It is caused by the apicomplexan protozoal parasite Cytauxzoon felis and transmitted primarily by Amblyomma americanum ticks in the North America. To date, there is no effective treatment and no vaccine available for prevention, and the infected cats usually have rapid disease progression and succumb to infection. Thus, the early diagnosis and effective therapy is crucial for the survival of these cats.

In this thesis, two novel diagnostics including a C. felis probe-based droplet digital PCR (ddPCR) and a C. felis IgM ELISA were developed and validated for early detection of feline acute cytauxzoonosis. The probe-based ddPCR was designed using the hydrolysis probe and droplet digital technology. The assay is highly sensitive and specific that can detect infection in all clinical cats presented with acute cytauxzoonosis and had 100% agreement with blood smear evaluation. Infections were also detected in experimentally infected cats as early as one day prior to developing clinical signs. Additionally, it can monitor parasite loads over time to assess therapeutic efficacy. An indirect ELISA was developed utilizing the recombinant schizogenous antigen contig88 as coating protein to capture C. felis-specific IgM in infected cat plasma samples. IgM was detected by goat-anti-cat IgM antibody-HRP. The absorbance was measured at 450 nm. The IgM ELISA had 100% specificity and 86.21% sensitivity for strong positive samples and 75.86% specificity and 100% sensitivity in weak positive samples.

Lastly, a clinical study was conducted evaluating the efficacy of combined immunomodulatory therapy (atovaquone + azithromycin + dexamethasone) in treating cats with acute cytauxzoonosis. Although there was no significant difference in survival (p=0.8343), increased expression of pro-inflammatory cytokines (IFNɣ, IL-1β, IL-8, IL-12, RANTES and Flt-3L) were detected in cats with acute cytauxzoonosis compared to healthy cats (p<0.05), while expression of PDGF-BB, SDF-1, MCP-1 and IL-18 was decreased in C. felis infected cats (p<0.05).

Collectively, these studies developed novel, applicable diagnostics for cytauxzoonosis and insight on the immunologic alteration during acute C. felis infection, which has the potential to become therapeutic targets in the future.