A Serodiagnostic IgM ELISA to Detect Acute Cytauxzoonosis

Abstract

Cytauxzoonosis is a tick-borne infectious disease affecting domestic cats with high mortality and limited treatment modalities. Because early diagnosis and therapeutic intervention are crucial to survival of infected cats, the objective of this study was to develop an ELISA capable of detecting cytauxzoonosis and differentiating acute vs. chronic infection in clinical feline blood samples. A microsphere immunoassay (MIA) was developed to evaluate the production of Cytauxzoon felis-specific IgM and IgG antibodies in serial plasma samples from cats with experimental C. felis infection by targeting a C. felis-specific transmembrane protein (c88). Recombinant c88 protein was utilized to develop indirect ELISAs to detect IgM and IgG antibodies in clinical plasma samples from: PCR-positive cats with acute C. felis infection (n = 36), C. felis-negative cats with pyrexia (n = 10), healthy C. felis-negative cats (n = 22), and chronic C. felis carriers (n = 4). Anti-c88 IgM antibodies were detectable at day 12 post-tick infestation in cats with experimental C. felis infection (within 24 hours of developing clinical signs), while anti-c88 IgG was detectable at day 15 post-tick infestation – indicating IgM could be used to detect early infection. Using a cut-off value of 19.85 percent positive, the C. felis IgM ELISA detected acute cytauxzoonosis in 94.44% (34/36) of cats presented with clinical signs of acute cytauxzoonosis with 100% specificity (indicating a “Strong Positive” result). When a lower cutoff of 8.60 percent positive was used, cytauxzoonosis was detected in the 2 remaining PCR-positive cats with 87.88% specificity (indicating of a “Weak Positive” result). One C. felis-negative, febrile cat had high IgG, and chronic carriers had variable IgM and IgG results. Combined interpretation of IgM and IgG ELISAs did not reliably differentiate acute vs. chronic infection. While further validation on assay performance is needed, the C. felis IgM ELISA is a promising test to detect acute cytauxzoonosis and can be utilized to develop a point-of-care test for clinical use.

Cytauxzoonosis in Indiana, USA: a case series of cats infected with Cytauxzoon felis (2018–2022)

“…This method has been validated against other infectious agents that infect cats and specifically detects C felis DNA to as low as 0.0000231 ng DNA/reaction in cases of acute cytauxzoonosis and 0.00232 ng DNA/reaction in cases of chronic cytauxzoonosis. 21 ELISA methods that detect C felis -specific IgM and IgG, and can differentiate between acute and chronic cytauxzoonosis, have been developed 32 but are not commercially available. A point-of-care test for cytauxzoonosis that could provide accurate, real-time results would be a tremendous asset and allow veterinary practitioners to interpret clinical signs and reach a definitive diagnosis to initiate antiprotozoal therapy promptly.…”

A Novel Vaccine Strategy to Prevent Cytauxzoonosis in Domestic Cats

“…Indirect ELISAs were performed to detect anti-c88 and anti-cf76 IgM and IgG antibodies in response to vaccination and C. felis infection using plasma separated from EDTA-treated blood samples. Anti-c88 specific IgM and IgG levels were measured using a previously published assay [ 42 ]. Briefly, 96-well Microplates (Corning Inc. Glendale, AZ, USA) were coated with c88 protein (5 μg/mL for IgM, 2.5 μg/mL for IgG; 100 μL/well) diluted in carbonate buffer (4 °C, overnight).…”

“…A notable anti-cf76 IgG response was not observed in response to vaccination cf76. Dashed horizontal line in ( a ) indicates the strong positive cutoff, as reported in [ 42 ]. Upper dashed horizontal line in ( b ) indicates strong positive cutoff, while the lower dashed horizontal line indicates weak positive cutoff, as reported in [ 42 ].…”

“…Dashed horizontal line in ( a ) indicates the strong positive cutoff, as reported in [ 42 ]. Upper dashed horizontal line in ( b ) indicates strong positive cutoff, while the lower dashed horizontal line indicates weak positive cutoff, as reported in [ 42 ]. Dashed horizontal lines in ( c , d ) indicate positive cut-off values established as previously described [ 43 ].…”