An indirect ELISA for detection of Encephalitozoon cuniculi infection in farmed blue foxes (Alopex lagopus)

Abstract

Infection with the intracellular microsporidium Encephalitozoon cuniculi can cause serious disease, encephalitozoonosis, in the blue fox (Alopex lagopus). The disease diagnosis is based on clinical signs and pathological findings, and detection of E. cuniculi or circulating antibodies directed against the parasite. Indirect immunofluorescence (IFAT) and carbon immunoassay (CIA) are the most commonly used serological methods for diagnosis in this species. In the present study, an indirect ELISA (enzyme linked immunosorbent assay) was established and evaluated against IFAT by testing of 205 field samples from blue foxes. There was high agreement between the results of the ELISA and CIA (kappa=0.99), and the ELISA and IFAT (kappa=0.958). There was no significant statistical difference between the tests (p>0.05). It was concluded that the ELISA could be used to identify seropositive farmed blue foxes. The advantage of the ELISA lies in the potential of screening large numbers of animals with the goal of eradicating E. cuniculi infection in the farms.

Figure 1

ELISA values for positive reference serum and soluble E. cuniculi antigen diluted 2-fold from 1:1000. Each line represents the following coating buffers: -■- carbonate bicarbonate (100 mM, pH 9.6) -▲- citrate phosphate (70 mM, pH 4.0) -◆- distilled water -●- phosphate buffered saline (PBS; 170 mM, pH 7.4) -▼- citrate phosphate (150 mM, pH 5.0) -□- ethanol (70% v/w).

Figure 2

Two-graph receiver operating curve (TG-ROC) analysis for 205 sera from blue foxes tested in the E. cuniculi ELISA. Based on CIA results, the 2 graphs represent sensitivity (solid line) and specificity (dotted line) for thresholds of the ELISA S:P values, where the intersection is the point were sensitivity and specificity are equal. An intermediate range lies between 2 cut-off values were sensitivity and specificity are 0.95, respectively. ELISA results below the lower cut-off value are considered negative, and S:P values above the upper cut-off value are positive. The S:P values represent only wells coated with E. cuniculi.