Serodiagnosis of nasal myasis in camels (Camelus dromedaries) in Egypt using third larval instar affinity-purified glycoprotein

Abstract

The larvae of Cephalopina titillator cause nasopharyngeal myiasis in camels, which parasitize the living tissues of the nasal and paranasal sinuses, pharynx, and larynx. C. titillator infestation adversely affects camel health, meat, and milk production, and can even cause death. In our study, to improve the immunodiagnosis of camel nasal myiasis, a sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed and evaluated using the Concanavalin-A (Con-A) affinity purification for the C. titillator-N-acetylglucosamine (Ct-GlucNAc) glycoprotein fraction from third larval instars as an antigen for detecting C. titillator antibodies. Crude antigens were prepared from larval instars of C. titillator and evaluated by indirect ELISA. The third C. titillator larval antigen (L₃Ct) had the highest protein content (P < 0.001) and the best diagnostic value; chi-square = 235 (P < 0.001). Four glycoprotein fractions were purified separately from the L₃Ct antigen by Con-A purification and evaluated. The Ct-GlucNAc glycoprotein fraction was the fraction of choice with the highest diagnostic accuracy (P < 0.05). Using Ct-GlucNAc as a coating antigen, indirect ELISA showed a 99.3% sensitivity for positive results in camel myiasis samples and 100% specificity for negative results in healthy camel samples. The diagnostic accuracy was 99.7%, and no cross reactivity was detected for other parasitic diseases. The indirect ELISA results were confirmed by the western immunoblotting which was characterized by comparing sera from naturally infested dromedary camels with C. titillator, sera from healthy camels and sera from camels with other parasitic infections (Echinococcus granulosus, Fasciola gigantica, Hard ticks; Hyalomma dromedarii, Trichostronglid sp., Eimeria spp., and Cryptosporidium sp.). Immunoreactive antigenic bands of 63, 50, 30 and 18 kDa were predominantly detected in sera from camels with nasopharyngeal myiasis and didn't react with healthy and camel's sera from other parasitic infections. However, seven immunoreactive bands appeared at 120, 70, 63, 48, 35, 29, and 19 kDa in the crude L₃Ct antigen. In addition, a positive rate of C. titillator immunodiagnosis was detected by indirect ELISA (48.6%, chi-square = 483, P < 0.001), which was significantly greater than that of postmortem diagnosis (31%). In conclusion, the current study introduces a new diagnostic immunoaffinity glycoprotein fraction of C. titillator 3rd larval instar-based ELISA as a highly accurate, simple and fast method to detect specific antibodies of nasal myiasis in camels.

Keywords: Cephalopina Titillator; Con-A affinity chromatography; ELISA; Glycoprotein fractions; SDS‒PAGE; Western blot.

Fig. 1

A The nasopharyngeal region of infested camel head by C. titillator, congested with dark color, swollen, hemorrhagic, and edematous mucous. B The three instars of C. titillator larvae from infested camel heads

Fig. 2

Evaluation and comparison of the immunodiagnostic potency of the three prepared C. titillator crude larval antigens in the detection of nasal myiasis infestation in naturally infected camel sera and negative sera, where the OD value of the cutoff was 0.219 (red line) according to indirect ELISA

Fig. 3

SDS‒polyacrylamide gel electrophoretic profile of the Cephalopina titillator third-stage larval crude antigen (Lane 1), and four isolated glycoprotein fractions stained with Coomassie blue; Ct-GlucNAc (Lane 2), Ct-Gluc (Lane 3), Ct-Gal (Lane 4) and Ct-Lac (Lane 5). The protein molecular weight standard is shown in kDa (Lane Mr)

Fig. 4

Diagnostic profile of four glycoprotein fractions compared to crude 3rd instar C. titillator larvae as coating antigens in naturally infested camels

Fig. 5

Immunoblot analysis showing the presence of specific and cross-reactive antigenic bands in sera from camels with C. titillator and those infected with other parasitic diseases, including the crude third larval L₃Ct (C) antigen and Ct-GlucNAc glycoprotein fraction (F). Control negative camel sera are shown on the right (Strips 15 and 16). The standard protein molecular weight is in kDa (Strip St. MW).

Fig. 6

Diagnosis of camel C. titillator nasal myiasis by indirect ELISA based on the purified Ct-GlucNAc glycoprotein fraction in collected camel sera. The red line represents the cutoff (OD value = 0.258)