Veterinary world · 2025
Comparative immunodiagnostic evaluation of recombinant serine proteases from Trichinella spiralis and Trichinella nativa for early detection of trichinellosis.
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Zhumalin Aibek, Gajimuradova Aisarat, Bulashev Aitbay, Akibekov Orken, Syzdykova Alfiya, Gubaidullin Nurtai, Tosini Fabio, Zhagipar Fariza, Aydin Ali, Askarova Nasipkhan
Аннотация
BACKGROUND AND AIM: Trichinellosis, a major zoonotic foodborne disease caused by Trichinella spp., remains challenging to diagnose in its early stages due to low antibody titers and limitations of conventional excretory-secretory (ES) antigens. Recombinant antigens, particularly serine proteases expressed throughout infection, represent promising candidates for specific and sensitive diagnostics. This study aimed to clone, express, and evaluate recombinant serine proteases from Trichinella spiralis (rTsp-LE) to Trichinella nativa (rTnsp-4E), and to assess their diagnostic performance compared with ES antigens and a commercial kit.
MATERIALS AND METHODS: Serine protease genes of T. spiralis and T. nativa were amplified, cloned into pET28c+ vectors, expressed in Escherichia coli, and purified under denaturing conditions. Antigenicity was tested by immunoblotting and an indirect enzyme-linked immunosorbent assay using sera from experimentally infected rabbits, naturally infected pigs, and immunized BALB/c mice. Antibody dynamics were monitored over 30 day post-infection (dpi), and results were compared with ES antigens and the VetLine Trichinella kit. Statistical analyses were performed using one- and two-way analysis of variance.
RESULTS: Both recombinant proteins were successfully expressed at expected sizes (10.8 kDa for rTsp-LE; 6.8 kDa for rTnsp-4E) and induced strong, specific antibody responses. Antibodies against rTsp-LE and rTnsp-4E were detectable as early as 7 dpi, earlier than ES antigens (14-21 dpi). Immunized mice showed significant titers (up to 1:5700 for rTsp-LE and 1:10400 for rTnsp-4E by day 30). No cross-reactivity was observed with Echinococcus antigens. In rabbits, rTsp-LE showed the highest titers for T. spiralis infection, while rTnsp-4E was more specific for T. nativa. Overall sensitivity and specificity reached 100% and 88.2%, respectively.
CONCLUSION: Recombinant serine proteases rTsp-LE and rTnsp-4E demonstrated high sensitivity, species-specific reactivity, and early antibody detection, outperforming ES antigens. These findings support their potential as reliable candidates for serological assays, contributing to earlier and more accurate trichinellosis diagnosis and improved epidemiological surveillance.
MATERIALS AND METHODS: Serine protease genes of T. spiralis and T. nativa were amplified, cloned into pET28c+ vectors, expressed in Escherichia coli, and purified under denaturing conditions. Antigenicity was tested by immunoblotting and an indirect enzyme-linked immunosorbent assay using sera from experimentally infected rabbits, naturally infected pigs, and immunized BALB/c mice. Antibody dynamics were monitored over 30 day post-infection (dpi), and results were compared with ES antigens and the VetLine Trichinella kit. Statistical analyses were performed using one- and two-way analysis of variance.
RESULTS: Both recombinant proteins were successfully expressed at expected sizes (10.8 kDa for rTsp-LE; 6.8 kDa for rTnsp-4E) and induced strong, specific antibody responses. Antibodies against rTsp-LE and rTnsp-4E were detectable as early as 7 dpi, earlier than ES antigens (14-21 dpi). Immunized mice showed significant titers (up to 1:5700 for rTsp-LE and 1:10400 for rTnsp-4E by day 30). No cross-reactivity was observed with Echinococcus antigens. In rabbits, rTsp-LE showed the highest titers for T. spiralis infection, while rTnsp-4E was more specific for T. nativa. Overall sensitivity and specificity reached 100% and 88.2%, respectively.
CONCLUSION: Recombinant serine proteases rTsp-LE and rTnsp-4E demonstrated high sensitivity, species-specific reactivity, and early antibody detection, outperforming ES antigens. These findings support their potential as reliable candidates for serological assays, contributing to earlier and more accurate trichinellosis diagnosis and improved epidemiological surveillance.