Development of a Colloidal Gold Immunochromatographic Strip Based on GAPDH for Pentatrichomonas hominis in Dogs

Abstract

Pentatrichomonas hominis is a zoonotic protozoan that primarily inhabits the cecum and colon of dogs, with infection rates as high as 47.4%, posing a significant public health risk due to close human contact. Current detection methods, including direct smear microscopy and PCR, have limitations in sensitivity, equipment requirements, and operational complexity. To address the need for a practical on-site detection method, we employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify specific antigens from P. hominis excretory-secretory (ES) proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was selected as a candidate antigen due to its high immunogenicity and species specificity. The GAPDH gene was cloned and expressed in E. coli, and the recombinant protein was purified. Mouse anti-GAPDH serum was generated, and its reactivity was confirmed by Western blot and indirect ELISA (titer 1:102,400). Immunofluorescence localization showed GAPDH in the cytoplasm of P. hominis trophozoites. Based on this antigen, we developed a colloidal gold immunochromatographic strip for rapid detection of P. hominis in dogs. The strip demonstrated high sensitivity and specificity, providing a practical tool for veterinary diagnosis and epidemiological surveillance. This study is the first to report an immunochromatographic strip for P. hominis detection, offering a rapid, user-friendly alternative to existing methods.

1. Introduction

Pentatrichomonas hominis is a zoonotic protozoan belonging to the family Trichomonadidae that primarily inhabits the cecum and colon [1]. Although traditionally regarded as an opportunistic pathogen, P. hominis is increasingly recognized for its pathogenic potential, including roles in animal diarrheal disease and the induction of intestinal epithelial damage and chronic inflammation in mice [2,3]. A previous study further identified a significant correlation between P. hominis infection and colorectal cancer, underscoring its growing clinical and public health significance [4]. This parasite infects a wide range of hosts, among which dogs, displaying infection rates as high as 47.4%, are regarded as a potential zoonotic reservoir because of their close contact with humans [5]. Consequently, establishing reliable detection methods for P. hominis in dogs is essential for veterinary practice and public health surveillance.

Current methods for detecting P. hominis infections in dogs mainly include direct smear microscopy and polymerase chain reaction (PCR)-based techniques. Although direct smear microscopy is straightforward, it frequently exhibits low sensitivity. In comparison, PCR demonstrates high sensitivity and specificity, yet it relies on specialized equipment, trained operators, and extended processing time. Recently, reported nucleic acid detection approaches, such as recombinase polymerase amplification coupled with lateral flow dipstick (RPA-LFD) and RPA-CRISPR/Cas12a assays, have enhanced the efficiency and accessibility of molecular detection for P. hominis [6,7]. However, these methods still necessitate nucleic acid extraction, controlled temperature conditions, and operational complexity. In addition, some emerging detection technologies, such as microfluidic chips and nanozyme-based detection systems, offer advantages of high throughput and sensitivity but have not yet been widely applied in the field of detection of parasitic infection [8–10]. Although immunoassays, including enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunochromatographic strips, have been successfully used for the detection of infections of some intestinal protozoans, such as Giardia, there have been no reports on their application for detecting P. hominis infections, primarily due to the lack of specific detection antigens.