Development of a colloidal gold immunochromatographic strip based on GAPDH for Pentatrichomonas hominis in dogs

Abstract

Pentatrichomonas hominis is a zoonotic protozoan belonging to the family Trichomonadidae that primarily inhabits the cecum and colon. 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. A previous study further identified a significant correlation between P. hominis infection and colorectal cancer, underscoring its growing clinical and public health significance. 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. 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. 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. 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. To address the detection need, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used as the primary discovery tool to identify specific detected antigens. Using this targeted proteomics approach, we precisely identified immunoreactive proteins and selected glyceraldehyde 3 phosphate dehydrogenase (GAPDH), an immunogenic and species-specific antigen in related parasites, as the candidate antigen. Based on this identified antigen, we subsequently developed and evaluated the detection performance in both indirect ELISA and colloidal gold immunochromatographic strips using recombinant GAPDH. This study aimed to identify novel antigens for immunodetection of P. hominis and to establish a practical, on-site method for detecting dog infections, thereby facilitating further epidemiological and clinical research.

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.