Early diagnosis of Trichinella spiralis and Trichinella nativa: Expression of the serine protease gene at the invasive intestinal and muscular larva stages.

Abstract

Background and aim: Diagnosis of trichinellosis at the intestinal stage during larval development is the primary challenge in the early detection and treatment of trichinellosis. The use of serine protease as a diagnostic marker for serological tests has been the subject of various studies, but data on Trichinella nativa serine protease in the intestinal phase are still insufficient for a proper diagnosis. This study aimed to establish the duration of the intestinal phase for early diagnosis and to determine the level of expression of the serine protease gene in T. nativa and Trichinella spiralis larvae.

Materials and methods: We used European isolates from T. spiralis pigs and T. nativa larvae isolated from spontaneously infected wild carnivorous animals (wolf, Karaganda region) in Central Kazakhstan. Isolation of larvae from the meat of infected animals was carried out using the compressor method. For two species of Trichinella, 36 mice (in each group 18 mice) were infected with 250 larvae and euthanized by intramuscular injection of xylazine followed by an intravenous overdose of anestofol at 3, 5, 7, 14, 21, and 30 dpi (each day 3 infected mice) and one control group (3 mice). Sequencing and bioinformatics methods were used to determine the DNA and cDNA of the serine protease gene, and molecular methods (DNA extraction, reverse transcription polymerase chain reaction, and sequence) were used to measure the accumulation of serine protease transcripts in isolated larvae.

Results: The results showed differences in the duration of intestinal phase between T. spiralis and T. nativa. The intestinal larvae of T. nativa were observed from 7 to 30 dpi, and the intensity of invasion increased up to 30 dpi (p < 0.001), while in the case of T. spiralis, the increase in larval growth in the intestinal phase decreased to 21 dpi, and only an increase of 1.6 ± 0.88 (p < 0.01) was detected at 30 dpi. T. nativa muscle larvae were detected at 21 dpi, compared with T. spiralis at 14 dpi. This characteristic was also reflected in the levels of serine protease transcripts in the samples. Accumulation was observed in both cases higher in the muscular stage of development, whereas the duration of the intestinal stage of T. nativa made it possible to detect serine protease at 30 dpi.

Conclusion: The intestinal stage of T. nativa lasts for 30 days, indicating that the use of T. nativa serine protease is useful for the identification of intestinal infection. Furthermore, this protein can be used to identify T. spiralis and T. nativa in laboratory samples. Serine protease can be used as a marker for serological diagnosis. Within the framework of the research topic, it is important to conduct further studies on the species specificity of the obtained recombinant protein. It is necessary to focus on identifying highly specific Trichinella proteins for early disease detection.