关键词: 广州管圆线虫, 实时荧光定量PCR, DNA载量, 组织分布

Abstract:

Objective To investigate the dynamic distribution and changes in DNA load of Angiostrongylus cantonensis in rats, so as to provide experimental evidence for clinical diagnosis and disease control. Methods Fifty-five SD rats were randomly assigned to a control group (n = 10) and an infection group (n = 45). The infection group was subdivided into a low-dose group (20 larvae/rat), a medium-dose group (60 larvae/rat), and a high-dose group (100 larvae/rat), with 15 rats per subgroup. Rats in the infection groups were infected with third-stage larvae of A. cantonensis by oral gavage; the control group received an equal volume of sterile physiological saline by gavage. Cerebrospinal fluid, whole blood, serum, brain tissue, heart, and lung tissue were collected on days 3, 7, 14, 28, and 35 post-infection. Brain, heart, and lung tissues were examined under a dissecting microscope and visible worms were removed. All valid samples were tested for the ITS2 sequence with conventional PCR assay and ITS1 sequence with TaqMan probe-based qPCR assay, and positive PCR amplification products were subjected to bidirectional sequencing, followed by sequence alignment using the BLAST program. The limit of detection was determined by both conventional PCR and qPCR using serially diluted standard plasmids of A. cantonensis (positive standard), and the specificity was assessed using genomic DNA from Trichinella spiralis, Cysticercus cellulosae, Necator americanus, and rats as templates, while repeatability was evaluated across a range of 1.67 × 10⁷ to 1.67 × 10² copies/μL. All statistical analyses were performed using the software SPSS 26.0. Results Visible parasites were detected in 45 rats from the infection groups by dissection microscopy, with a 100% (45/45) percentage of successful infections. A total of 270 samples were collected, including 245 valid samples. Conventional PCR assay yielded no specific bands, with a detection rate of 0 (0/245), while the detection rate of qPCR assay was 67.35% (165/245). The sensitivity of the qPCR assay was 10 copies/μL, and no cross-reactivity was observed with genomic DNA from T. spiralis, C. cellulosae, N. americanus, or rats. The detection rates showed a tendency towards a rise in the low-, medium-, and high-dose infection groups over time (χ² = 11.82, 13.57, 14.84, all P < 0.01), with 7/18, 5/18, and 11/18 on day 3 post-infection, 8/18, 11/18, and 11/18 on day 7 post-infection, 6/13, 7/11, and 13/15 on day 14 post-infection, 11/17, 12/15, and 15/15 on day 28 post-infection and 15/16, 16/18, and 17/17 on day 35 post-infection, while no A. cantonensis DNA was detected in samples from the negative control group at any time point. The highest detection rate of A. cantonensis was seen in brain tissues (88.37%, 38/43) using qPCR assay, followed by in heart (74.42%, 32/43), lung tissue (72.09%, 31/43), cerebrospinal fluid (65.79%, 25/38), serum (52.94%, 18/34), and whole blood (47.73%, 21/44). Three-way analysis of variance (ANOVA) revealed that tissue type (F = 22.110, P < 0.01), dose of infection (F = 3.762, P < 0.05), and time of infection (F = 34.768, P < 0.01) significantly affected DNA load, with a significant interaction between tissue and time (F = 4.295, P < 0.01). DNA signals were predominantly concentrated in brain tissues at early stage of infection (3 to 7 days post-infection), and the DNA load increased rapidly in lung and heart since day 14 post-infection. High levels of DNA load were seen in brain, lung, and heart on days 28 to 35 post-infection, and the DNA load was higher in lung tissues than in brain tissues on day 35 post-infection. Conclusion Residual DNA of A. cantonensis is predominantly distributed in brain and lung tissues of rats and evolves dynamically over the course of infection, with the period from 14 to 35 days post-infection representing the peak of residual DNA load.

Key words: Angiostrongylus cantonensis, Real-time fluorescent quantitative PCR, DNA load, Tissue distribution