Angiostrongylus cantonensis is a well-known pathogen causing eosinophilic meningitis associated with angiostrongyliasis. Humans, as accidental hosts, are infected by consuming undercooked snails containing third-stage larvae. A. malaysiensis is closely related to A. cantonensis and has been described as a potential human pathogen. The two species distribution was recently reported to overlap in the same endemic area, particularly in the Indochina Peninsula. Similar morphological characteristics of the third-stage larva in the snail-intermediate host often lead to misidentification of the two species. Thus, we aimed to develop a sensitive and specific method to detect and discriminate Angiostrongylus third-stage larva by designing species-specific primers based on the mitochondrial cytochrome b gene. We developed the SYBR Green quantitative real-time PCR (qPCR) method for two species-specific detection assays, which could be conducted simultaneously. The method was subsequently employed to detect and identify third-stage larvae of Angiostrongylus isolated from infected Achatina fulica collected from six public parks in Bangkok Metropolitan, Thailand. The method was also a preliminary applied to detect parasite tissue debris in the patients' cerebrospinal fluid (CSF). SYBR Green qPCRs quantitatively detected approximately 10−4 ng of genomic DNA from one larva, facilitating species-specific detection. Based on the pools of third-stage larvae isolated individually from the tissue of each infected A. fulica collected from the public parks, the qPCR results revealed that A. malaysiensis was the predominant species infecting 5.26% of the collected snails. In comparison, coinfection between A. malaysiensis and A. cantonensis was 5.97%, and no single infection of A. cantonensis was detected in A. fulica. Our SYBR Green qPCR method is a useful and inexpensive technique for A. cantonensis and A. malaysiensis discrimination, and the method has sufficient sensitivity to detect isolated larvae from a snail-intermediate host. The ratio of A. cantonensis and A. malaysiensis larvae infecting the snails can also be estimated simultaneously. Our qPCRs can be employed in a molecular survey of A. cantonensis and A. malaysiensis within intermediate hosts and for clinical diagnosis of angiostrongyliasis with CSF specimens in future studies.

Oyster health is important for aquaculture productivity and sustainability. In Thailand, the white scar oyster, Crassostrea belcheri, is being promoted for cultivation, yet its health status has not been compared between research breeding and community farming sites. This study evaluated histopathological features, ultrastructure, apoptosis, and defender against apoptotic death 1 (dad1) gene expression in sexually mature C. belcheri collected from these two sites. Gill tissues were examined by histology, transmission electron microscopy, TUNEL assay, and gene expression analysis, while organ condition was assessed using a Health Assessment Index (HAI). The proportion of TUNEL-positive cells in the gills and mantle differed significantly between sites (p < 0.05), with higher levels in oysters from the farming site. In contrast, TUNEL-positive cells in the digestive gland did not differ significantly between sites, although brown cells were observed only in the digestive gland of oysters from the breeding site, suggesting possible physiological stress. To assess the expression level of dad1 in oysters cultured under different conditions, RT-qPCR revealed no significant difference between the two sites. The breeding site also had lower temperature and salinity than the farming site. Overall, these findings suggest that site-specific environmental conditions may influence gill health and stress-related responses in C. belcheri, providing baseline information for oyster health assessment and aquaculture management.