Blastocystis sp. is the most common enteric protist colonizing a wide range of host and is characterized by wide genotypic diversity. Heat Shock Protein 70 (HSP70), a common stress-response protein has been associated with increased proliferation in Blastocystis sp. under thermal stress. However, the extent of HSP70 genetic variation and its link to thermostability remains unexplored. Our study aims to elucidate HSP70 gene variants among different Blastocystis sp. subtypes, analyze their responses to thermal stress, and evaluate their protease activity following the thermal stress. Heat Shock Protein 70 gene from eighteen Blastocystis sp. isolates of varying subtype – ST1, ST3, ST4, ST6, and ST7 – were amplified, sequenced and phylogenetically analyzed. The cells were then subjected to thermal stress at 42°C for 24 hours and the cell viability was monitored for seven days. Protease activity in solubilized antigens (SA) from purified Blastocystis sp. cells was evaluated following exposure to thermal stress. Phylogenetic analysis revealed two distinct clades: ST4 and ST7 (Clade 1) which are mostly thermotolerant, while ST1, ST3, and ST6 (Clade 2) exhibited lower tolerance. Total protease activity was similar across subtypes, suggesting need for further sub-class specific analysis. Our findings suggests that thermal stress adaptation is subtype dependent and may be influenced by HSP70 variation. This insight highlights the importance of HSP70 as a potential molecular marker for stress adaptation in Blastocystis sp. and underscore the need for deeper investigation of its functional role in parasite persistence and pathogenic potential.

Blastocystis sp. is a unicellular, anaerobic intestinal protist regularly reported in humans and various animals worldwide. There seems to be little research on Blastocystis infection in poultry in Malaysia, and none on Blastocystis in quail specifically. In Malaysia, the consumption of quail meat and eggs is rapidly gaining popularity as a significant source of protein. It is, therefore, essential to explore the presence of Blastocystis in Malaysian quails in order to aid in the understanding of Blastocystis in this group of birds and their role in its transmission. Intestinal contents were collected from 90 commercial quails raised on two farms in Penang, Malaysia, in a multi-layer cage system with adequate farm management. Detection of Blastocystis sp. was by cultivation in modified Jones’ medium supplemented with 10% horse serum. Giemsa-stained slides made from positive cultures were used for morphological studies whereas Blastocystis subtyping was conducted by using Polymerase Chain Reaction (PCR). A prevalence of 17.8% (16/90) was recorded for Blastocystis sp. in quail in this study. The most common forms detected in the in vitro culture medium were vacuolar and granular forms with cell diameters ranging from 9.09μm to 33.33μm. None of the quail birds screened had any visible gastrointestinal symptoms or signs. All successfully sequenced isolates were identified as Blastocystis sp. ST6, one of the potentially zoonotic subtypes of Blastocystis. This study posits that the quail birds may serve as reservoirs of zoonotic subtypes of Blastocystis. More studies are required to understand the source of Blastocystis infection to poultry under intensive care and the role of poultry animals in the transmission of Blastocystis to humans.