Blastocystis sp. is an eukaryotic protozoan with 17 distinctive subtypes discovered in animals and humans worldwide. Cattle is one of important source of animal protein, which shown an increasing demand for its produce due to increased in growth of population especially in developing countries. However, the production is likely to be affected by infection and disease management of this animal. Currently, the zoonotic pathogen of Blastocystis sp. from cattle was identified with multiple concurrent infections with rates of infection as high as 80%. Unlike human, animal particularly cattle infected with Blastocystis sp. are commonly healthy carriers and serve as a main reservoir in transmitting the infection to human. Though, Blastocystis exhibited protease activity that suggest its pathogenicity, but its effect on host remain unclear. Thus, this study was aimed to determine occurrence of Blastocystis sp. isolated from cattle and subtypes variation in the protease activity for better understanding of the host-parasite relationships and effect of location and farm management on Blastocystis infection. A total of 120 faecal samples of cattle were collected from three farms in Pahang, Malaysia for in-vitro cultivation and microscopy identification. The gender and age of the cattle as well as management system of the farms were also noted during the sampling. Later, DNA extracted from the faecal were subjected to genotyping analysis before protease activity of three selected subtypes were determined using azocasein assay of colorimetric quantification method. The study found 30 out of 120 (25%) cattle infected with Blastocystis sp. with vacuolar as the dominant form observed during cultivation. While gender has no association with the occurrence of Blastocystis sp. and cattle of age below 3 months as well as Muazam Shah farm with integrated system were significantly (p<0.05) associated with the infection. Molecular genotyping revealed that Blastocystis STlO (21.3%) occurred predominantly in the cattle with STl (2.5%), ST3 (7.5%), ST4 (2.5%), ST5 (8.8%) and ST14 (1.3%). Phylogenetic analysis found that these subtypes were closely related and had shared common ancestors with high homologous sequences of genetic relation. ST3, ST5 and STl 0 exhibited inter-and intra-subtype quantitative protease activity variation, which mainly expressed cysteine protease and partially serine protease, aspartic protease and metallo-protease in the respective subtypes. In conclusion, moderate rates of Blastocystis sp. infection with six different subtypes were identified in the cattle. The farm management systems, cleaning and sanitation as well as segregation condition influence the distribution of Blastocystis sp. infection which significantly associated with age and condition of the farms. While, protease activity were commonly been reported in ST3, ST4 and ST?, this study has discovered in the ST3, ST5 and STl 0 of Blastocystis. The variant observed in the protease activity indicate that isolates of different subtypes may exhibit different pathogenic condition upon exhibition of diseases, yet suggests for more studies needed in the future. Nevertheless, this study presented updates on the occurrence of Blastocystis sp. in cattle from Pahang, Malaysia. This information is important in understanding host-parasite relationship associated with gastrointestinal diseases and identification of possible virulence factor in the future.

Cryptosporidium parvum is a widespread pathogenic parasite which causes cryptosporidiosis in human. The host cell-C. parvum interaction causes alteration of expression of a series of microRNAs or miRNAs within the host cells due to activation of defense mechanism. In immunocompromised host cells, miRNAs play a key role in post-transcriptional gene regulation. The regulation of miRNAs in the infected cells may be identified as possible biomarkers in cancer onset and progression. Upregulation of oncomicroRNAs in the host epithelial cells due to the C. parvum infection may lead to colorectal cancer initiation and progression in human. HCT-8 and HT-29 were grown in different L-glutamine concentration and pH to observe the relative growth. The study was aimed to delineate the host epithelial colorectal cancer cells-C. parvum interaction and its associated miRNAs involvement in the immunocompromised host cells in vitro. The differential expression level of oncomiRNA, miR-21 and tumor suppressor miRNA, miR-145 in host cells upon C. parvum infection was observed in order to establish a relationship between C. parvum infection and colorectal cancer in humans. As Cryptosporidium mainly infects the epithelial cells of the colorectal region, the host cells of interest in this study are HCT-8 and HT-29 cell lines. The direct immunofluorescent staining method has been applied using fluorescein conjugated Vicia Villosa Lection (VVL) to observe the C. parvum infection sites on HCT-8 and HT-29 cell lines after 2.5 hours of infection period. In addition, scanning electron microscopy has been used to observe the surface micrographs of different life stages of C. parvum adhered to the cell lines. The real time quantitative PCR (R T-qPCR) was performed to observe the relative expression ofmiR-21 and miR-145 in infected HCT- 8 and HT-29 cell lines. Data normalization of the miRNA expression was carried out using reference gene RNU44. The result showed that, the cell growth was optimum for cell lines in higher L-glutamine concentration and higher pH. In the immunofluorescent staining assay, the micrographs show multiple sites of C. parvum infection on both HCT-8 and HT-29 cell lines. Meanwhile, scanning electron micrographs demonstrated the adherence of C. parvum oocysts in both HCT-8 and HT-29 cell lines after 24 hours of inoculation. Along with the surface meront I, several numbers of early trophozoites in their developmental stages were confined to the apical surfaces of HCT-8 and HT- 29 cell lines. Besides~ a few sporozoites have been observed to be attached on the HCT- 8 and HT-29 cell lines. The normalized expression quantification data obtained from RT-qPCR has shown upregulation of miR-21 expression and significant downregulation of miR-145 expression in HCT-8 and HT-29 cell lines upon C. parvum infection. The rniR-21 expression showed significantly higher upregulation in infected HCT-8 than in infected HT-29. On the other hand, miR-145 downregulation was significantly lower in HT-29 cells than in HCT-8 cells upon C. parvum infection. In this study, immunofluorescent micrographs and scanning electron micrographs indicated that C. parvum effective infects human colorectal cancer cell lines. The upregulation of oncomiRNA and downregulation of tumor suppressor miRNA in infected HCT-8 and HT-29 cells possibly indicates the influence of C. parvum in cancer progression. However, the findings are less sufficient to confirm the C. parvum as a pathogenic parasite for cancer progression. Future studies should be aimed to observe more targeted genomic and transcriptomic alterations, relative expression of sets of targeted miRNAs to absolutely verify the progression of colorectal cancer by C. parvum infection.