A rapid and simple single-step method for the purification of Toxoplasma gondii tachyzoites and bradyzoites.

This study describes a simple method for the large-scale isolation of pure Toxoplasma gondii tachyzoites and bradyzoites. T. gondii tachyzoites were obtained from infected human foreskin fibroblasts (HFFs) and peritoneal exudates of mice, while tissue cysts containing bradyzoites were collected from chronically infected mice. Harvested cells and brain tissues were incubated in Hanks balanced salt solution (HBSS), containing 0.25% trypsin and 0.5% taurodeoxycholic acid (TDC) for 5 min. Subsequent washes in phosphate buffered saline (PBS) were conducted, and the cell viability of the preparations was good, as determined by flow cytometry and ability to reinfect HFF cells and propagate in mice. The purification procedure allowed for a rapid preparation of pure T. gondii tachyzoites and bradyzoites in sufficient quantity that can be used for downstream procedures. The advantage of the new method is that it is convenient and inexpensive.

An ex vivo abomasal ovine model to study the immediate immune response in the context of Haemonchus contortus larval-stage.

We have set up an ex vivo ovine abomasal model, which can mimic the multicellular process to explore the early steps in haemonchine nematode infection using RNA-seq technology. Ovine abomasal explants were collected for histological and transcriptional analysis and supernatants collected to quantitate lactate dehydrogenase (LDH) enzymes. Atotal of 233 were substantially induced genes between L4-inoculated and uninoculated-control tissues, respectively. However, a total of 14 were considerably down-regulated genes between the 51 aforementioned tissues. Fifteen pathways were annotated by Kyoto Encyclopedia of Genes, and Genomes pathway analysis accounted for the significant percentage in immediate response to larval-stage of H. contortus. Key genes upregulated in response to the addition of L4-inoculum of H. contortus were IL-6, IL-8, C1q, Atypical chemokine receptor-3, chemokine ligand-2, manganese superoxide dismutase, integrin alpha-7, -8, -9, integrin subunit beta-1, integrin subunit beta 6, intercellular adhesion molecule-1 and actin alpha-1. This study shows for the first time that galectin-1 is up-regulated in an ex vivo abomasal segment model exposed to L4-inoculum of H. contortus following 6 h of incubation. The abomasal segment model has been shown to be a suitable tool to study the haemonchine larval-stage effects on the ovine abomasal tissues prior to in vivo assessment.

An ex vivo ruminal ovine model to study the immediate immune response in the context of bacterial lipopolysaccharide.

We have set up an ex vivo ovine ruminal model, which can mimic the multicellular process to explore the early steps in Salmonella typhimurium lipopolysaccharide (LPS) stimulation using RNA-seq technology. Ovine ruminal explants were collected for histological and transcriptional analysis and supernatants collected to quantitate lactate dehydrogenase (LDH) enzymes. A total of 8 and 523 genes were significantly over-expressed between LPS-treated and control tissues at 6 and 12 h, respectively. However, six and seven hundred and thirteen genes were substantially repressed between the aforementioned tissues, correspondingly. Key genes up-regulated in response to the addition of LPS were tumor necrosis factor (TNF), interlukin (IL)-1 beta(b), IL-6, IL-8, IL-17B, IL-19, MMP-1, MMP-3, and integrin alpha 2 (ITGA8, 9). This study shows for the first time that galectin-1 is up-regulated in an ex vivo ruminal segment model exposed to bacterial lipopolysaccharide following 6 h of incubation. The ruminal segment model has been shown to be a suitable tool to study the bacterial lipopolysaccharide effects on the ovine ruminal tissues prior to in vivo assessment.

Microbial community and ovine host response varies with early and late stages of Haemonchus contortus infection.

The interactions between gastric microbiota, ovine host, and Haemonchus contortus portray the ovine gastric environment as a complex ecosystem, where all factors play a pertinent role in fine-tuning each other and in haemeostasis. We delineated the impact of early and late Haemonchus infection on abomasal and ruminal microbial community, as well as the ovine host. Twelve, parasite-naive lambs were divided into four groups, 7 days post-infection (dpi) and time-matched uninfected-control groups; 50 dpi and time-matched uninfected control groups were used for the experiment. Six sheep were inoculated with 5000 H. contortus infective larvae and followed for 7 or 50 days with their corresponding uninfected-control ones. Ovine abomasal tissues were collected for histological analysis and gastric fluids were collected for PH value measurements, microbial community isolation and Illumina MiSeq platform and bioinformatic analysis. Our results showed that Haemonchus infection increased the abomasal gastric pH (P = 0.05) and resulted in necrotizing and inflammatory changes that were more severe during acute infection. Furthermore, infection increased the abomasal bacterial load and decreased the ruminal microbiome. A 7-day infection of sheep with H. contortus significantly altered approximately 98% and 94% of genera in the abomasal and ruminal bacterial profile, respectively (P = 0.04-0.05). However, the approximate altered genera 50 days after infection in the ovine abomasal and ruminal microbiome were about 62% and 69%, correspondingly (P = 0.04-0.05) with increase in some bacteria and decrease in others. Overall, these results indicate that Haemonchus infection plays a crucial role in shaping stomach microbial community composition, and diversity.