Methods of releasing sporocysts from oocysts using controlled shear forces
A technology of shearing force and oocysts, applied in the field of oocysts, can solve the problems of ineffectiveness and achieve a consistent yield of sporocysts
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Embodiment 1
[0053] A series of experiments were carried out aiming at the recovery of spores and the rupture of oocysts within a wide range of parameter values. Each experiment uses a total of approximately 2 x 10 in 500 mL HBSS 8 Sporulated oocysts (4×10 per mL 5 sporulated oocysts); a portion of the sample was counted prior to processing in the Microfluidizer processor. The diameter of the chamber to be tested varies according to the species: 1) Eimeria maxima: 200, 300, 400 micron diameter chamber; 2) E. tenella: 200, 300 micron diameter chamber; Coccidia: 125 micron chamber. Use one-pass mode. Pressures from 2,000 psig to 6,000 psig are used, depending on the study. After passing through the microfluidic homogenizer processor, the total volume was adjusted to 1 L with HBSS, the samples were mixed, and aliquots were taken for counting. Typically, three replicate experiments were performed using each condition.
[0054] Determine the percentage of sporocysts recovered and the perc...
Embodiment 2
[0081] The release of sporocysts using cell disruption equipment requires optimization of the release conditions to ensure the viability of the released sporocysts. Activity can be assessed by providing the birds with a dose of sporocysts and by counting the associated oocysts produced as a result of the infection. Efficient release of sporocysts from oocysts in an inviable state is possible under a variety of conditions varying in cell disruption device chamber geometry and pressure. That is, the infection in birds resulting from the application of the sporocyst can be reduced compared to the infection achieved using the sporocyst released by traditional methods such as glass beads. Chamber geometry and pressure conditions must be carefully evaluated to ensure production of viable induments.
[0082]The sporocysts were released from the Eimeria oocysts at 2000 psi or 5000 psi using glass beads or a Microfluidizer processor with a 100Z chamber configuration. The sporocysts r...
Embodiment 3
[0088] observed that smaller Eimeria species, including Eimeria spp (approx. (approximately 20 microns in length) are less susceptible to shear. Microscopic yields of sporocysts from oocysts can be improved by higher levels of shear produced by increased pressure in the microfluidizer system for any species, but efficient release is especially needed for smaller species; however, increasing Stress can also reduce activity. Lowering pressure to increase activity inherently sacrifices yield. Pretreatments have been developed to condition the oocyst wall while providing improved microscopy yield and improved viability.
[0089] A study was conducted to understand the effect of pretreatment of oocysts with a combination of bile salts (taurodeoxycholic acid (TDCA)), anaerobic environment (bubbling carbon dioxide) and warm temperature (at 37°C for 1 hour). The aim of the study was to determine the in vitro recovery (by microscopy) and in vivo activity (by produced oocysts) of the...
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