Experimental materials
Experimental oocysts Foshan Zhengdian Biotechnology Co., Ltd. donated the oocysts of Isospora suis useed in this study, while Yannong Farm, Chaolong Farm, and Jinsha Farm provided clinical samples of piglet feces.
Reagents Shandong Baiqian Chemical Co., Ltd. offered pure analytical urea; Ketuo Experimental Consumables Sales Center provided pure analytical NaCl; Guangzhou Zhichun Chemical Co., Ltd. supplied soap powder; Guangdong Fengqi Instruments Co., Ltd. provided silicate defoaming agent.
Equipment Micropipettes (10 µL and 200 µL), inculation ring with a bent end(1 cm diameter), water bath, centrifuge, microscope, counting plate, beaker, fecal cup, funnel, filter, timer, density meter, stirrer, plastic dropper, glass slide, and cover glass.
Solution preparation To create a saturated salt solution, 380 g of salt were mixed with 1000 mL of water and stirred until the salt was dissolved. However, the urea flotation solution was made by dissolving 200 g of analytically pure urea in 200 mL of water, heating it to dissolve it, and cooling it to room temperature.
Procedure
Flotation solutions (urea, soap powder, and silicate defoamer) Twelve grams of fatty and dense piglet feces were mixed uniformly and divided into four equal parts in a fecal examination cup. The first flotation solution received 30 mL of saturated salt solution, the second flotation solution received 50% urea solution, the third flotation solution received 50% urea solution and a few soap powder, and the fourth flotation solution included 50% urea and a few soap powder, one minute later, a defoamer was added. Following 10 min, the flotation solutions were compared by microscopic observation using an inoculation loop with a bent end (1 cm ring diameter).
Optimal amount of soap powder Bubbles formed during the stirring process because the fatty acid salt, the major component of soap powder, was a surface-active substance. As a consequence, it was required to investigate the amount of soap powder. The steps were: (1) Eighteen grams of fat and sticky piglet feces were well mixed, then split into six 100 mL fecal inspection cups, numbered 1–6, and 30 mL of urea solution were thoroughly mixed until the feces were completely dissolved, then filtered. (2) The quantity of soap powder was fixed at 0 g, 0.1 g, 0.3 g, 0.5 g, and 0.7 g, and then put into the beakers of groups 2–6, respectively. The saturated salt solution group served as a control, and the mixture was well agitated. (3) Using the liquid taking ring (1 cm diameter inculation ring with a bent end) a ring of the upper layer liquid was taken into the glass slide for microscopic examination.
Optimal flotation time Because the urea solution (50%) has a different specific gravity (approximately 1.14) than the saturated salt solution (1.20), the flotation time must be recalculated. The procedure was carried out:(1) The flotation time was set to 0 min, 5 min, 10 min, 15 min, 20 min, and 25 min. ༈2༉ Each of the six 100 mL beakers was filled with 30 mL of urea solution. ༈3༉ Each beaker received 10,000 Isospora oocysts, which were mixed well and timed simultaneously. ༈4༉ A ring of suspension was taken from the center and area of the top layer of the cup every 5 min and was placed on the glass slide. A cover glass was placed, and Isospora oocysts were calculated.
Laboratory examination (1)Thirty grams of intestinal content from piglets were well mixed, and six fecal examination cups containing three grams of fecal samples were obtained and separated into two groups (traditional and modified saturated salt solution groups). Three parallel controls (numbers 1–3) were used in each group. ༈2༉ Each cup in the traditional saturated salt solution group received thirty millimeters of saturated salt solution, while the modified saturated salt solution group received thirty milliliters of urea solution and 0.1 g of soap powder; Ten thousand oocysts were added to each cup, mixed, and filtered. After 1 min, a drop of the defoaming agent was added to each cup of the modified saturated salt solution group, gently swirling the liquid level, and set aside for 15 min. Three rings were removed from the top fluid in each group for microscopic analysis.
Field examination Twenty samples of piglet manure were collected from three pig farms: Yannong (This site is located at 113º30'34.44"E, 23º21'03.41"N), Chaolong (This site is located at 113º34'04.50"E, 23º20'34.59"N), and Jinsha (This site is located at 112º57'13.80"E, 23º04'36.56"N) regions, properly mixed. We prepared 120 fecal examination cups and divided them into two groups. The first group was designated as the saturated salt solution group and the second as the modified group. Each sample was separated into two equal portions and examined. The first group (traditional method) received 30 mL of saturated salt solution, while the second group (modified approach) received 30 mL of urea + soap powder solution, which was properly mixed. In the second group, a drop of defoaming agent was added to each cup after 1 minute, and it was allowed to stand for 15 min.