Detection of yeasts in the pig microbiota
Nine pigs, from seven different farms, were submitted for necropsy to the Veterinary Diagnostic Laboratory (VDL) at the University of Illinois and sampled to detect yeasts. Swabs from the oral cavity, large colon and rectum were plated on Sabouraud agar (L−1: 40 g glucose, 10 g peptone and 20 g Bacto agar) with chloramphenicol (20 μg mL−1; Sab + Cml), and incubated for 24 h at 37°C. Pure cultures were derived for colonies with yeast-like cellular morphology. Each isolate was streaked onto CHROMagar Candida (www.chromagar.com) and tested for germ tube formation by inoculation into RPMI 1640 medium (Gibco catalog no. 11875-085). Yeasts were identified by PCR of genomic DNA using primers ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) and ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) that amplify the internal transcribed spacer region of the nuclear-encoded rRNA genes (White et al., 2011). DNA sequences were aligned against the non-redundant nucleotide database using blast. Candida albicans isolates were analysed using multilocus sequence typing (mlst) as described previously (Bougnoux et al., 2003; Wrobel et al., 2008). Oral and rectal swabs were also collected from normally healthy pigs on the University of Illinois Veterinary Research Farm (VRF). Thirty-four pigs were sampled from a herd of c. 200. Fourteen pigs were from the nursery unit (15–40 lbs), 10 were growers (40–100 lbs) and 10 were from the finishing unit (100–280 lbs). Swabs were plated on Sab + Cml and yeasts were identified as described above. Animal experiments were conducted with the approval of the University of Illinois Institutional Animal Care and Use Committee.
Farrowing and artificial rearing of piglets
VRF piglets are an outbred population that includes a mix of the Yorkshire, Landrace and Duroc breeds. Piglets were farrowed normally and housed with the sow in farrowing crates to ensure intake of colostrum. On day 4, piglets were moved to an artificial rearing environment. Piglets were handled minimally: they were not subjected to any of the normal processing of VRF piglets that typically includes ear notching, removal of needle teeth, tail docking, iron dextran injection (100 mg; Aspen Veterinary Resources), penicillin G injection (300 000 U; Butler Schein) and castration of males. Approximately equal numbers of male and female piglets were used in our studies.
Piglets were moved to 4 × 4-ft raised pens constructed from painted steel hog panels, lined with solid plastic sheeting to prevent animal-to-animal contact. Pens had plastic slotted flooring over a flush pan. All material was easily disinfected. Piglets had 24-h access to water via a nipple-triggered drinking cup. Rooms were ventilated to outdoor air by a variable-speed, 12-in exhaust fan. Room temperature was controlled by use of a room heater or an air conditioning unit. Heat lamps were lowered over each pen, when additional heat was required. Housing met or exceeded the standards and recommendations of the National Research Council (2011).
Piglets were fed Ralco-Birthright Acidified Baby Pig Milk Replacer (Ralco Nutrition, Inc.; five cups per gallon of water; 360 mL kg−1 body weight, up to 2000 mL daily). In the initial study, milk replacer was provided hourly using a timer and pump system. The pig's bowl, stock bottles and tubing were washed and bleached daily, and always returned to the same pig's feeding system to prevent cross-contamination between animals. Subsequent work showed that milk replacer can be provided in a plastic baby pig waterer (see Miller Little Giant item number 291-25). Half of the total daily volume of milk replacer was provided twice daily. Plastic waterers were washed and bleached daily, then returned to the same pen. Research workers wore clean coveralls and boots. Gloves were changed between handling animals and materials in different pens. A bleach-filled boot wash outside each room was used to control spread of C. albicans in the animal facility.
Inoculation of piglets with C. albicans and assessment of colonization
Piglets and their environment were confirmed C. albicans-free before each study. Oral swabs sampled liberally over all oral surfaces. For rectal swabs, the swab tip was inserted into the pig's rectum and withdrawn, often covered in fecal material. Environmental swabs were wet in the piglet's drinking cup, then rubbed thoroughly across all pen surfaces. Swabs were plated on Sab + Cml as described above.
After 1 day of acclimation, pigs were inoculated orally with 109 C. albicans cells. One of three strains was used: SC5314 (the most commonly studied isolate, for which the genome sequence is known; Jones et al., 2004), 216 (an oral isolate from a normally healthy human; Wrobel et al., 2008) or 3317 (collected from the oral cavity of a VDL necropsy pig evaluated for lameness; see above). Candida albicans strains were taken from −80 °C storage and streaked onto YPD plates (L−1: 10 g yeast extract, 20 g peptone, 20 g glucose and 20 g Bacto agar). Plates were incubated at 37 °C for 24 h then stored at 4 °C for ≤ 1 week. An isolated, representative colony was inoculated into 20 mL YPD and incubated for 16 h at 30 °C with shaking (200 r.p.m.). Yeasts were collected by centrifugation, washed twice in Dulbecco's phosphate-buffered saline without calcium or magnesium (DPBS) and counted using a hemocytometer. The yeast inoculum was resuspended in 1 mL DPBS. Piglets readily drank the inoculum or DPBS control from a needleless tuberculin syringe.
Oral, rectal and environmental swabs were collected 8 h following inoculation, and once every 24 h thereafter. Swabs were plated on Sab + Cml. Plates were incubated for 24 h at 37 °C and C. albicans colonies counted. A seven-category scale was used to report the plating results: 0 (no growth), 1 (1–25 colonies), 2 (26–99 colonies), 3 (100–499 colonies), 4 (500–1000 colonies), 5 (> 1000 colonies, with distinct colony growth still visible) and 6 (lawn of growth). Piglets were weighed daily to monitor growth.
Two independent experiments were conducted. In the first, C. albicans isolates were tested using one pig per strain. A DPBS-inoculated pig was maintained in the same room to control for unintentional C. albicans transfer. Pigs were monitored for 21 days, including 15 days on milk replacer and 6 days on solid feed (pelleted Diet 111 from the University of Illinois Department of Animal Sciences feed mill; per ton: 625.2 lbs corn, 500 lbs whey, 404.8 lbs soybean meal, 200 lbs lactose, 150 lbs appetein, 60 lbs fat, 22.8 lbs lime, 12.6 lbs dicalcium phosphate, 8 lbs zinc oxide, 7 lbs swine trace minerals, 4 lbs Vitamix ADEK, 2.8 lbs dl-methionine, 2 lbs iodized salt and 0.8 lbs lysine). In the second experiment, C. albicans isolates were tested using two pigs per strain. Pigs were housed individually, with complete experimental replicates in separate rooms, and monitored for 13 (1st group) or 14 days (2nd group) while fed milk replacer. An uninoculated control pig was placed in each room. Because duplicate animals were used for each treatment, results from the second experiment are featured here. However, results were reproducible across both experiments.
Pigs were anesthetized with an intramuscular injection of telazol/ketamine/xylazine (4.4 mg kg−1), then euthanized by intracardiac overdose of sodium pentobarbital. Oral and rectal swabs were plated on Sab + Cml. Tissue sections were collected from the esophagus, stomach, duodenum, proximal jejunum, mid jejunum, distal jejunum, ileum, cecum, spiral colon and large colon. Candida albicans associated with the GIT mucosa was evaluated by collecting scrapings from the lumen of each tissue using the edge of a sterile glass microscope slide. A portion of the mucosa was weighed and homogenized in DPBS. Serial 10-fold dilutions were plated on Sab + Cml and incubated for 24 h at 37 °C to count CFU. Another portion of the mucosa was baked overnight in a vacuum oven at 80 °C. The ratio of dry weight to wet weight was calculated, then multiplied by the mass of the original mucosal sample that was homogenized and plated. This final dry weight equivalent was used as a divisor to calculate CFU g−1 dry weight. Reported results were rounded to the nearest order of magnitude.
In addition to the piglets housed in the artificial rearing environment, a piglet of similar age was selected from the VRF farrowing unit and necropsied as a control. All samples from this animal were negative for C. albicans (data not shown).