Rotifers Ingest Giardia Cysts

Seven species of rotifers representing 6 genera, Epiphanes, Plationus, Asplanchna, Philodina species A, Philodina species B, Pla- tyias, and Brachionus, were exposed to Giardia cysts isolated from the feces of experimentally infected holstein calves. Giardia cysts were prestained with a fluorescein isothiocyanate-conjugated monoclonal an- tibody and mixed with viable rotifers on 3-well Teflon-coated micro- scope slides. Organisms were observed with phase-contrast, differential interference contrast, and fluorescence microscopy. Five rotifer species, Epiphanes brachionus, Plationus patulus, Philodina (both A and B), and Platyias quadricornis, ingested varying numbers of cysts, which were retained within the rotifers' bodies throughout the observation period. Rotifer ingestion of Giardia cysts may represent a means of reducing water contamination. Rotifers are a large group of free-living microscopic organisms con- sisting of more than 1,850 species that may be free-swimming, sessile, or benthic (Segers, 2002). They are found in diverse locations such as freshwater lakes and ponds, brackish and salt water, and virtually any other place where water accumulates, such as puddles, damp soil, and even birdbaths (Nogrady et al., 1993). Rotifers thrive in highly eutro- phic environments such as wastewater and domestic sewage, which are not conducive to the survival of their major predators, copepods. Rang- ing in length from 50-2,000 mm, rotifers possess a ciliated corona at their anterior end that is used for locomotion and food gathering (Wal- lace and Snell, 2001). Although the structure of the corona varies widely among species, the beating cilia sweep water and suspended particles toward a central oral cavity where a second set of cilia reject particles unsuitable for ingestion (Gilbert and Starkweather, 1977, 1978; Wallace and Snell, 2001). Particles that enter the oral cavity pass into the mastax, an organ with chitinous plates that chops food and pushes it into the esophagus. From the esophagus, particles move into the stomach and then into an intestinal tube (Nogrady et al., 1993). One rotifer species was reported to have 5 digestive glands (Schramm, 1978). Digestive enzymes, such as glycosidases and endoglucanases, were identified in homogenates from another species (Kuhle and Kleinow, 1990; Chun et al., 1997). Giardia includes a number of species capable of causing infection in a variety of vertebrate hosts, including humans. Human giardiasis is the most commonly diagnosed protozoan intestinal disease worldwide. In- fection is often accompanied by intestinal distress, diarrhea, malabsorp- tion, and impaired growth (Farthing, 1994). The infective stage, an en- vironmentally resistant cyst, can be found in surface waters throughout the world. Giardia spp. cysts are similar in size to the food particles ingested by rotifers, and because rotifers were reported to ingest oocysts of the protozoan parasite Cryptosporidium parvum(Fayer et al., 2000), the present study was undertaken to determine if rotifers would also ingest Giardia spp. cysts. If rotifers do indeed ingest cysts, this could provide clues to the environmental fate of cysts and suggest potential biological control strategies for reducing Giardia spp. contamination of surface water. Giardia spp. cysts were isolated from the feces of experimentally infected neonatal holstein calves and used within 2 wk. Briefly, feces were passed through a series of screens of increasingly finer mesh and further purified by centrifugation over a discontinuous CsCl gradient (Kilani and Sekla, 1987). This procedure is routinely used in our lab- oratory to isolate cysts from feces and results in a highly purified, viable population of cysts that are then used to infect subsequent calves to maintain continuous propagation of this Giardia isolate. Purified cysts were washed extensively to remove residual CsCl and were then labeled with a fluorescein isothiocyanate (FITC)-conjugated monoclonal anti- body (mAb) (MerIFluor y, Meridian Biosciences, Inc., Cincinnati, Ohio) by suspending them in 200 ml of the detection reagent. After 30 min of incubation at room temperature, cysts were washed thoroughly to remove excess antibody and then resuspended in springwater at a con- centration of 4,000 cysts/ml. Different rotifer species were either col- lected from nature or purchased (Carolina Biological Supply Co., Bur- lington, North Carolina) (Table I). The rotifers were maintained in 100- ml polyethlyene screw-capped jars and were fed approximately every 2 days with a mixture of Chlamydomonas reinhardtii(UTEX-90) and Ankistrodesmus falcatus(UTEX-749). No effort was made to starve the rotifers before testing. Cultures were monitored daily using an Olympus CK2 inverted microscope equipped with both 10 3 and 203 phase- contrast objectives. To obtain rotifers for testing, 5-15 ml was aspirated with a pipet from heavily populated areas of the cultures, or in the case of sparsely populated cultures, individual organisms were located with the inverted microscope and removed using a pipet. Approximately 5- ml aliquots containing rotifers were placed into a 11-mm-diameter well of a heavy teflon y