Single-dose Metronidazole Clears Opalina sp. from Juvenile Bufo woodhousii
Author(s): Devin R. Nickol and Danielle M. TuftsSource: Journal of Parasitology, 99(3):573-575. 2013.
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J. Parasitol., 99(3), 2013, pp. 573–575 Ó American Society of Parasitologists 2013 Single-dose Metronidazole Clears Opalina sp. from Juvenile Bufo woodhousii Devin R. Nickol and Danielle M. Tufts*, University of Nebraska Medical Center, 985185 Nebraska Medical Center, Omaha, Nebraska 68198-5185;*University of Nebraska–Lincoln, 323 Manter Hall, Lincoln, Nebraska 68599. Correspondence should be sent to: dnickol@unmc.edu ABSTRACT: Protozoans of the family Opalinidae are intestinal commensals may interfere with normal host behavior, growth, and development in amphibians. To test the hypothesis that these organisms are susceptible (Hegner, 1923; Nathan and James, 1972). Little is known about the role to the antiprotozoal antibiotic metronidazole, we randomly assigned 60 opalinids play within fish hosts, but Foissner and colleagues (1979) juvenile Woodhouse's toads (Bufo woodhousii) to receive a single oral dose showed increased mortality of infected Symphysodon aequifasciata of metronidazole or water. In pilot trials, the prevalence of opalinids in individuals. Additionally, under certain experimental conditions, some untreated members of this population was over 70%. One-third of the protozoans may compete with their hosts for various resources (Nathan study population was dissected at each of 3 time points: 18 hr, 1 wk, and 2 and James, 1972). For example, Hegner (1923) found that tadpoles heavily wk post-treatment. An examiner blinded to the toad's treatment history infected with a euglenoid flagellate did not reach the same body size as determined the presence or absence of opalinids using a dissecting non-infected tadpoles, and heavily infected tadpoles never completed microscope. Opalinids were found in 3/10 toads in the treatment group metamorphosis, implying that there may be some type of interspecific and 9/10 in the control group after 18 hr (P , 0.02), in none of the competition for food or resources between the anuran host and the treatment group and 8/10 in the control group after 1 wk (P , 0.001), and protozoan, especially in malnourished individuals. In settings where these in none of the treatment group and 10/10 in the control group after 2 wk types of interactions may confound experiments performed with infected (P , 0.0001). These results suggest that a single-dose of metronidazole individuals, a parasite-free population would be beneficial (Kessel, 1930; quickly and reliably clears opalinids from juvenile Woodhouse's toads Cairns, 1953).
with no evidence of short-term recurrence. The treatment was well Metronidazole (Flagylt) is a commercially available antibiotic used to tolerated, with no apparent morbidity and no mortality in either group.
eliminate anaerobic bacteria and protozoans from human and animal Future exploration of opalinid-related host fitness consequences may be hosts. We hypothesized that administration of metronidazole to an facilitated by this simple method of developing a protozoan-free host amphibian host would result in the clearance of its opalinid population.
The objectives of this study were to (1) determine if metronidazole couldclear all opalinid protozoans from B. woodhousii, (2) determine theapproximate amount of time needed for clearance, and (3) confirm the Organisms of the genus Opalina are commensal protozoans of reptiles, short-term durability of treatment. Although metronidazole has been used amphibians, fish (marine and fresh water), mollusks, and termites; to treat a variety of infections in livestock, companion animals, and however, they are most commonly found in anurans and have a wide captive reptiles and amphibians, to our knowledge this is the first geographic distribution (Metcalf, 1929; Sandon, 1976). Opalinids are evaluation of its effectiveness against opalinids.
multinucleated, mouth-less, and covered with rows of flagella (Wenrich, In July 2010 we collected 60 juvenile B. woodhousii from the North 1935; Mignot, 1994; Mitchell, 2007). Opalinids were first discovered by Platte River near Paxton, Nebraska (418100520 0N, 1018210560 0W). Toads Leeuwenhoek in 1683; approximately 200 species of opalines comprising were immediately transported to the laboratory in 5-gal plastic buckets 5 genera are currently recognized (Delvinquier and Patterson, 1993): and randomized by coin flip into treatment and control groups of 30 Cepedea (Metcalf, 1920), Opalina (Purkinje and Valentin, 1835), individuals each. Pilot studies supported the effectiveness of a single, 10- Protoopalina (Metcalf, 1918), Protozelleriella (Delvinquier et al., 1991), mg dose of metronidazole. We prepared a suspension by finely crushing and Zelleriella (Metcalf, 1920). Most hosts harbor 2–5 different species two 500-mg tablets of metronidazole (USP) and suspended the powder in of these protozoans; however, specific combinations are more prevalent 40 ml of bottled spring water. Each toad in the treatment group received (Nathan and James, 1972; Wilbert and Schmeier, 1982; Schorr et al., 400 ll of the suspension orally by micropipette for an approximate dose of 1990). The opaline protozoans generally occur in the large intestine and 10 mg of metronidazole. The flask containing the suspension was stirred cloaca of their hosts (Kudo, 1922; Wenrich, 1935; Schorr et al., 1990).
prior to each dosing because of the poor water solubility of the antibiotic.
Amphibians are known to host a wide variety of other micro- and We attempted to inject the suspension orally by micropipette into each macroparasites in addition to opalinids and have been model systems for toad's posterior oropharynx to minimize the possibility of the suspension field and laboratory research in the biomedical, teaching, animal being regurgitated, but some variability in dosing almost certainly behavior, life history, toxicology, physiology, evolutionary, and repro- occurred. Toads in the control group received an equivalent oral volume ductive fields (Goater and Goater, 2001; Mitchell, 2007). In amphibians of spring water by micropipette.
infection occurs during the tadpole stage of development. In their Throughout the experiment, all toads were housed in 10-gal aquaria aquatic environment tadpoles ingest opaline cysts released into the water lined with coconut fiber substrate (Zoo Med Eco Earth, San Luis Obispo, with the feces from infected adults or other tadpoles (Brumpt, 1915; California) and covered by a mesh screen. Treatment and control groups Metcalf, 1928, 1940; Mofty and Smyth, 1960; Delvinquier and Freeland, were housed separately. Each aquarium housed 10 toads, included rock 1988). The Woodhouse's toad (Bufo woodhousii) from North America is and plant cover, and a clean, standard-size glass petri dish (100 mm 3 15 known to harbor many different types of parasites (Goldberg et al., mm) filled with bottled spring water each day. The aquaria were warmed 1996; Bolek and Janovy, 2007). Individuals from our study area were during the day by incandescent light from a desk lamp. Ambient room most commonly infected with protozoans (Opalina and Nyctotherus temperatures ranged from 22 to 24 C. The toads were fed daily with a cordiformis) and cestodes (Distoichometra bufonis) (D. Nickol and D.
variety of commercially raised crickets (Fluker Farms, Port Allen, Tufts, pers. obs.; Hardin and Janovy, 1988).
Louisiana; Ghann's, Augusta, Georgia; Petco, San Diego, California).
Although opalinids are usually described as commensal protozoans that One-third of the study population was dissected at each of 3 time do not harm the host, some studies suggest that high infection intensity points: 18 hr, 1 wk, and 2 wk post-treatment. At each time point, 10treatment and 10 control toads were selected in random order by coin flip.
DOI: 10.1645/12-76.1 After selection each toad was passed to the primary author, who remained THE JOURNAL OF PARASITOLOGY, VOL. 99, NO. 3, JUNE 2013 unaware of its treatment status. After the toad's length was measured it TABLE I. Prevalence of opalinids in Bufo woodhousii, treatment efficacy, was pithed, the abdomen was opened with scissors, and the gastrointes- and number needed to treat (NNT) at each study time point.
tinal tract removed en bloc from just proximal to the stomach to justproximal to the anus. This section was subjected to blunt dissection with forceps under the dissecting microscope. A small amount of water wasadded to the dissection dish, and the presence or absence of opalinids was determined by microscopic examination. Incidental notice was also made of the presence or absence of Nyctotherus cordiformis and adults or proglottids of the cestode Distoichometra bufonis (Dickey, 1921). The findings were relayed verbally to the investigator responsible for selectingthe toads who recorded the body length, dissection results, and treatment * Two of these 3 toads had a small number (,10) of immobile, dead- status for each toad.
We used Fisher's exact test (2-tailed) to calculate P-values for the difference in opalinid prevalence (proportion of the population infected)between treatment and control groups at each time point. We used a The treatment and control solutions were well tolerated by our study Student's t-test (unpaired, 2-tailed) to calculate the P-value for the population; however, some mortality had been observed during earlier difference in toad body length between the groups. Treatment efficacy was pilot trials. The observation that deaths occurred no sooner than 5–7 days after treatment, and were evenly distributed between the treatment andcontrol groups, led us to suspect that some factor other than antibiotic ðprevalence in controlsprevalence in treatedÞ : toxicity was most likely responsible (i.e., water quality, temperature, prevalence in controls enclosure environment, etc.). This conclusion was reinforced by the lack of The number needed to treat (NNT) to clear one toad of opalinids was mortality once modifications were made to the study animal habitat, including the change from plastic terraria to larger glass aquaria for theformal trial. Because no changes were made from pilot studies to the dose or delivery of the treatment and control solutions, we concluded that earlier ðprevalence in controlsprevalence in treatedÞ observed mortality was unlikely to be a result of the study medication.
We were initially concerned that the poor water solubility of the All toads in both study groups appeared healthy throughout the study antibiotic and the challenges of oral administration would result in a lack period, and there were no deaths prior to dissection. Average toad length of dosing precision that might compromise treatment efficacy. Our results at the time of dissection was 3.16 cm in the treatment group (2.4–4.2 cm) suggest that, while some variability in dosing almost certainly occurred, and 3.15 cm in the control group (2.4–4.0 cm) (df ¼ 58, P ¼ 0.95).
the overall effectiveness of the antibiotic was sufficient to overcome this Opalinids were present in 3/10 toads in the treatment group and 9/10 in variation and led to reliable clearance of the target organism.
the control group after 18 hr (P , 0.02), in none of the treatment group The effectiveness of our intervention reached 100% by 1 wk and and 8/10 in the control group after 1 wk (P , 0.001), and in none of the remained 100% at 2 wk. The effectiveness at 18 hr would have been higher treatment group and 10/10 in the control group after 2 wk (P , 0.0001).
(89%) had we included the 2 treated toads found to have only a few, The NNT was 1.7 at 18 hr, 1.3 at 1 wk, and 1.0 at 2 wk (Table I). Unless immobile opalinids into the ‘‘no opalinids'' group. The high opaline otherwise noted, when opalinids were present they were motile and too prevalence in our study population, combined with the high efficacy of our numerous to count.
treatment, resulted in a low NNT of ,2 toads at all time points.
Significant differences in opalinid prevalence between the treatment and Multiplying this low NNT by the low cost of metronidazole per treated control groups were present at all time points measured. There was no toad (approximately US$0.02) suggests that creation of sizable opalinid- significant difference in average toad size between the treatment and free research populations should be cost-effective.
control groups at the time of dissection. All toads in the trial survived to The results of our study suggest several areas for further research. It the time of dissection and appeared healthy. Nyctotherus cordiformis and would be interesting to study this method of protozoan clearance in other D. bufonis were incidentally noted in both treatment and control animals.
host species that harbor opalinids or other protozoan species. Within B.
Our findings indicate that treating juvenile B. woodhousii with a single woodhousii, the effect of metronidazole on Nyctotherus cordiformis and D.
oral dose of metronidazole results in rapid, reliable, and well-tolerated bufonis remains unclear, as our anecdotal observations did not suggest clearance of opalinids from the gastrointestinal tract. Anecdotal obvious reductions in prevalence. If these organisms are not susceptible to observations at the time of dissection suggested that the prevalence and metronidazole but are susceptible to other agents, selective manipulation intensity of Nyctotherus cordiformis and D. bufonis were not obviously to the various protozoan and cestode populations within the host might be decreased in the treated toads as compared to the control population.
possible. The relative impact of each parasite on overall host fitness might Future research could assess the effectiveness of agents other than then be clarified.
metronidazole in the clearance of these organisms.
The authors thank Dr. Matthew Bolek for his assistance and Limitations of our study include the relatively short 2-wk follow-up encouragement with this project.
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