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Persistence of Two Trichomonas gallinae Isolates in Chlorinated and DistilledWater With or Without Organic MaterialAuthor(s): Richard W. Gerhold , Lauren P. Maestas , and Philip M. HarnageSource: Avian Diseases, 57(3):681-683. 2013.Published By: American Association of Avian PathologistsDOI: http://dx.doi.org/10.1637/10518-022213-ResNote.1URL: http://www.bioone.org/doi/full/10.1637/10518-022213-ResNote.1

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Research Note—

Persistence of Two Trichomonas gallinae Isolates in Chlorinated and Distilled WaterWith or Without Organic Material

Richard W. Gerhold,ABC Lauren P. Maestas,A and Philip M. HarnageA

AThe Center for Wildlife Health, Department of Forestry, Wildlife, and Fisheries, University of Tennessee, Knoxville, TN 37996BDepartment of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996

Received 26 February 2013; Accepted 7 May 2013; Published ahead of print 8 May 2013

SUMMARY. Trichomonas gallinae is a protozoan parasite commonly found in columbids, passerines, and raptors. In passerinesand columbids, trichomonosis causes significant morbidity and mortality associated with contaminated bird feeders and waters.However, there has been little work on the persistence of T. gallinae in water to determine if artificial waters are a likely source ofinfection for naıve birds. To examine drinking water as a source of T. gallinae transmission, we inoculated 1 3 106 trichomonadsinto containers with 500 ml of either distilled or chlorinated water. In addition, we inoculated the same number of trichomonads indistilled or chlorinated water contaminated with 15 g organic matter. Aliquots of 0.5 ml were collected from each container at 0,0.5, 1, 5, 10, or 20 min; inoculated into a Trichomonas culture packet; and incubated at 37 C for 6 days. Survival was best in thepresence of organic matter, with either distilled or chlorinated water. Uncontaminated chlorinated water did not allow survival atany sampling period.

RESUMEN. Nota de Investigacion—Persistencia de dos aislamientos de Trichomonas gallinae en agua clorada y destilada con osin materia organica.

Trichomonas gallinae es un parasito protozoario que se encuentra comunmente en los columbidos, paseriformes y aves rapaces.En paseriformes y en columbidos, la trichomonosis causa una morbilidad importante y mortalidad asociada con comederos ybebederos contaminados. Sin embargo, ha habido muy poco trabajo sobre la persistencia de T. gallinae en agua para determinar silas aguas artificiales son una fuente probable de infeccion para las aves susceptibles. Para examinar el agua potable como una fuentede transmision de T. gallinae, se inocularon 13106 trichomonas en recipientes con 500 ml de agua destilada o tratada con cloro.Ademas, se inocularon el mismo numero de trichomonas en agua destilada o agua clorada contaminadas con 15 g de materiaorganica. Las alıcuotas de 0.5 ml se recogieron de cada recipiente a los 0, 0.5, 1, 5, 10, o 20 minutos se inocularon en un paquete decultivo de trichomonas y se incubaron a 37C durante seis dıas. La supervivencia fue mejor en presencia de materia organica, ya seacon agua destilada o tratada con cloro. El agua clorada no contaminada no permitio la supervivencia en ningun periodo demuestreo.

Key words: persistence, Trichomonas gallinae, trichomonosis

Trichomonas gallinae is a protozoan parasite commonly found incolumbids, passerines, and raptors (5,16). This parasite infects the oralcavity, proventriculus, and liver of affected birds and can havesignificant population impacts (8,13,15). In Europe, trichomonosishas been associated with significant and rapid decline in garden birdpopulations (15). Additionally, trichomonosis outbreaks have beendocumented in Canada, Fennoscandia, and the United States (1,6,14).All these outbreaks were clustered around contaminated bird feedersand waters. Little is known about the persistence of T. gallinae andother flagellated protozoa in water, feed, or other materials as apotential source of infection of young birds (3,11,12). We examinedthe persistence of T. gallinae in chlorinated and distilled water with orwithout contaminating organic material to simulate poor sanitationconditions often found in backyard water troughs or pans.

MATERIALS AND METHODS

Isolates. Two T. gallinae isolates were used. The isolates werepreviously collected from clinically affected birds, including a broad-

winged hawk (Buteo platypterus) from Florida and a Cooper’s hawk(Accipiter cooperii) from Arizona (9). The concentration of culturedtrichomonads (approximately 1 3 106 axenic trichomonads from eachisolate) was determined using a hemocytometer.

Water treatments. Five hundred milliliters of either chlorinated ordistilled water was placed in a 5.68-L plastic container and kept at aconstant temperature of 30 C. For each T. gallinae isolate, four treatmentswith two replications were used for a total of eight containers. The fourtreatments included clean distilled water, clean chlorinated water, distilledwater with 15 g of nonautoclaved organic matter (detritus, leaves, andsoil), and chlorinated water with 15 g of nonautoclaved organic matter.The chlorinated water was obtained from a municipal drinking watersource in Knoxville, TN. The chlorine concentration used at themunicipal plant varies from 2.2 to 2.3 ppm. The distilled water waspurchased commercially. Organic material was used to simulate drinkingwater with poor sanitation. The water and organic material were placedinto the containers approximately 18 hr prior to inoculation with thetrichomonads and stirred using a clean plastic stir rod to homogenize themixture. Following the addition of the trichomonads, the contents of thecontainers were stirred in the same manner as before and the containerscovered to minimize chlorine dissipation.

Isolate persistence. The persistence of both isolates for the varioustreatments was determined at six different time points, includingimmediately after inoculation (time 0), and at 0.5, 1, 5, 10, and 20 minpostinoculation. Samples consisted of 0.5 ml from each container. Theremoved sample was immediately placed into a TF InPouch (BioMed

CCorresponding author. Department of Biomedical and DiagnosticSciences, College of Veterinary Medicine, University of Tennessee, 2407River Dr., Knoxville, TN 37996. E-mail: rgerhold@utk.edu

AVIAN DISEASES 57:681–683, 2013

681

Diagnostics, White City, OR) and incubated at 37 C. Pouches wereexamined for trichomonad growth daily for 6 consecutive days,beginning 18 hr after sample collection, using light microscopy at1003magnification. The TF InPouch packets have been shown to be assensitive as Diamond’s media and are more practical for field work (4).Positive growth was indicated if at least one motile trichomonad wasobserved during any of the observation periods during the 6-day TFInPouch examination.

Verification of results. For some samples, PCR was used to verify theisolate in the container to ensure contamination of isolates did notoccur. PCR and sequencing were done as previously described (9). Thesequences were aligned and compared to the previously publishedsequences of isolates 1 and 2 used in this study (9).

RESULTS

No trichomonad growth was detected in the noncontaminatedchlorinated water treatment for either isolate in any sample. In cleandistilled water, isolate 1 showed no persistence, whereas isolate 2survived up to 5 min in one replication (Table 1). Results forchlorinated water with organic matter were variable, with positivetrichomonad growth detected at 0 and 1 min in replicate 1 of isolate 1(Table 1). Replicate 2 of the same isolate was positive at 20 min,whereas samples collected between 0 to 10 min remained negative.For isolate 2, positives were detected in replicate 1 at time point 0, andpositives were detected in replicate 2 at time points 0, 1, and 5 min. Inthe distilled water with organic material, the majority of samples weretrichomonad-positive for isolate 1, but all samples for bothreplications of isolate 2 were positive (Table 1). The rRNA-ITSsequence of the trichomonads recovered from the 20-min time pointfor isolate 1 replicate 2 (I1 R2 TP 20) was compared to the previouslypublished sequences of isolate 1 and isolate 2 (9); the comparisonshowed a 99% identity to isolate 1 and a 92% identity to isolate 2.

DISCUSSION

Although the transmission of T. gallinae is facilitated incolumbids via behaviors including feeding nestlings, sloughing offthe lining of the crop, and a mating behavior called billing (16), recentoutbreaks of trichomonosis have potentially been associated withcontaminated bird baths and feeders (6,14,15). Affected birds arefrequently found near feeders and are emaciated and weak. Our resultsdemonstrate that T. gallinae can persist in water for variable periods,depending on presence of organic material. In particular, the resultsdisclosed that the two examined isolates of T. gallinaedid not survive in clean chlorinated water; however, the additionof organic matter increased the persistence of trichomonads, as

observed with both tested isolates (Table 1). Similarly, increasedpersistence of T. gallinae was observed in distilled water supplementedwith organic material as compared to clean distilled water. Overall, thepersistence of T. gallinae was greater in distilled water compared tochlorinated water, suggesting that bird water sources filled with wellwater or rain water may have greater transmission risk than watersources filled with chlorinated water from municipal water sources.

The time points for sampling were arbitrarily decided by theresearchers. Previous research of trichomonad persistence in water wasnot available; thus the authors elected to use time points that mayreflect avian presence at feeders and waters. Our findings allow for afoundation for future studies examining trichomonad persistence.

Interestingly, the results found that treatment 2, which includedchlorinated water supplemented with organic matter, disclosed apositive sample at 20 min for I1 R2 TP 20, whereas all other samplesfrom this replicate were negative. Although unlikely, we wereconcerned if the positive result for I1 R2 TP20 in the clean distilledtreatment was potentially due to contamination of a recentinoculation of isolate 2 into an adjacent container. To determineif the positive result was due to contamination, PCR and sequenceanalysis of the I1 R2 TP 20 sample was conducted. Sequenceanalysis confirmed the presence of T. gallinae isolate 1 in the TFInPouch. Although there were only a few trichomonads in theInPouch, previous research has demonstrated that experimentalinfection of birds with just one live trichomonad can result inmortality (17). The finding of the positive sample at 20 min and notbefore may be because only a few trichomonads were persisting inthe chlorinated water with organic material, and only 0.1% of thetotal volume (0.5 ml sampled from 500 ml container) was beingsampled at one time.

Future research should examine the effects of variable levels oforganic material on persistence of T. gallinae isolates inoculated intowater or feed. Other variables that could influence survival of T.gallinae in water or feed include temperature, pH, and osmolality, allof which need to be investigated further.

Species within the order Trichomonadida do not have a true cyststage and are thought to survive for only a short time when expelledfrom the host (18). Understanding persistence and means oftransmission of T. gallinae in wild birds is important for diseasemanagement and prevention. Artificial bird baths and feeders causean aggregation of wild birds in a single location, increasing thepotential of disease transmission (15). Our results suggest thatpractices including routine cleaning of bird baths and limitingorganic material concentrations in bird baths would be beneficial inreducing trichomonosis outbreaks.

The exact mechanism associated with increased persistence of T.gallinae in organic material–supplemented water is unknown,

Table 1. Trichomonas gallinae persistence over time in various water treatments. Detectable growth, was determined by light microscopydetection of at least one motile trichomonad.

Distilled waterDistilled water and

organic material Chlorinated waterChlorinated water and organic

material

Samplingtimes (min)

Isolate 1 Isolate 2 Isolate 1 Isolate 2 Isolate 1 Isolate 2 Isolate 1 Isolate 2

Rep 1A Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2

0 2 2 + + + + + + 2 2 2 2 + 2 + +0.5 2 2 + 2 + 2 + + 2 2 2 2 2 2 2 21 2 2 + 2 + + + + 2 2 2 2 + 2 2 +5 2 2 + 2 2 + + + 2 2 2 2 2 2 2 +10 2 2 2 2 + + + + 2 2 2 2 2 2 2 220 2 2 2 2 + 2 + + 2 2 2 2 2 + 2 2

ARep 1 5 replication 1, Rep 2 5 replication 2, + 5 detectable growth; 2 5 no detectable growth.

682 R. W. Gerhold et al.

although it is well known that the effectiveness of chlorine and someother disinfectants is limited by such contamination. Severalprotozoa in the family Trichomonadidae are known to phagocytizebacteria for growth and reproduction (2,10). The presence of theorganic material may provide bacteria and other nutrients needed forthe trichomonads to persist for longer time periods than they wouldin clean water. Recent studies have focused on the variablesassociated with protozoal persistence in various media or material(7,12). However, more research is needed to understand themechanisms of protozoal persistence.

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ACKNOWLEDGMENTS

We thank the University of Tennessee Advanced Wildlife Health classmembers (WFS 301) for assistance in conducting the experiment.Funding for this project was through the University of Tennessee’sDepartment of Forestry, Wildlife, and Fisheries.

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