Diversity and seasonal impact of Acanthamoeba species in a subtropical rivershed.

This study evaluated the presence of Acanthamoeba species in the Puzih River watershed, which features typical subtropical monsoon climate and is located just above the Tropic of Cancer in Taiwan. The relationship between the seasonal and geographical distributions of Acanthamoeba species in this rivershed was also investigated. Acanthamoeba species were detected in water samples using the amoebal enrichment culture method and confirmed by PCR. A total of 136 water samples were included in this study, 16 (11.7%) of which contained Acanthamoeba species. Samples with the highest percentage of Acanthamoeba (32.4%) were obtained during the summer season, mainly from upstream areas. The identified species in the four seasons included Acanthamoeba palestinensis (T2), Acanthamoeba sp. IS2/T4 (T4), Acanthamoeba lenticulata (T5), Acanthamoeba hatchetti (T11), Acanthamoeba healyi (T12), and Acanthamoeba jacobsi (T15). The most frequently identified Acanthamoeba genotype was T4 (68.7%). Acanthamoeba genotype T4 is responsible for Acanthamoeba keratitis and should be considered for associated human health risk potential in the rivershed.


Introduction
Acanthamoeba has been isolated from a variety of environmental sources [1]. It is classified into three distinct morphological groups (I-III) based largely on the cyst morphology of the species and encompassing more than 25 nominal species [2,3]. The developments in the subgenus classification and taxonomy of Acanthamoeba have been currently classified into 17 different genotypes (T1-T17) based on 18S rRNA gene sequencing by molecular techniques. Acanthamoeba causes serious human diseases, such as Acanthamoeba keratitis and granulomatous amoebic encephalitis [4]. Acanthamoeba genotype T4 is the most prevalent type causing disease in human [5], the main Acanthamoeba keratitis-related genotype worldwide.
The main objectives of this study were to evaluate the effects of seasonal variations on the diversity of Acanthamoeba species and the presence/absence of these microbes in a subtropical river throughout a cycle of one year. Water quality parameters were also measured, the results of which are discussed with respect to the risk of infection in a given season.
Physical water quality parameters were measured for each water sample on location at the time of sample collection. Water temperature and pH level were measured in situ using a thermometer and a portable pH meter (D-24E, Horiba Co., Kyoto, Japan). Turbidity was measured in situ using a ratio turbidimeter (Turb555, WTW, Germany). Additional water samples were taken from each sampling location in 300 mL sampling bags (Nasco Whirl-Pak, Salida, CA, USA) for microbiological water quality parameters. The samples were kept in coolers during transportation to the laboratory for subsequent analyses within 24 h. Heterotrophic bacteria were cultured on the M-(HPC) heterotrophic plate count agar base measured by the spread plate method (Methods 9215 C) [30]. In addition, total coliforms were measured by membrane filtration and a differential medium described in the standard method for the examination of water and wastewater (Methods 9222 B) [30]. The total coliform cultures were placed in m-Endo LES agar (Difco, USA) at 36 ± 1 ∘ C for 24 h before counting. The potential effect of water quality parameters on the presence/absence of Acanthamoeba species was evaluated using STATISTICA version 6.0 (StatSoft, Inc., Tulsa, OK, USA).

Sample Pretreatment.
For each collected water sample, 2 L was concentrated via membrane filtration method using GN-6 Metricel mixed cellulose ester (MCE) membranes (0.45 m pore diameter, Pall, Port Washington, NY, USA). After filtration, the membranes were scraped, and the collected material was washed with 100 mL eluted fluid consisting of phosphate-buffered saline (PBS; 7.5 mM Na 2 HPO 4 , 3.3 mM NaH 2 PO 4 , and 108 mM NaCl, pH 7.2), 1% Tween 80, and 1% sodium dodecyl sulfate. The resulting solution was then transferred into two 50 mL conical centrifuge tubes and centrifuged at 2,600 ×g for 30 min. Subsequently, the top 45 mL supernatant fluid was removed, and the remaining 5 mL pellet was preserved with PBS at 4 ∘ C for further culture procedure and PCR analyses.

Culture and DNA Extraction for Acanthamoeba Species.
The resulting pellet was transferred to a culture plate containing 1.5% nonnutrient agar (NNA) and a lawn of heat killed Escherichia coli; then, the plates were sealed and incubated at 30 ∘ C, relative humidity of 85% for 2 weeks. During incubation time, the Acanthamoeba candidates were transferred onto new NNA plates containing a lawn of heat killed E. coli 1-3 times to avoid fungal contamination. The transfer times were determined by considering the fungal growth situation. The Acanthamoeba candidates on the plate were transferred to 5 mL glass tubes with PYG medium consisting of 2% proteose peptone, 0.2% yeast extract, 0.1 M glucose, and 1% Gibco antibiotic-antimycotic (Cat. No. 15240-06, USA) and incubated at 32 ∘ C for 3-4 days. The samples that yielded Acanthamoeba species were harvested and then were confirmed by molecular taxonomic identification method.
DNA extraction was done with the concentrated pellet using MagNA Pure LC instruments (Roche, USA) with MagNA Pure LC DNA isolation kits III (Roche, USA) following automated mode specified by the kit manufacturer's instructions manual. The resulting solution was analyzed for the presence of Acanthamoeba species specific genes with PCR assays.

PCR Conditions, Gel Electrophoresis, and Gene Sequence
Analysis. The PCR assay genus-specific primers' sets JDP1 and JDP2 used in this study were designed for Acanthamoeba genotyping as previously described [31]. They were established as the following sequences: 5 -GGCCCAGATCGT-TTACCGTGAA-3 and 5 -TCTCACAAGCTGCTAGGG-GAGTCA-3 , to amplify a 450 bp fragment of the 18S rDNA stretch Acanthamoeba-specific amplimer ASA.S1 of Acanthamoeba genotypes.
PCR reaction solution was prepared with 5 L of the DNA templates together with the PCR mixture to create a total volume of 25 L. The PCR mixture had 0.5 L PCR buffer (25 Mm MgCl 2 ), 0.5 L dNTP Mix (10 Mm of each dNTP), 1 L each of the oligonucleotide primers, and 0.1 L VioTaqTM DNA Polymerase (VIOGENE, 5 U/ L), as well as DNase-free deionized water. Cycling conditions were as follows: 95 ∘ C for 5 min for the initial denaturation step, followed by 35 cycles of 15 s at 95 ∘ C for denaturation, 15 s at 62 ∘ C for annealing, 3 s at 72 ∘ C for extension, and a final extension at 72 ∘ C for 10 min.
Negative DNA controls (template DNA replaced with distilled water) and positive controls (Acanthamoeba lenticulata ATCC30841) and sample DNA were analyzed in triplicates during each PCR run. PCR products of Acanthamoeba species were detected with gel electrophoresis on a 2% agarose gel (Biobasic Inc., Markham, ON, Canada) performed with 5 L of the reaction solution. The DNA fragments were confirmed using ethidium bromide staining (0.5 g/mL, 10 min). A 100 bp DNA ladder was used as a DNA size marker. The sequence analysis was done using a Bio-Dye terminator cycle sequencing kit (Applied Biosystems, Carlsbad, CA, USA). Phylogenetic construction produced gene trees by using neighbor-joining distance trees with a generation of 1,000 bootstrapped replicates. The 18S rRNA gene sequences were assigned to the GenBank database in order to allow BLAST searching and alignment using the MEGA software program version 5.0 (Mega Software, Tempe, AZ, USA). The highest percentage similarity was taken to identify the species.

Occurrence and Seasonal Distribution of Acanthamoeba
Species in a Subtropical River. A total of 136 water samples were collected in this study. The detection results of Acanthamoeba species from river waters are shown in Table 1. Acanthamoeba species were detected in 16 of the 136 samples (11.7%). The detection results for Acanthamoeba species were further analyzed with respect to detection rates by four sampling seasons, and the results are presented in Table 1. Seasonal detection rates of Acanthamoeba species were the spring (2.9%), summer (32.4%), autumn (2.9%), and winter (8.8%). The greatest percentage of Acanthamoeba species was detected during the summer. The results support a previous report that Acanthamoeba species were most prevalent in late summer in the aquatic environment [32,33]. The geographic distribution and occurrence of Acanthamoeba species along Puzih River were further compared by sampling seasons, and the results are presented in Figure 1. Geographic distribution was determined by the detection of Acanthamoeba species throughout the year. During the summer, Acanthamoeba was found in the upstream (7/11, 63.6%), midstream (2/11, 18.1%), and downstream (2/12, 16.6%) areas. In contrast, only single detection was obtained in the midstream areas during spring and autumn, respectively. Detection rates in the winter were 18.1% (2/11) and 8% (1/12) in the midstream and downstream areas, respectively. In Taiwan, rainfall mainly occurs in summer, with occasional typhoon and frequent thunderstorm. In the Puzih River, the total amount of rainfall in summer makes up roughly 90% of the annual rainfall amount for the study region. As shown in Figure 1, sampling locations with Acanthamoeba species detection principally occurred in the upstream areas. It is possible that water contaminants (such as livestock wastewater and sewage) may be carried into the river after rainfall [34,35] or riverbed sediment underwent seasonal rainfall in summer [32,33] that these sediments supported microbial growth [36]. In addition, weather events were found to play a major role in the presence/absence of Acanthamoeba species in the rivershed, with such changes probably due to resuspension of Acanthamoeba species from riverside or riverbed sediment by rainfall or wind action and input from the rivershed via runoff [32].
Quarterly monitoring revealed that the diversity of Acanthamoeba species varied according to season and sampling location and evidence further indicated that seasonal effects influence the diversity of Acanthamoeba species in the rivershed. In addition, the results of this study mainly indicate that Acanthamoeba species genotype T4 accounted for 68.7% of all positive samples. The highest proportion of Acanthamoeba keratitis cases was associated with the T4 genotype, possibly due to its greater virulence and greater transmissibility with respect to other genotypes. Therefore, the presence of pathogenic Acanthamoeba should be considered a potential health threat associated with human activities in Puzih River watershed.

Relations between Acanthamoeba Presence and Water
Quality Parameters. Water quality parameters were compared with respect to Acanthamoeba species detection results. The range, mean, and standard deviation of water quality parameters in four seasons are included in Table 2. Statistical analyses were performed to detect differences (Mann-Whitney test) and correlation coefficients (Spearman rank test) in water quality parameters between Acanthamoebapositive and negative samples. Results of the above nonparametric tests are presented in Table 3. No significant difference and correlation were observed between the presence/absence of Acanthamoeba species for all the water quality parameters in the total water samples. For sampling season impact, no significant difference and correlation were observed between the presence/absence of Acanthamoeba species for all the water quality parameters in spring, autumn, and winter. However, significant differences were observed only between the presence/absence of Acanthamoeba species and turbidity ( < 0.05), and significant correlation were found only between the presence/absence of Acanthamoeba species, heterotrophic plate count (HPC), and turbidity ( < 0.05) in summer. The correlations and differences between turbidity and Acanthamoeba species demonstrated that sediments supported microbial growth [36]. The HPC from the natural inhabitants in the aquatic environment may cause the predation of Acanthamoeba species [16]. It explains why the presence/absence of Acanthamoeba species had a significant