G. agilis trophozoites were collected from frogs and tadpoles. The frogs and tadpoles were anesthetized to death with 20% ethanol and then their intestines were removed and cut into 0.1 cm segments. The segments of intestines were collected into centrifuge tubes with amphibian normal saline (0.65% sodium chloride). The centrifuge tubes were chilled in ice for more than 30 min. The suspension was briefly centrifuged at 1,000× g for 1s to remove the precipitants of large fragments and the supernatant was collected into new centrifuge tubes. Then centrifuge tubes were centrifuged at 750× g for 5 min and the supernatant was discarded to concentrate the trophozoites. The sediment was re-suspended with amphibian normal saline and kept at room temperature for 30 min. Then the amphibian normal saline was replaced. The centrifuge tubes were chilled in ice for 30 min and centrifuged at 2,000× g for 5 min, to collect the trophozoites. The supernatant was discarded and the precipitated trophozoites were re-suspended with amphibian normal saline and were stored at 4 °C for later use.
The frog samples were collected in some selected areas of Yunnan, Sichuan, Gansu, and Shaanxi provinces, and Shanghai city of China at the time from March of 2014 to May of 2019. We followed the guidelines of The Animal Care and Use Committee of the American Society of Mammalogists for the use of wildlife in our research . All the experimental procedures and animal care were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the Kunming Institute of Zoology, Chinese Academy of Sciences (PAOKIZ140105, 01/2014).
Morphological identification of G. agilis
The slides were prepared with trophozoite supernatant in amphibian normal saline. All slides were examined under oil immersion by 40× and 100× HCX PL APO objectives on a Leica DM2500 microscope (Leica, Wetzlar, Germany). The images were captured by a Leica DFC450 C digital camera. Sample handling and photomicrographs of scanning electron microscope were processed at the Kunming Medical University (Kunming, China) using HITACHI 3700N (Tokyo, Japan).
Statistical analysis was performed using mid-P exact probability tests and 95% confidence interval (CI) value was calculated, differences were considered significant when p-values ≤ 0.05 were obtained (https://www.medcalc.org/).
DNA extraction, PCR amplification and sequencing
G. agilis genomic DNA was extracted from feces samples of tadpoles and frogs using the TIANamp Stolol DNA Kit (Tiangen, Beijing, China) according to the manufacturer's protocol. The primers specific to both ends of the sequences of the two genes, SSU rRNA and β-giardin, were designed according to the conserved sequences of these genes of G. intestinalis (50586 isolate) using PRIMER PREMIER program version 5.00 (Biosoft International) , and the expected product lengths of the SSU rRNA and β-giardin were 950 bp and 500 bp, respectively (see primer sequences in Additional file 1). The primers were used to amplify the sequences of the two genes from all fecal genomic DNA samples by PCR. The PCR reactions were set up in 25 μl 2× PCR Taq Plus MasterMix with dye (abm, Canada), 1 μM of each primer and 1-5 μl of DNA sample. Thermocycling conditions were as follows: 94 °C for 10 min followed by 30 cycles of 94 °C for 30 s, 52 °C for 30 s and 72 °C for 90 s, followed by 72 °C for 10 min. The PCR products were purified using the Wizard SV Gel and PCR clean-up system kit (Qiagen), and cloned into pMD-19T vectors using TaKaRa pMD-19T VectorCloning Kit (TaKaRa, Tokyo, Japan). The ligation products were transformed into DH5α chemically competent E. coli. Colony PCR was carried out with the vector-specific primers provided in the kit, and colonies were selected and Sanger-sequenced using vector-specific forward and reverse primers by TSINGKE Biological Technology (Kunming, China).
Molecular phylogenetic analysis
In order to perform the phylogenetic analysis, we sequenced, identified and retrieved the SSU rRNA and β-giardin of G. agilis (GenBank accession numbers: MN227552 and MG733773). The other sequences used in the analysis were all retrieved from the GenBank database (see their accession numbers in Additional file 2). The number of isolates of G. agilis for each locus sequenced is as follows: 5 for SSU rRNA and 7 for β-giardin. The maximum likelihood phylogenetic trees based on SSU rRNA and β-giardin DNA sequences were reconstructed by using the PhyML software . The sequence data of the two loci are from 9 and 10 Giardia species or isolates. Multiple sequence alignments were performed with ClustalW 2.0 program , and the alignments were visually inspected to eliminate poorly aligned positions. The best-fit DNA model used for reconstructing the maximum likelihood phylogeny was selected by the JModelTest software [15, 16]. Trees were constructed using the PhyML 3.0 based on 14 taxa with 817 nt positions for SSU rRNA gene and 16 taxa with 335 nt positions for β-giardin gene. Tree reliability was determined by using bootstrap analyses with 1000 replicates