Giardia, Kunstler, 1882

Delport, Tiffany C., Asher, Amy J., Beaumont, Linda J., Webster, Koa N., Harcourt, Robert G. & Power, Michelle L., 2014, Giardia duodenalis and Cryptosporidium occurrence in Australian sea lions (Neophoca cinerea) exposed to varied levels of human interaction, International Journal for Parasitology: Parasites and Wildlife 3 (3), pp. 269-275 : 272

3.1. Giardia detection and species identification

Screening of genomic DNA using a Giardia specific 18S rRNA protocol resulted in the detection of Giardia in 28 samples from wild sea lions (10.3%) and in seven samples from captive sea lions (36.8%). There was a significant difference in Giardia presence between wild and captive individuals (χ 2 = 11.758, df = 1, p = <0.001). In wild colonies, the distance from human settlement had a significant effect on the presence or absence of Giardia (Wald χ 2 = 39.078, df = 2, p = <0.001). Colonies less than 25 km from human settlements had a higher occurrence of Giardia than colonies more than 26 km away ( Table 1). There was no effect of sampling season on Giardia presence (Wald χ 2 = 6.112, df = 3, p = 0.106).

DNA sequences were obtained for 28 of the 35 18S rRNA positive samples. BlastN search identified 27 sequences as belonging to Giardia duodenalis assemblage B and one belonging to assemblage A. Analysis using the phyologentic framework clustered all samples from wild sea lions (n = 24) and three samples from captive animals within a clade that also contained the assemblage B reference sequence from GenBank ( Fig. 2 ). One captive sample clustered within a clade containing the reference sequences from GenBank for assemblages A, E and F. Analysis of clustalW alignment showed a 2 bp polymorphism between the assemblages with the captive sample most closely aligned to assemblage A. Alignment of the sample to representative sequences showed that the sample was 100% identical to assemblage A but not E or F.

18S rRNA positive samples (n = 35) failed to amplify at the gdh locus. Representative samples all produced a gdh amplicon when spiked with Giardia isolate DNA. The β-giardin locus amplified in one of 35 18S rRNA positive samples identified as assemblage B. A DNA sequence was obtained for the β-giardin positive sample and a BlastN search identified the sequence as belonging to Giardia duodenalis assemblage AI. The inferred phylogeny placed the sample within a clade that also contained the assemblage AI reference sequence from GenBank. All samples spiked with Giardia positive DNA produced an amplicon when screened using β-giardin PCR.

Figures

Fig. 2. Giardia duodenalis 18S rRNA phylogenetic tree.Phylogenetic analysis of Giardia duodenalis positive samples was performed using a fragment of 18S rRNA gene.Analysis within the phylogenetic framework placed sea lion samples within the assemblage B (n = 27) and assemblage A clades (n = 1). Branch values indicate percent bootstrapping using 1000 replicates.