Validation of the usefulness of 26S rDNA D1/D2, internal transcribed spacer, and intergenic spacer 1 for molecular epidemiological analysis of Macrorhabdus ornithogaster

Abstract

Macrorhabdus ornithogaster (MO) is an infectious fungus that causes gastric damage in birds. In this study, we established nested and seminested polymerase chain reaction (PCR) methods that specifically amplify the domain D1/D2 region (D1/D2) of 26S ribosomal DNA (rDNA), internal transcribed spacer (ITS) of rDNA, and intergenic spacer (IGS) 1 region from avian feces. Phylogenetic analysis of MO collected from Japanese pet birds showed little genetic variation; analysis based on these regions did not distinguish between host species order, differences in MO shape, or host gastrointestinal symptoms. These regions were found to be unsuitable for molecular epidemiological studies of MO and further investigation into other genetic regions is required.

Keywords: 26S rDNA D1/D2; Macrorhabdus ornithogaster; genotyping; intergenic space 1; internal transcribed spacer.

Fig. 1.

Microscopic morphology of cultured Macrorhabdus ornithogaster (scale bar=25 μm). The left panel shows a budgerigar-derived strain (SuMO2) and the right panel shows a cockatiel-derived strain (SuMO1). SuMO2 had an elongated rod-like morphology, while SuMO1 was thicker and shorter, often with a ball-like tip (similar to a drumstick).

Fig. 2.

In conventional PCR, to amplify the D1D2 and internal transcribed spacer (ITS) regions, primer set 1 (MO18SF2 and MO26SR3), primer set 2 (MO18SF2 and 26SR2), and primer set 3 (ITS3 and MO26SR3) were used. In nested PCR, primer set 1 was used for the first run, and primer sets 2 (ITS) and 3 (D1D2) were used for the second run (A). Primer set 4 (MO26SF2 and 26SR2) was used in conventional PCR to amplify the Macrorhabdus ornithogaster (MO) rDNA sequence containing the unknown intergenic spacer (IGS) region. In nested PCR, to amplify the IGS1 region, primer set 5 (MO26SF3 and MOIGS2R1) and primer set 7 (MO26SF3 and 5SR1) were used for the first run, and primer set 6 (MO26SF3 and MOIGSR1) and primer set 8 (MO26SF3 and 5SR1) were used for the second run (B).

Fig. 3.

Maximum likelihood tree of Macrorhabdus ornithogaster based on nucleotide sequences: (A) D1/D2, (B) internal transcribed spacer (ITS), and (C) intergenic spacer (IGS) regions. Saccharomyces cerevisiae, Kluyveromyces nonfermentans, and K. marxianus were analyzed together as an out-group. Bootstrap rate values are shown next to the branches. Accession number and bacterial name are followed by the scientific name of the sampled bird species in ( ). For samples analyzed in this study, the scientific name is followed by the sample number. In addition, samples of birds showing gastrointestinal symptoms are marked with ●, and the order name of the bird is marked with }. In (B), group A and B genotypes reported by Abdi-Hachesoo et al. are circled with dotted lines [1]. In the analysis of the D1/D2 region, 16 samples matched the Macrorhabdus ornithogaster (MO) sequence in the NCBI database (GenBank accession no. AF350243.1, KX426595, and KX426594) and were classified in the same group, while two samples from cockatiels (M55 and M69) formed a new group (A). Analysis of the ITS region showed that two MO samples of Passeriformes and 15 MO samples of Psittaciformes belonged to group B. Only one sample (M60) from a zebra finch belonged to group A. The groups were not separated by host bird order (B). In analysis of the IGS1 region, 17 samples were classified into the same group and only one sample (M60) was classified into a different group (C). No association with gastrointestinal symptoms was observed in any of the analyses.