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Identification of Austwickia chelonae as cause of cutaneous granuloma in endangered crocodile lizards using metataxonomics - PubMed

  • ️Tue Jan 01 2019

Identification of Austwickia chelonae as cause of cutaneous granuloma in endangered crocodile lizards using metataxonomics

Haiying Jiang et al. PeerJ. 2019.

Abstract

The crocodile lizard (Shinisaurus crocodilurus Ahl, 1930) is an endangered reptile species, and in recent years many have died from diseases, especially the rescued and breeding individuals. However, pathogens underlying these diseases are unclear. In this study, we report our effort in rapidly identifying and isolating the pathogen that causes high mortality in crocodile lizards from Guangdong Luokeng Shinisaurus crocodilurus National Nature Reserve. The typical symptom is cutaneous granuloma in the infected crocodile lizards. Metagenomic next-generation sequencing (mNGS) is a comprehensive approach for sequence-based identification of pathogenic microbes. In this study, 16S rDNA based mNGS was used for rapid identification of pathogens, and microscopy and microbe isolation were used to confirm the results. Austwickia chelonae was identified to be the dominant pathogen in the granuloma using 16S rDNA based mNGS. Chinese skinks were used as an animal model to verify the pathogenicity of A. chelonae to fulfill Koch's postulates. As expected, subcutaneous inoculation of A. chelonae induced granulomas in the healthy Chinese skinks and the A. chelonae was re-isolated from the induced granulomas. Therefore, A. chelonae was the primary pathogen that caused this high mortality disease, cutaneous granuloma, in crocodile lizards from Guangdong Luokeng Shinisaurus crocodilurus National Nature Reserve. Antibiotics analysis demonstrated that A. chelonae was sensitive to cephalothin, minocycline and ampicillin, but not to kanamycin, gentamicin, streptomycin and clarithromycin, suggesting a possible treatment for the infected crocodile lizards. However, surgical resection of the nodules as early as possible was recommended. This study is the first report of pathogenic analysis in crocodile lizards and provides a reference for disease control and conservations of the endangered crocodile lizards and other reptiles. In addition, this study indicated that mNGS of lesions could be used to detect the pathogens in animals with benefits in speed and convenient.

Keywords: Austwickia chelonae; Dermatosis; Granuloma; Infection; Lizard; Next-generation sequencing (NGS).

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Locations of cutaneous granulomas in crocodile lizards.

The arrows indicate the lesions. One or more nodules were found under the lesion. (A) Right forelimb. (B) Right hind limb. (C) Head. (D) Under the tongue. (E) Around cloaca. (F) The right of the lower jaw. (G) Lower jaw. (H) Submental triangle. Photo credit: (A–C, E–F) Jinping Chen. (D, G–H) Nan He.

Figure 2
Figure 2. Nodules in the lesions of crocodile lizards.

Photo credit: Jinping Chen.

Figure 3
Figure 3. Histological micrographs of nodules.

(A) The multi-layered structure of the nodule. H&E staining. Scale bar = 400 µm. (B) A high number of filamentous bacteria were stained in dark blue inside the nodule. H&E staining. Scale bar = 100 µm. (C) The membrane and multi-layered structure of the nodule. GMS staining. Scale bar = 400 µm. (D) Filamentous bacteria were stained in black. GMS staining. Scale bar = 100 µm.

Figure 4
Figure 4. SEM image of the inside of a nodule.

Scale bar = 15 µm.

Figure 5
Figure 5. Flower plot conducted based on OTUs.

The core number in the middle represents the number of OTUs common to all samples. The numbers on the petals represent the number of OTUs unique to the sample.

Figure 6
Figure 6. Relative abundances of nodule and environmental bacteria at the general level.
Figure 7
Figure 7. Agarose gel (2%) electrophoresis diagram of ITS1 gene amplicons.

M, marker; +, positive control (fungus Ophiocordyceps sinensis); 1, L.LK.4L; 2, L.LK.06; 3, L.LK.07; 4, L.LK.13; 5, L.LK16; 6, L.LK.17; 7, L.LK.18; 8, L.LK.19; 9, L.LK.20; 10, L.LK.21; 11, L.LK.22; −, negative control (water).

Figure 8
Figure 8. Agarose gel (2%) electrophoresis diagram of ranavirus specific MCP gene amplicons.

(A) Amplicons of primer set RanaM68F/ BIVMCP154. The expected size was 230 bp. M, marker; 1, L.LK.16; 2, L.LK.17; 3, L.LK.19; 4, L.LK.20; 5, L.LK21; 6, L.LK.22; 7, L.LK.07; 8, L.LK.13. (B) Amplicons of primer set RanaJP556F/ RanaJP 772R. The expected size was 217 bp. M, marker; 1, L.LK.16; 2, L.LK.17; 3, L.LK.19; 4, L.LK.20; 5, L.LK21; 6, L.LK.22; 7, L.LK.07; 8, L.LK.13; 9, L.LK.18. (C) Amplicons of primer set OIT1/ OIT2R. The expected size was 500 bp. M, marker; 1, L.LK.16; 2, L.LK.17; 3, L.LK.19; 4, L.LK.20; 5, L.LK21; 6, L.LK.22; 7, L.LK.07; 8, L.LK.13.

Figure 9
Figure 9. Beta hemolysis of Austwickia chelonae on a Columbia blood agar base plate.
Figure 10
Figure 10. Chinese skink hypodermically inoculated with Austwickia chelonae (photo time: 44 days after inoculation).

Arrows note the visible nodules on the left side compared with the right side. Photo credit: Haiying Jiang.

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Grants and funding

This project was supported by the Planning Funds of Science and Technology of Guangdong Province (2016B070701016), the Guangzhou Science Technology and Innovation Commission (201804020080), the Training Fund of Guangdong Institute of Applied Biological Resources for PhDs, Masters and Postdoctoral Researchers (GIABR-pyjj201604), the GDAS Special Project of Science and Technology Development (2019GDASYL-0105046, 2018GDASCX-0107 and 2017GDASCX-0107) and the Special Funds for Forestry Development and Protection of Guangdong Province (2017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.