Identification ofAustwickia chelonaeas cause of cutaneous granuloma in endangered crocodile lizards using metataxonomics

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.

Identification of Pseudomonas aeruginosa From the Skin Ulcer Disease of Crocodile Lizards (Shinisaurus crocodilurus) and Probiotics as the Control Measure

“…As crocodile lizards are a protected species, we chose closely related Chinese skinks ( Plestiodon chinensis ) for in vivo testing and constructed an artificial infection animal model by creating abrasions on the skin of the skinks ( 14 , 16 , 45 – 48 ). Jiang et al ( 14 ) also used Chinese skinks as an animal model for cutaneous granuloma identification in crocodile lizards. Here, all experimental skinks were adult males obtained from the Longmeng Lizard Breeding Station (Business license: 102620002940) in China.…”

“…Here, all experimental skinks were adult males obtained from the Longmeng Lizard Breeding Station (Business license: 102620002940) in China. A scalpel was first used to make a small round wound (0.5 cm in diameter) under sterile conditions, after which P. aeruginosa fluid (1 ml, 1CFU:10 5 ml in solution) was administered to the wounds ( 14 , 16 , 33 , 41 ). Two control groups were established, including a no-operation group and a wound-only group.…”

“…Two control groups were established, including a no-operation group and a wound-only group. Two separate wounds were created on each of the eight skinks ( 14 , 16 ).…”

“…In addition to the high mortality rate, the disease also causes stunted growth, further undermining attempts to recover wild population. Of concern, the release of these captive-bred lizards may introduce the disease to unprotected wild populations, further impacting restoration efforts and population decline ( 2 , 14 – 16 ). Several reports also suggest that humans are at risk of zoonotic infections from reptiles ( 6 , 8 , 17 – 19 ).…”

Skin Microbiota Was Altered in Crocodile Lizards (Shinisaurus crocodilurus) With Skin Ulcer

“…There are ∼800 captive crocodile lizards globally. However, in recent years, they have been affected by various diseases, such as cutaneous granuloma, skin ulceration, early death in newborns, malformation, incomplete absorption of yolk, and seizure (21)(22)(23), thus impeding conservation programs and increasing the risk of transmission to the wild population. Among these, the most widespread and serious are skin diseases.…”