Detection and Identification of Mycobacteria in Alien (Including Invasive) Turtle Species in Poland
Department of Microbiology, National Reference Laboratory for Bovine Tuberculosis, National Veterinary Research Institute, Pulawy, Poland
In the available literature, there are few reports on mycobacteriosis in reptiles, including various species of sea turtles and aquatic freshwater turtles, both captive or wild.1-3 The occurrence of Mycobacterium species in clinically healthy chelonians were studied in Poland during the period 2015–2017. The samples of 134 chelonians were examined bacteriologically. A total of 382 microbiological tests were performed. Most of the turtles captured represented subspecies of Trachemys scripta: red-eared slider (Trachemys scripta elegans; n=76), yellow-eared turtle (Trachemys scripta troosti; n=11), and yellow-bellied slider (Trachemys scripta scripta; n=32). The remaining chelonians were false map turtles (Graptemys pseudogeographica; n=3), a single common snapping turtle (Chelydra serpentina), and hybrids of different slider species. Eighty-five turtles (63%) were female and 49 (37%) males. All were caught in ETT (Epictares Turtle Trap) spaced at freshwater reservoirs in eastern Poland. After euthanasia and anatomopathological examinations, the necessary samples were collected for laboratory tests. First a microscopic stain from internal organs was prepared using the Ziehl-Neelsen method with carbolic fuchsin and malachite green. Classical microbiological tests for the presence of mycobacteria were carried out with swabs from the mouth cavity, internal organs (liver, spleen, kidneys), scrapes from plastron and carapace, and from skin swabs. The dissected tissue was soaked and homogenized in a 5% solution of oxalic acid. The resulting supernatant was removed and the pellet was washed twice with sterile physiological saline. The rinsed pellet was used for inoculation of culture media including 3 slants of Stonenbrink’s, Petragnani’s, and Loewenstein-Jensen’s media which were incubated at 37°C. Simultaneously, samples were tested in Mycobacteria Growth Indicator Tube (MGIT) system. All samples were prepared with MycoPrep kit used for the preparation of specimens containing mycobacteria, according to the manufacturer’s recommendations. Pre-prepared samples were incubated for 6 weeks in MGIT 320 incubator. For identification of potentially isolated strains, GenoType Mycobacterium CM (for common mycobacteria) and AS (for additional species), Hain Lifescience tests were prepared. The trial to determine membership of the isolated species using MALDI-TOF technique was also conducted.
In studies performed with classical microbiological methods, 34 positive results were obtained, which constituted 9.0% of all cultures. All positive results in classical studies were confirmed by positive results in MGIT studies. The most positive mycobacteria growth results were obtained from plastron and carapace, whereas fewer positive results were obtained from inoculations of internal organs. Only 2 positive cultures were obtained in the group of samples being swabs from the skin and from the mouth cavity. To date, 9 species of turtles have been identified. Six of them were defined as Mycobacterium fortuitum, 2 as Mycobacterium nonchromogenicum, and 1 as Mycobacterium neoaurum. Eight strains were unclassified, absent from the database. Studies are underway to determine the remaining 16 strains. Preliminary results indicate that turtles with positive results were mechanical carriers of mycobacteria because their internal organs lacked lesions to suggest the existence of mycobacteriosis. However, there is a certain correlation between the presence of mycobacteria and incorrect carapace construction.
This work was supported by the National Science Centre project “Invasive turtle species as a source and vector of animal and human pathogens” (Grant No. 2013/11/B/NZ7/01690). We would like to thank all colleagues involved in aforementioned studies.
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