Paramecium caudatum enhances transmission and infectivity of Mycobacterium marinum and M. chelonae in zebrafish Danio rerio

Abstract

Mycobacterial infections in laboratory zebrafish Danio rerio are common and widespread in research colonies. Mycobacteria within free-living amoebae have been shown to be transmission vectors for mycobacteriosis. Paramecium caudatum are commonly used as a first food for zebrafish, and we investigated this ciliate's potential to serve as a vector of Mycobacterium marinum and M. chelonae. The ability of live P. caudatum to transmit these mycobacteria to larval, juvenile and adult zebrafish was evaluated. Infections were defined by histologic observation of granulomas containing acid-fast bacteria in extraintestinal locations. In both experiments, fish fed paramecia containing mycobacteria became infected at a higher incidence than controls. Larvae (exposed at 4 d post hatch) fed paramecia with M. marinum exhibited an incidence of 30% (24/80) and juveniles (exposed at 21 d post hatch) showed 31% incidence (14/45). Adult fish fed a gelatin food matrix containing mycobacteria within paramecia or mycobacteria alone for 2 wk resulted in infections when examined 8 wk after exposure as follows: M. marinum OSU 214 47% (21/45), M. marinum CH 47% (9/19), and M. chelonae 38% (5/13). In contrast, fish feed mycobacteria alone in this diet did not become infected, except for 2 fish (5%) in the M. marinum OSU 214 low-dose group. These results demonstrate that P. caudatum can act as a vector for mycobacteria. This provides a useful animal model for evaluation of natural mycobacterial infections and demonstrates the possibility of mycobacterial transmission in zebrafish facilities via contaminated paramecia cultures.

Figure 1

. Danio rerio. Histological presentations in zebrafish exposed to Mycobacterium marinum and Mycobacterium chelonae residing within paramecia food vacuoles. (a) Paramecium caudatum cultured with M. marinum OSU 214 for 8 h, with numerous acid-fast bacteria in food vacuoles. Kinyoun’s acid-fast. Scale bar = 10 µm. (b) Mycobacteria (arrow) in pneumatic duct of larval fish exposed to M. chelonae. Ziehl-Neelsen acid-fast. Scale bar = 10 µm. (c) M. chelonae aggregate in larval fish swim bladder (arrows). Ziehl-Neelsen acid-fast. Scale bar = 25 µm. (d) Mycobacteria (arrow) in the intestinal lumen of an adult fish. Kinyoun’s acid-fast. Scale bar = 25 µm. (e) Liver (L) and spleen (S) granulomas in a juvenile fish fed paramecia containing M. marinum OSU 214. Arrow = granuloma demonstrated in Fig. 1f. Hematoxylin and eosin. Scale bar = 100 µm. (f) High magnification of spleen granuloma with numerous mycobacteria. Kinyoun’s acid-fast. Scale bar = 25 µm.

Figure 2

Prevalence (%) of extraintestinal mycobacteriosis (white bars) and presence of mycobacteria in the intestinal lumen (gray bars). Data points indicate results from each of the for four tanks for each treatment regime. A. Mycobacterium marinum OSU 214 at 8wk pe. B. Mycobacterium marinum CH at 8 wk pe. C. Mycobacterium chelonae at 8 wk. D. M. chelonae at 16 wk pe.