Histología Pez Dorado.

Goldfish, Carassius auratus, a Novel Animal Model for the Study

ofMycobacterium marinum Pathogenesis

Adel M. Talaat,1 Renate Reimschuessel,2 Steven S. Wasserman,1 and Michele Trucksis1,3,*

ABSTRACT

Although there has been some progress in developing genetic systems to

study Mycobacterium tuberculosis(2, 4, 12, 21), its slow growth rate (a generation time of

more than 20 h) and the necessity of working in a biosafety level-3 facility has led

investigators (17, 18) to explore surrogate mycobacterial model systems to study the

molecular pathogenesis of this organism.

Mycobacterium marinum, first isolated from saltwater fish in 1926 (1), is an agent of fish

tuberculosis. Fish tuberculosis is a disseminated infection reported in more than 150 species

of fish (15). The disease is usually accompanied by emaciation and deaths in the infected

fish population over a period of months to years. The typical lesion seen with

histopathological examination is the granuloma, which may be present in any internal organ

(24). The histopathology of the formed granuloma in fish tuberculosis (8, 11, 24) is similar

to the histopathology seen in human tuberculosis (10, 13, 14). Unlike M. tuberculosis, M.

marinum can be studied in a biosafety level-2 laboratory with standard bacteriological

protocols. The generation time for M. marinumis about 4 h in the laboratory (5). Based on

an analysis of 16S rRNA sequences of 19 mycobacterial species,M. marinum is the

mycobacterial species closest to the M. tuberculosis complex, with a sequence homology of

99.4% (22).

Two animal models have been described that utilize M. marinum. The first is the mouse

footpad infection model, which was used to simulate Mycobacterium leprae infection (6).

Attempts to produce a systemic infection in mice failed even when the inoculum was

administered intravenously (5). More recently, a frog (Rana pipiens) model for M.

marinum infection has been proposed (17, 18). With the frog model, granulomas were

reported in the livers and spleens of animals sacrificed at 6 weeks postinoculation. No

animals died of M. marinum infection in the 40-week observation period. Moreover, the M.

marinum strain used to induce chronic disease in the frog failed to induce overt signs of

disease when tested in fish (18). In 1963, fish were among 50 species of poikilothermic

animals that were found to be susceptible to experimental infection with M. marinum. In

the model described in this report, the goldfish, Carassius auratus, is used to study the

pathogenesis of M. marinum. Systemic granuloma formation is the characteristic finding in

our model of a chronic progressive disease which parallels the pathology seen in human

tuberculosis. At higher doses (108 to 109 CFU), our experimental mycobacterial fish

infection becomes an acute model, with systemic dissemination, necrosis, and

inflammation, which results in death in 4 to 17 days. The minimum dose to produce

systemic granulomas within 8 weeks was 600 CFU per fish. This model should prove

useful for studying mycobacterial pathogenesis and for identifying avirulent mutant strains.

MATERIALS AND METHODS

Fish.

Goldfish, C. auratus (20 to 30 g), were obtained from a local commercial fish farm

(Hunting Creek Fisheries, Hunting Creek, Md.). They were acclimated to their new

environment (20-gal flowthrough aquaria with a water temperature of 20 ± 2°C and a

photoperiod of 16 h light and 8 h dark) in the quarantine area of the fish facility in the

Aquatic Pathobiology Center, University of Maryland. After 2 weeks of acclimation, the

fish were moved to a negative-air-pressure room, where the experimental infection with

mycobacteria was performed. Skin scrapes, gill biopsies, and fecal examinations were

performed on a representative sample of fish to determine that they were free of parasitic

infestation prior to infection by mycobacteria. The fish were treated with a prophylactic

dose, 100 ppm, of formalin to prevent parasitic infestation. The fish were fed pellet trout

grower (30% protein; Ziegler Bros., Gardner, Pa.) 3 days a week. Fish inoculated with

different doses of mycobacteria and control fish inoculated with phosphate-buffered saline

(PBS) were housed in separate aquaria.

Bacteria.

The M. marinum strain ATCC 927 (fish isolate) was obtained from the American Type

Culture Collection (Rockville, Md.). M. marinum M (human isolate) was from Lalita

Ramakrishnan, Stanford University (17), while M. marinum F-110 was isolated

from Cichlid sp. fish in the Aquatic Pathobiology Center, University of Maryland (23). All

strains were grown with shaking at 30°C as a dispersed culture in 7H9 (Difco, Detroit,

Mich.) broth with 10% albumin-dextrose complex enrichment (12). Animal inocula were

obtained from mid-exponential-phase cultures (optical density at 600 nm, ∼1.0) and

adjusted to the appropriate dose. The number of CFU per milliliter was determined by

plating on Middlebrook 7H10 agar (Difco). Prior to inoculation in animals, the inocula

were disaggregated by sonication for 3 min (power level 3) while cooling, using a cup horn

accessory attached to a cell disrupter (model W-220 F; Heat Systems- Ultrasonics, Inc.,

Farmingdale, N.Y.).

Diagnostic PCR.

In brief, the PCR protocol uses genus-specific primers designed from conserved regions of

the 16S rRNA sequence of mycobacteria. A 924-bp DNA fragment is amplified from the

mycobacterial species known to cause fish mycobacteriosis (M. marinum, Mycobacterium

fortuitum, and Mycobacterium chelonae). Following amplification, the DNA product was

digested with restriction enzymes, BanI (NEB, Beverly, Mass.) and ApaI (GIBCO BRL,

Gaithersburg, Md.), to yield unique restriction patterns for each of the mycobacterial

species (23).

Animal inoculation.

Fish were inoculated intraperitoneally through the lateral abdominal musculature with 0.5

ml of various concentrations of M. marinum organisms by using a 25-gauge needle and

tuberculin syringe.

Negative-control fish groups were inoculated with sterile PBS coincidentally with the

experimentally infected fish to control for environmental conditions (parasitic infestation,

changes in water temperature, etc.) in the aquaria.

Fish tissue processing.

Fish were sacrificed either in a moribund state

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