Mycobacterium marinum Causes a Latent Infection that Can Be Reactivated by Gamma Irradiation in Adult Zebrafish

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (~35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5–10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.Public Library of Science open acces

Gamma irradiation induces reactivation resulting in increased mortality due to uncontrolled growth of mycobacteria.

<p>(A–C) Zebrafish (n = 17) with a latent <i>M. marinum</i> infection were irradiated twice with 25 Gy with one month between the irradiations. Twice irradiated, non-infected zebrafish (n = 23) as well as zebrafish with a latent infection (n = 14) were included as controls. (A) Survival was followed for 30 days after the second dose. *P<0.05. (B) During this period, moribund or recently dead fish were collected 15–22 days after the second radiation dose. Bacterial loads were compared with those of similarly infected, non-irradiated control fish that were collected at the end-point of the experiment. *P<0.05 (C&D) A representative Ziehl-Neelsen stained sample from a reactivated fish showing large numbers of free mycobacteria (purple areas) in the zebrafish body cavity (C). The sides of the body cavity are marked with arrowheads O = ovary, P = pancreas, L = liver, G = gut, K = kidney. (D) A picture taken with a higher magnification showing individual rods (few examples pointed out with arrows). (E) Four groups of 4 adult zebrafish (1 rag2-gfp, 1 lck-gfp and 2 wild-type groups) were γ-irradiated with 25 Gy. Similar control groups were left untreated. Kidneys were collected 8 d post irradiation, pooled and analyzed by FCM. FSC-SSC -plots were gated based on cell size and granularity as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002944#ppat.1002944-Traver2" target="_blank">[56]</a> (gates shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002944#ppat.1002944.s003" target="_blank">Figure S3</a>) to assess the effect of irradiation on leukocyte populations. *P<0.05. For further verification of the effect of radiation on lymphocytes, a GFP gate was used for the rag2 and lck groups expressing GFP in B and T cells, or T cells, respectively. (F) Adult non-infected wt zebrafish were irradiated with 25 Gy once (grey bars) (n = 3) or twice (n = 7) (black bars) with one month between the doses. Leukocyte recovery and re-depletion were assessed by FCM. Non-irradiated fish (n = 4) were used as controls. *P<0.05 (G) Fish with a latent infection (n = 7) were irradiated twice with 25 Gy with one month between the doses and plated +/− Rpf for 18 d after the second radiation dose. (H) Fish (n = 6) with a latent infection were plated +/− Rpf.</p

A major part of the mycobacteria are in a dormant state in latent infection.

<p>(A) Parallel dilutions of fresh logarithmic or old plateau phase <i>M. marinum</i> cultures were plated +/− Rpf to show the resuscitating effect of <i>Micrococcus luteus</i> Rpf on dormant <i>M. marinum</i>. (B) Parallel homogenate sample dilutions from low-dose (34±15 cfu) infected fish (wt or rag1 (−/−)) were plated at different time points +/− Rpf to detect dormant mycobacteria. (C) <i>GltA1</i> expression was measured from low-dose infected rag1 (−/−) and wt fish and high-dose infected wt fish and normalized to the total <i>M. marinum</i> load in each fish measured by qPCR. *P<0.05.</p

Zebrafish mortality, the development of bacterial load and the number of lesions have dose-dependent patterns.

<p>Adult zebrafish were i.p. infected with either a low (34±15 cfu) (n = 180) or a high dose (2029±709 cfu) (n = 104) of <i>M. marinum</i>. (A) Survival was followed for 32 weeks. * P<0.05 (B) The figure shows the average loads for 5 fish (except 32 wk high dose, n = 2). Low-dose statistics: * sig. diff. from 1 wk, ** sig. diff. from 1 and 2 wk. High-dose statistics: *** sig. diff. from 1, 2, 8, 11 and 20 wk. Low-dose vs. high-dose statistics: loads at time-points marked with † are sig. diff. (C) By default, 4 individuals per dose were analyzed by Ziehl-Neelsen staining (except 20 wk high dose, n = 3) per time-point The gonads, pancreas, liver, muscle, mesentery, spleen, gut and kidney were assessed and the number of organs with visible bacteria was determined. *P<0.05. (D) The total number of granulomas in a sample set for each individual was counted. * P<0.05.</p

Bacterial dose and the presence of functional adaptive immunity define the outcome of mycobacterial infection.

<p>(A) The early cytokine response at 1 d post infection was measured from wt fish infected with a high (2029±709 cfu) or a low (34±15 cfu) dose or injected with sterile PBS buffer (n in each group 10–20). *P<0.05 (B) Wt fish were infected with a high or a low dose or sterile PBS buffer (for early time-points), and rag1 (−/−) fish were infected with a low dose Nos2b expression was measured with q-RT-PCR (n in each group was 9–20/time point). *P<0.05 (C) Fish were infected as in (B) and <i>IFNγ1–2</i> was measured with q-RT-PCR. *P<0.05. (D) Adult wt and rag1 (−/−) zebrafish were infected with a low dose (n = 30) and followed for survival. *P<0.05 (E) Adult wt and rag1 (−/−) fish were infected with a low dose. Average mycobacterial load was measured by qPCR at 2, 4, and 7 wpi (n = 10 per time point). *P<0.05.</p

<i>M. marinum</i> induces the formation of granulomas that mature into well-defined structures during an infection.

<p>In fish infected with a low dose (34±15 cfu) of <i>M. marinum</i>, Ziehl-Neelsen staining at 2 wpi commonly reveals areas with free bacteria (C). Some slightly better formed and restricted areas containing bacteria, here referred to as early granulomas, are also seen (A), but as shown in (B) trichrome staining of the adjacent slide, encapsulation around the mycobacterial lesions is absent at the early stage of infection. At 20 weeks, fish that have survived have mature granulomas (D–F) many of which are multicentric surrounded by a fibrous capsule (D&E). (E) Trichrome staining shows the fibrous capsule in blue (F). The amount of bacteria inside granulomas has increased from the earliest time-points.</p

Adequate Th2-Type Response Associates with Restricted Bacterial Growth in Latent Mycobacterial Infection of Zebrafish

Tuberculosis is still a major health problem worldwide. Currently it is not known what kind of immune responses lead to successful control and clearance of Mycobacterium tuberculosis. This gap in knowledge is reflected by the inability to develop sufficient diagnostic and therapeutic tools to fight tuberculosis. We have used the Mycobacterium marinum infection model in the adult zebrafish and taken advantage of heterogeneity of zebrafish population to dissect the characteristics of adaptive immune responses, some of which are associated with well-controlled latency or bacterial clearance while others with progressive infection. Differences in T cell responses between subpopulations were measured at the transcriptional level. It was discovered that a high total T cell level was usually associated with lower bacterial loads alongside with a T helper 2 (Th2)-type gene expression signature. At late time points, spontaneous reactivation with apparent symptoms was characterized by a low Th2/Th1 marker ratio and a substantial induction of foxp3 reflecting the level of regulatory T cells. Characteristic gata3/tbx21 has potential as a biomarker for the status of mycobacterial disease.Public Library of Science open acces