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.

Intelectin 3 is dispensable for resistance against a mycobacterial infection in zebrafish (Danio rerio).

Tuberculosis is a multifactorial bacterial disease, which can be modeled in the zebrafish (Danio rerio). Abdominal cavity infection with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, leads to a granulomatous disease in adult zebrafish, which replicates the different phases of human tuberculosis, including primary infection, latency and spontaneous reactivation. Here, we have carried out a transcriptional analysis of zebrafish challenged with low-dose of M. marinum, and identified intelectin 3 (itln3) among the highly up-regulated genes. In order to clarify the in vivo significance of Itln3 in immunity, we created nonsense itln3 mutant zebrafish by CRISPR/Cas9 mutagenesis and analyzed the outcome of M. marinum infection in both zebrafish embryos and adult fish. The lack of functional itln3 did not affect survival or the mycobacterial burden in the zebrafish. Furthermore, embryonic survival was not affected when another mycobacterial challenge responsive intelectin, itln1, was silenced using morpholinos either in the WT or itln3 mutant fish. In addition, M. marinum infection in dexamethasone-treated adult zebrafish, which have lowered lymphocyte counts, resulted in similar bacterial burden in both WT fish and homozygous itln3 mutants. Collectively, although itln3 expression is induced upon M. marinum infection in zebrafish, it is dispensable for protective mycobacterial immune response.

DNA vaccination boosts Bacillus Calmette-Guérin protection against mycobacterial infection in zebrafish.

Despite the widespread use of the current Bacillus Calmette-Guérin (BCG) vaccine, tuberculosis is still a major cause of morbidity and mortality worldwide. Vaccination with BCG does not prevent a Mycobacterium tuberculosis infection, nor does it inhibit the reactivation of latent tuberculosis. Here, we show that adult zebrafish are modestly and variably protected from a mycobacterial infection by BCG vaccination. An intraperitoneal (i.p.) BCG vaccination was associated with enhanced survival upon a high-dose (20,000 bacteria) Mycobacterium marinum infection. In addition, BCG-vaccinated fish were more able to restrict a low-dose (30 bacteria) intraperitoneal infection with M. marinum, as indicated by lower bacterial loads at six weeks post infection (wpi). However, the vaccination could not completely prevent an infection. A qRT-PCR analysis comparing BCG-vaccinated and unvaccinated fish upon a mycobacterial infection indicated that the induction of Tumor necrosis factor (TNF) was more modest in vaccinated fish. The partial protection gained by BCG could be boosted by a DNA vaccine combining Ag85B, ESAT6 and a resuscitation-related gene RpfE, suggesting that this combination of antigens could be useful for a future BCG booster vaccine. We conclude that zebrafish is a useful early-phase preclinical model for studying subunit vaccines designed for boosting the effects of BCG.

Animal models in tuberculosis research - where is the beef?

INTRODUCTION: Tuberculosis (TB) is a major global health problem, and new drugs and vaccines are urgently needed. As clinical trials in humans require tremendous resources, preclinical drug and vaccine development largely relies on valid animal models that recapitulate the pathology of human disease and the immune responses of the host as closely as possible. AREAS COVERED: This review describes the animal models used in TB research, the most widely used being mice, guinea pigs and nonhuman primates. In addition, rabbits and cattle provide models with a disease pathology resembling that of humans. Invertebrate models, including the fruit fly and the Dictyostelium amoeba, have also been used to study mycobacterial infections. Recently, the zebrafish has emerged as a promising model for studying mycobacterial infections. The zebrafish model also facilitates the large-scale screening of drug and vaccine candidates. EXPERT OPINION: Animal models are needed for TB research and provide valuable information on the mechanisms of the disease and on ways of preventing it. However, the data obtained in animal studies need to be carefully interpreted and evaluated before making assumptions concerning humans. With an increasing understanding of disease mechanisms, animal models can be further improved to best serve research goals.

An adult zebrafish model for preclinical tuberculosis vaccine development.

Tuberculosis remains a major global health challenge despite extensive vaccination schemes with the current live vaccine, Bacillus Calmette-Guérin. Tuberculosis vaccine research has been hampered by a scarcity of animal models which replicate human disease and are suitable for large-scale studies. We have shown recently that Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, causes an infection resembling human tuberculosis in adult zebrafish (Danio rerio). In the present study we use this model to show that BCG vaccination as well as DNA vaccination with selected mycobacterial antigens (Ag85B, CFP-10 and ESAT-6) protects adult zebrafish from mycobacterial infection. Using a low-dose (∼20-30 bacteria) intraperitoneal M. marinum infection, both the number of granulomas and the amount of infected organs were reduced in the DNA vaccinated fish. Likewise, when infecting with a lethal infection dose (∼20,000-27,000 bacteria), vaccination significantly reduced both mortality and bacterial counts in a manner dependent on the adaptive immune response. Protective effects of vaccination were associated with enhanced expression of interferon gamma. Our results indicate that the zebrafish is a promising new model for preclinical tuberculosis vaccine research.