Mycobacteriosis, a chronic bacterial infection, has been associated with severe losses in some zebrafish facilities and low-level mortalities and unknown impacts in others. The occurrence of at least six different described species (Mycobacterium abscessus, M. chelonae, M. fortuitum, M. haemophilum, M. marinum, M. peregrinum) from zebrafish complicates diagnosis and control because each species is unique. As a generalization, mycobacteria are often considered opportunists, but M. haemophilum and M. marinum appear to be more virulent. Background genetics of zebrafish and environmental conditions influence the susceptibility of fish and progression of disease, emphasizing the importance of regular monitoring and good husbandry practices. A combined approach to diagnostics is ultimately the most informative, with histology as a first-level screen, polymerase chain reaction for rapid detection and species identification, and culture for strain differentiation. Occurrence of identical strains of Mycobacterium in both fish and biofilms in zebrafish systems suggests transmission can occur when fish feed on infected tissues or tank detritus containing mycobacteria. Within a facility, good husbandry practices and sentinel programs are essential for minimizing the impacts of mycobacteria. In addition, quarantine and screening of animals coming into a facility is important for eliminating the introduction of the more severe pathogens. Elimination of mycobacteria from an aquatic system is likely not feasible because these species readily establish biofilms on surfaces even in extremely low nutrient conditions. Risks associated with each commonly encountered species need to be identified and informed management plans developed. Basic research on the growth characteristics, disinfection, and pathogenesis of zebrafish mycobacteria is critical moving forward.

We report the first cases of Edwardsiella ictaluri causing epizootics in laboratory populations of Zebrafish Danio rerio. Edwardsiella ictaluri is primarily recognized as a disease of catfish species and is known to cause an economically important bacterial disease of farm-raised catfish in the USA and abroad; however, it has been isolated on occasion from 10 other genera of nonictalurid fishes. We isolated E. ictaluri from moribund Zebrafish held in quarantine at two different universities in two states and from a research facility in a third state between February 23 and December 6, 2011. Edwardsiellosis in Zebrafish can be described as a severe systemic disease characterized by tissue necrosis and the presence of large numbers of extracellular and intracellular bacteria, often within macrophages. The kidneys (pronephros and mesonephros), spleen, nares, and forebrain were the most commonly and severely affected tissues. In outbreaks, mortality was acute and numerous fish died over a 1–2 week period. Mortality continued until the majority of the population was lost, at which time the remaining fish were euthanized. In addition to these cases, four cultures of bacteria isolated from Zebrafish by another diagnostic laboratory were submitted to the Louisiana Aquatic Diagnostic Laboratory for identification and were confirmed as E. ictaluri. In total, eight cultures of E. ictaluri from Zebrafish from Louisiana, Massachusetts, Pennsylvania, and Florida were identified. The isolates were confirmed as E. ictaluri by biochemical phenotype, API 20E (bioMérieux), and amplification and sequencing of a portion of the 16S rRNA gene. Edwardsiella ictaluri isolates from Zebrafish are believed to comprise a unique group and were differentiated from catfish isolates by exhibiting weaker motility, autoaggregation in broth, a different plasmid profile (two plasmids of 4.0 and 3.5 kb), a different API 20E code (4204000), and lack of lipopolysaccharide recognition with Mab Ed9.

Despite the prevalence of gamma delta T cells in mucosae that are typically colonized by Candida albicans, little is known of the possible role of these cells in resistance to candidiasis. A sharp increase in the number of gamma delta T cells and macrophages following intraperitoneal inoculation of mice with C. albicans led us to examine the role of these cells in the immune response to C. albicans. We show that the gamma delta T cells enhance macrophage nitric oxide (NO) production and anti-candida activity, in vitro. We also propose that the gamma delta T cells regulate macrophage function during candidiasis in vivo as well, because depletion of these cells abrogated inducible NO synthase expression in mucosae and enhanced murine susceptibility to candidiasis.

BackgroundVaginal epithelial cells have receptors, signal transduction mechanisms, and cytokine secretion capabilities to recruit host defenses against Candida albicans infections. This research evaluates how probiotic lactobacilli affect the defensive epithelial response.MethodsThis study used quantitative reverse transcription-polymerase chain reaction assay (qRT-PCR), flow cytometry, and a multiplex immunoassay to observe changes in the regulation of gene expression related to cytokine responses in the VK2 (E6/E7) vaginal epithelial cell line treated with 17β-estradiol, exposed to probiotic Lactobacillus rhamnosus GR-1® and Lactobacillus reuteri RC-14® and challenged with C. albicans. Data were statistically evaluated by repeated measures analysis of variance and paired t-tests where appropriate.ResultsC. albicans induced mRNA expression of genes related to inflammatory cytokine responses associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signal transduction pathways. 17β-estradiol suppressed expression of interleukin-1α (IL-1α), IL-6, IL-8, and tumor necrosis factor alpha (TNFα) mRNA. Probiotic lactobacilli suppressed C. albicans-induced nuclear factor-kappa B inhibitor kinase kinase alpha (Iκκα), Toll-like receptor-2 (TLR2), TLR6, IL-8, and TNFα, also suggesting inhibition of NF-κB signaling. The lactobacilli induced expression of IL-1α, and IL-1β mRNA, which was not inhibited by curcumin, suggesting that they induce an alternate inflammatory signal transduction pathway to NF-κB, such as the mitogen activated protein kinase and activator protein-1 (MAPK/AP-1) signal transduction pathway. Curcumin inhibited IL-13 secretion, suggesting that expression of this cytokine is mainly regulated by NF-κB signaling in VK2 cells.ConclusionsThe results suggest that C. albicans infection induces pro-inflammatory responses in vaginal epithelial cells, and estrogen and lactobacilli suppress expression of NF-κB-related inflammatory genes. Probiotic lactobacilli may induce IL-1α and IL-1β expression by an alternate signal transduction pathway, such as MAPK/AP-1. Activation of alternate signaling mechanisms by lactobacilli to modify epithelial cell cytokine production may be a mechanism for probiotic modulation of morbidity in vulvovaginal candidiasis.

Four species of probiotic bacteria were assessed for their capacities to protect athymic bg/bg-nu/nu and euthymic bg/bg-nu/؉ mice from mucosal and systemic candidiasis. Each bacterial species and Candida albicans colonized the gastrointestinal tracts of both strains of mice. The presence of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei GG, or Bifidobacterium animalis) in the gastrointestinal tracts prolonged the survival of adult and neonatal bg/bg-nu/nu mice compared to that of isogenic mice colonized with C. albicans alone. The incidence of systemic candidiasis in bg/bg-nu/nu mice was significantly reduced by each of the four probiotic bacterial species. The numbers of C. albicans present in the alimentary tracts of euthymic bg/bg-nu/؉ mice were significantly reduced by L. casei GG and B. animalis. None of the probiotic bacteria species completely prevented mucosal candidiasis, but B. animalis reduced its incidence and severity. Probiotic bacteria also modulated antibody-and cell-mediated immune responses to C. albicans. The prolonged survival of mice, decreased severity of mucosal and systemic candidiasis, modulation of immune responses, decreased number of C. albicans in the alimentary tract, and reduced numbers of orogastric infections demonstrated not only that probiotic bacteria have biotherapeutic potential for prophylaxis against and therapy of this fungal disease but also that probiotic bacteria protect mice from candidiasis by a variety of immunologic (thymic and extrathymic) and nonimmunologic mechanisms in this model.

Probiotic bacteria can protect immunodeficient mice from orogastric candidiasis but cause some pathology of their own. Severely immunodeficient patients may be at risk if fed viable probiotics, so this study evaluated the probiotic potential of nonviable probiotic bacteria to protect immunodeficient mice from Candida albicans infections. Heat-killed probiotic bacteria were fed to gnotobiotic bg/bg-nu/nu and bg/bg-nu/+ mice to ascertain if they could protect the mice from mucosal and systemic candidiasis. Both heat-killed Lactobacillus acidophilus (HKLA) and heat-killed Lactobacillus casei (HKLC), in comparison to control mice not fed the probiotic bacteria but challenged (oral) with C. albicans, suppressed the severity of orogastric candidiasis in bg/bg-nu/nu mice at 2 weeks after colonization with C. albicans, inhibited disseminated candidiasis in C. albicans-colonized bg/bg-nu/+ mice at 4 weeks after colonization, and suppressed the number of viable C. albicans in the alimentary tract. HKLA, but not HKLC, treatment inhibited disseminated candidiasis in bg/bg-nu/nu mice at 2 weeks after oral challenge and enhanced the proliferative responses of splenocytes from C. albicans-colonized bg/bg-nu/+ mice to C. albicans antigens. Neither HKLA nor HKLC were able to prolong the survival of gnotobiotic bg/bg-nu/nu mice after oral challenge with C. albicans. These results demonstrate that heat-killed lactobacilli can induce some (limited) protection (probiotic effect) against candidiasis in mice.

A defined human microbiota-associated (HMA) mouse model in BALB/c and immunodeficient Tgepsilon26 mice was used to assess the ability of probiotic lactobacilli and bifidobacteria to enhance colonization resistance to gastrointestinal (GI) tract pathogens. Probiotic bacteria (1x10(8) colony forming unit (CFU)/mL) successfully excluded Campylobacter jejuni from both strains of mice 7 days after challenge. The probiotic bacteria also reduced the number of Salmonella in the large intestines of both mouse strains. The nylon wool fractionated spleen lymphocyte populations were incubated with Salmonella or C. jejuni antigens. The probiotic treatments did not affect lymphocyte proliferation to C. jejuni antigens, but significantly increased proliferation of lymphocytes to Salmonella antigens by 68 and 55%, respectively, over untreated mice. Caspase 3/7 activation was significantly reduced 33 and 38% in the T and B lymphocyte fractions, respectively, of probiotic-treated, Salmonella-challenged HMA BALB/c mice, suggesting that lymphocyte rescue from apoptosis was occurring as a result of probiotic bacteria activity. These results revealed an immunosuppressive activity by Salmonella that was inhibited by the presence of probiotic bacteria. In summary, lactobacilli and bifidobacteria competitively excluded C. jejuni from immunocompetent and immunodeficient mice and antagonized an observable Salmonella-induced immunosuppression in immunocompetent mice.

We assessed the capacity of four probiotic bacteria (Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus casei GG, and Bifidobacterium animalis) to colonize, infect, stimulate immune responses in, and affect the growth and survival of congenitally immunodeficient gnotobiotic beige-athymic (bg/bg-nu/nu) and beige-euthymic (bg/bg-nu/؉) mice. The bacteria colonized and persisted, in pure culture, in the alimentary tracts of both mouse strains for the entire study period (12 weeks). Although all adult and neonatal beige-euthymic mice survived probiotic colonization, some infant mortality occurred in beige-athymic pups born to mothers colonized with pure cultures of L. reuteri or L. casei GG. The probiotic bacteria manifested different capacities to adhere to epithelial surfaces, disseminate to internal organs, affect the body weight of adult mice and the growth of neonatal mice, and stimulate immune responses. Although the probiotic species were innocuous for adults, these results suggest that caution and further studies to assess the safety of probiotic bacteria for immunodeficient hosts, especially neonates, are required.