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A zebrafish model was developed to study AIEC colonization, invasion, and inflammation. This model can also be used to study the beneficial effects of a probiotic on AIEC infection of adult zebrafish. Bacteria are grown in vitro and then fish are infected with AIEC by immersion. Subsequently, colonization and inflammation can be assessed. Exposing fish to probiotic at different time points relative to AIEC can determine beneficial effects of probiotics as prophylactics or therapeutics against AIEC. For complete details on the use and execution of this protocol, please refer to Nag et al. (2022).
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Infecciones por Escherichia coli , Pez Cebra , Animales , Escherichia coli , Adhesión Bacteriana , Infecciones por Escherichia coli/terapia , Inflamación/microbiologíaRESUMEN
Adherent-invasive Escherichia coli (AIEC) is an opportunistic pathogen associated with major inflammatory bowel disease, Crohn disease, and ulcerative colitis. Unfavorable conditions push commensal AIEC to induce gut inflammation, sometimes progressing to inflammation-induced colon cancer. Recently, zebrafish have emerged as a useful model to study human intestinal pathogens. Here, a zebrafish model to study AIEC infection was developed. Bath inoculation with AIEC resulted in colonization and tissue disruption in the zebrafish intestine. Gene expression of pro-inflammatory markers including interleukin-1ß (IL-1ß), tumor necrosis factor alpha (TNFα), interferon-γ (IFNγ), and S100A-10b (akin to human calprotectin) in the zebrafish intestine was significantly induced by AIEC infection. The probiotic E. coli Nissle 1917 (EcN) was tested as a therapeutic and prophylactic against AIEC infection and reduced AIEC colonization, tissue damage, and pro-inflammatory responses in zebrafish. Furthermore, EcN diminished the propionic-acid-augmented hyperinfection of AIEC in zebrafish. Thus, this study shows the efficacy of EcN against AIEC in an AIEC-zebrafish model.
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Vibrio cholerae, the cause of human cholera, is an aquatic bacterium found in association with a variety of animals in the environment, including many teleost fish species. V. cholerae infection induces a proinflammatory response followed by a noninflammatory convalescent phase. Neutrophils are integral to this early immune response. However, the relationship between the neutrophil-associated protein calprotectin and V. cholerae has not been investigated, nor have the effects of limiting transition metals on V. cholerae growth. Zebrafish are useful as a natural V. cholerae model as the entire infectious cycle can be recapitulated in the presence of an intact intestinal microbiome and mature immune responses. Here, we demonstrate that zebrafish produce a significant neutrophil, interleukin 8 (IL-8), and calprotectin response following V. cholerae infection. Bacterial growth was completely inhibited by purified calprotectin protein or the chemical chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), but growth was recovered by the addition of the transition metals zinc and manganese. The expression of downstream calprotectin targets was also significantly increased in the zebrafish. These findings illuminate the role of host calprotectin in combating V. cholerae infection. Inhibition of V. cholerae growth through metal limitation may provide new approaches in the development of anti-V. cholerae therapeutics. This study also establishes a major role for calprotectin in combating infectious diseases in zebrafish.
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Cólera , Vibrio cholerae , Animales , Cólera/microbiología , Complejo de Antígeno L1 de Leucocito , Neutrófilos , Vibrio cholerae/fisiología , Pez CebraRESUMEN
The gram-negative bacterium Vibrio cholerae causes the life-threatening diarrheal disease cholera, which is spread through the ingestion of contaminated food or water. Cholera epidemics occur largely in developing countries that lack proper infrastructure to treat sewage and provide clean water. Numerous vertebrate fish species have been found to be natural V. cholerae hosts. Based on these findings, zebrafish (Danio rerio) have been developed as a natural host model for V. cholerae. Diarrheal symptoms similar to those seen in humans are seen in zebrafish as early as 6 hours after exposure. Our understanding of basic zebrafish immunology is currently rudimentary, and no research has been done to date exploring the immune response of zebrafish to V. cholerae infection. In the present study, zebrafish were infected with either pandemic El Tor or non-pandemic, environmental V. cholerae strains and select immunological markers were assessed to determine cellular immunity and humoral immunity. Significant increases in the gene expression of two transcription factors, T-bet and GATA3, were observed in response to infection with both V. cholerae strains, as were levels of mucosal related antibodies. Additionally, the cytokine IL-13 was shown to be significantly elevated and paralleled the mucin output in zebrafish excretions, strengthening our knowledge of IL-13 induced mucin production in cholera. The data presented here further solidify the relevancy of the zebrafish model in studying V. cholerae, as well as expanding its utility in the field of cholera immunology.
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Cólera , Vibrio cholerae , Animales , Diarrea , Humanos , Inmunidad , Pez CebraRESUMEN
Vibrio is a large and diverse genus of bacteria, of which most are nonpathogenic species found in the aquatic environment. However, a subset of the Vibrio genus includes several species that are highly pathogenic, either to humans or to aquatic animals. In recent years, Danio rerio, commonly known as the zebrafish, has emerged as a major animal model used for studying nearly every aspect of biology, including infectious diseases. Zebrafish are especially useful because the embryos are transparent, larvae are small and facilitate imaging studies, and numerous transgenic fish strains have been constructed. Zebrafish models for several pathogenic Vibrio species have been described, and indeed a fish model is highly relevant for the study of aquatic bacterial pathogens. Here, we summarize the zebrafish models that have been used to study pathogenic Vibrio species to date.
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Modelos Animales de Enfermedad , Vibriosis/microbiología , Vibrio/fisiología , Pez Cebra , Animales , Humanos , Vibrio/clasificación , Vibrio/genética , Vibrio/patogenicidad , Virulencia , Pez Cebra/microbiologíaRESUMEN
The authors wish to correct the following erratum in this paper [...].
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PURPOSE: Zika virus (ZIKV) has emerged as an important human pathogen causing ocular complications. There have been reports of the shedding of ZIKV in human as well as animal tears. In this study, we investigated the infectivity of ZIKV in corneal epithelial cells and their antiviral immune response. METHODS: Primary human corneal epithelial cells (Pr. HCECs) and an immortalized cell line (HUCL) were infected with two different strains of ZIKV (PRVABC59 & BeH823339) or dengue virus (DENV, serotypes 1-4). Viral infectivity was assessed by immunostaining of viral antigen and plaque assay. qRT-PCR and immunoblot analyses were used to assess the expression of innate inflammatory and antiviral genes. Supplementation of recombinant ISG15 (rISG15) and gene silencing approaches were used to elucidate the role of ISG15 in corneal antiviral defense. RESULTS: Pr. HCECs, but not the HUCL cells, were permissive to both ZIKV strains and specifically to DENV3 infection. ZIKV induced the expression of viral recognition receptors (TLR3, RIG-I, &MDA5), and genes involved in inflammatory (CXCL10 & CCL5) and antiviral (IFNs, MX1, OAS2, ISG15) responses in Pr. HCECs. Furthermore, ZIKV infection caused Pr. HCECs cell death, as evidenced by TUNEL staining. Silencing of ISG15 increased ZIKV infectivity while supplementation with rISG15 reduced ZIKV infection by direct inactivation of ZIKV and inhibiting its entry. CONCLUSIONS: Our study demonstrates for the first time, that ZIKV can readily infect and replicate in Pr. HCECs. Therefore, ZIKV may persist in the cornea and pose the potential risk of transmission via corneal transplantation.