RESUMO
We tested the efficiency of 2 different antibiotics, rifampicin and oxolinic acid, against an established infection caused by fish pathogen Francisella noatunensis ssp. orientalis (F.n.o.) in zebrafish. The drugs were tested in the free form as well as encapsulated into biodegradable nanoparticles, either polylactic-co-glycolic acid (PLGA) nanoparticles or nanostructured lipid carriers. The most promising therapies were PLGA-rifampicin nanoparticles and free oxolinic acid; the PLGA nanoparticles significantly delayed embryo mortality while free oxolinic acid prevented it. Encapsulation of rifampicin in both PLGA and nanostructured lipid carriers enhanced its efficiency against F.n.o. infection relative to the free drug. We propose that the zebrafish model is a robust, rapid system for initial testing of different treatments of bacterial diseases important for aquaculture.
Assuntos
Antibacterianos/uso terapêutico , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/veterinária , Ácido Láctico/química , Lipídeos/química , Nanopartículas/química , Ácido Poliglicólico/química , Animais , Antibacterianos/administração & dosagem , Doenças dos Peixes/tratamento farmacológico , Francisella , Ácido Oxolínico/administração & dosagem , Ácido Oxolínico/uso terapêutico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Rifampina/administração & dosagem , Rifampina/uso terapêutico , Peixe-ZebraRESUMO
There is an urgent need for more efficient viral vaccines in finfish aquaculture worldwide. Here, we report the use of poly(I:C) stabilized with chitosan as an adjuvant for development of better finfish vaccines. The adjuvant was co-injected with inactivated viral hemorrhagic septicemia virus (VHSV) (CSpIC+iV vaccine) in adult zebrafish and its efficiency in protection against VHSV infection was compared to a live, attenuated VHS virus vaccine (aV). Both free and stabilized poly(I:C) were strong inducers of an antiviral state, measured by transcriptional activation of the genes of viral sensors: toll-like receptors, interferons, and interferon-stimulated genes, such as MXa within 48 h after injection. Both the CSpIC+iV and the aV formulations provided a significant protection against VHSV-induced mortality. However, when plasma from survivors was tested for neutralizing antibodies in an in vitro protection assay, we could not demonstrate any protective effect. On the contrary, plasma from aV vaccinated fish enhanced cytopathic effects, indicating that antibody-dependent entry may play a role in this system. Our results show that poly(I:C) is a promising candidate as an adjuvant for fish vaccination against viral pathogens, and that the zebrafish is a promising model for aquaculture-relevant vaccination studies.
Assuntos
Quitosana/química , Septicemia Hemorrágica Viral/prevenção & controle , Novirhabdovirus/imunologia , Polinucleotídeos/imunologia , Vacinas Virais/imunologia , Peixe-Zebra , Adjuvantes Imunológicos , Animais , Células Cultivadas , Rim Cefálico/citologia , Rim Cefálico/metabolismo , Septicemia Hemorrágica Viral/virologia , Poli I-C , Polinucleotídeos/químicaRESUMO
Nanoparticles (NPs) enclosing antibiotics have provided promising therapy against Mycobacterium tuberculosis (Mtb) in different mammalian models. However, the NPs were not visualized in any of these animal studies. Here, we introduce the transparent zebrafish embryo as a system for noninvasive, simultaneous imaging of fluorescent NPs and the fish tuberculosis (TB) agent Mycobacterium marinum (Mm). The study was facilitated by the use of transgenic lines of macrophages, neutrophils, and endothelial cells expressing fluorescent markers readily visible in the live vertebrate. Intravenous injection of Mm led to phagocytosis by blood macrophages. These remained within the vasculature until 3 days postinfection where they started to extravasate and form aggregates of infected cells. Correlative light/electron microscopy revealed that these granuloma-like structures had significant access to the vasculature. Injection of NPs induced rapid uptake by both infected and uninfected macrophages, the latter being actively recruited to the site of infection, thereby providing an efficient targeting into granulomas. Rifampicin-loaded NPs significantly improved embryo survival and lowered bacterial load, as shown by quantitative fluorescence analysis. Our results argue that zebrafish embryos offer a powerful system for monitoring NPs in vivo and rationalize why NP therapy was so effective against Mtb in earlier studies; bacteria and NPs share the same cellular niche.
Assuntos
Portadores de Fármacos/química , Embrião não Mamífero/microbiologia , Mycobacterium marinum/efeitos dos fármacos , Nanopartículas/química , Imagem Óptica , Peixe-Zebra/embriologia , Peixe-Zebra/microbiologia , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Transporte Biológico , Cumarínicos/química , Portadores de Fármacos/metabolismo , Granuloma/microbiologia , Ácido Láctico/química , Macrófagos/microbiologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/fisiologia , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Rodaminas/química , Rifampina/química , Rifampina/farmacologia , Tiazóis/química , Tuberculose/microbiologia , Tuberculose/veterináriaRESUMO
Francisella spp. are facultative intracellular pathogens identified in increasingly diverse hosts, including mammals. F. noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis infect fish inhabiting warm and cold waters, respectively, while F. tularensis subsp. novicida is highly infectious for mice and has been widely used as a model for the human pathogen F. tularensis. Here, we established zebrafish embryo infection models of fluorescently labeled F. noatunensis subsp. noatunensis, F. noatunensis subsp. orientalis, and F. tularensis subsp. novicida at 22, 28, and 32°C, respectively. All infections led to significant bacterial growth, as shown by reverse transcription-quantitative PCR (RT-qPCR), and to a robust proinflammatory immune response, dominated by increased transcription of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß). F. noatunensis subsp. orientalis was the most virulent, F. noatunensis subsp. noatunensis caused chronic infection, and F. tularensis subsp. novicida showed moderate virulence and led to formation of relatively small granuloma-like structures. The use of transgenic zebrafish strains with enhanced green fluorescent protein (EGFP)-labeled immune cells revealed their detailed interactions with Francisella species. All three strains entered preferentially into macrophages, which eventually assembled into granuloma-like structures. Entry into neutrophils was also observed, though the efficiency of this event depended on the route of infection. The results demonstrate the usefulness of the zebrafish embryo model for studying infections caused by different Francisella species at a wide range of temperatures and highlight their interactions with immune cells.
Assuntos
Modelos Animais de Doenças , Doenças dos Peixes/microbiologia , Francisella , Infecções por Bactérias Gram-Negativas/microbiologia , Temperatura , Peixe-Zebra/embriologia , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/patologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/patologiaRESUMO
Nanoparticles (NPs) are increasingly used as biodegradable vehicles to selectively deliver therapeutic agents such as drugs or antigens to cells. The most widely used vehicle for this purpose is based on copolymers of lactic acid and glycolic acid (PLGA) and has been extensively used in experiments aimed at delivering antibiotics against Mycobacterium tuberculosis in animal models of tuberculosis. Here, we describe fabrication of PLGA NPs containing either a high concentration of rifampicin or detectable levels of the green fluorescent dye, coumarin-6. Our goal here was twofold: first to resolve the controversial issue of whether, after phagocytic uptake, PLGA NPs remain membrane-bound or whether they escape into the cytoplasm, as has been widely claimed. Second, we sought to make NPs that enclosed sufficient rifampicin to efficiently clear macrophages of infection with Mycobacterium bovis BCG. Using fluorescence microscopy and immuno-electron microscopy, in combination with markers for lysosomes, we show that BCG bacteria, as expected, localized to early phagosomes, but that at least 90% of PLGA particles were targeted to, and remained in, low pH, hydrolase-rich phago-lysosomes. Our data collectively argue that PLGA NPs remain membrane-enclosed in macrophages for at least 13 days and degrade slowly. Importantly, provided that the NPs are fabricated with sufficient antibiotic, one dose given after infection is sufficient to efficiently clear the BCG infection after 9-12 days of treatment, as shown by estimates of the number of bacterial colonies in vitro.