Current Protocols in Microbiology最新文献

Immunoelectron microscopy is a powerful technique for identifying viral antigens and determining their structural localization and organization within vaccines and viruses. While traditional negative staining transmission electron microscopy provides structural information, identity of components within a sample may be confounding. Immunoelectron microscopy allows for identification and visualization of antigens and their relative positions within a particulate sample. This allows for simple qualitative analysis of samples including whole virus, viral components, and viral-like particles. This article describes methods for immunogold labeling of viral antigens in a liquid suspension, with examples of immunogold-labeled influenza virus glycoproteins, and also discusses the important considerations for sample preparation and determination of morphologies. Together, these methods allow for understanding the antigenic makeup of viral particulate samples, which have important implications for molecular virology and vaccine development. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cover: In Frazer et al. (https://doi.org/10.1002/cpmc.76), Alternative media for white-opaque switching assays. (A) On YPD + phloxine B opaque cells take up the dye and show pink colonies after incubation for 7 days at room temperature. Scale bar, 2 mm. (B) White and opaque colony phenotypes on CHROMagar Candida medium. Scale bar, 1 mm. W, white; O, opaque.

Eimeria is a genus of apicomplexan parasites that contains a large number of species, most of which are absolutely host-specific. Seven species have been recognized to infect chickens. Infection of susceptible chickens results in an intestinal disease called coccidiosis, characterized by mucoid or hemorrhagic enteritis, which is associated with impaired feed conversion or mortality in severe cases. Intensive farming practices have increased the significance of coccidiosis since parasite transmission is favored by high-density housing of large numbers of susceptible chickens. Routine chemoprophylaxis and/or vaccination with live parasite vaccines provides effective control of Eimeria, although the emergence of drug resistance and the relative cost and production capacity of current vaccine lines can prove limiting. As pressure to reduce drug use in livestock production intensifies, novel vaccination strategies are needed. Development of effective protocols supporting genetic complementation of Eimeria species has until recently been hampered by their inability to replicate efficiently in vitro. Now, the availability of such protocols has raised the prospect of generating transgenic parasite lines that function as vaccine vectors to express and deliver heterologous antigens. For example, this technology has the potential to streamline the production of live anticoccidial vaccines through the generation of parasite lines that co-express immunoprotective antigens derived from multiple Eimeria species. In this paper we describe detailed protocols for genetic manipulation, laboratory growth, and in vivo propagation of Eimeria tenella parasites, which will encourage future work from other researchers to expand biological understanding of Eimeria through reverse genetics. © 2019 by John Wiley & Sons, Inc.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis, which kills 200,000 individuals worldwide each year. It is ubiquitous in the environment and is first inhaled into the lungs of the host, where it is taken up by phagocytes. The interaction of these fungal cells with host phagocytes, therefore, is a critical step in the pathogenesis of this disease. One characteristic of this initial step in host–pathogen interactions is the avidity with which fungal cells are taken up by phagocytes, described by the phagocytic index. In this chapter, we detail a high-throughput method of directly assessing the phagocytic index of fungal cells using an imaging-based paradigm. By automating image collection and processing, this method permits rapid assessment of this critical host interaction. © 2019 by John Wiley & Sons, Inc.

Cryptococcus neoformans is an environmental yeast found worldwide that causes lethal brain infections, particularly in immunocompromised hosts. In 2016, there were 280,000 cases of cryptococcal meningitis in the HIV+ population, two-thirds of them fatal; other immunocompromised patients are also affected. The burden of cryptococcal disease and the limits of current chemotherapy create a pressing need for improved treatment. One hindrance to the development of new therapies is lack of understanding of how this pathogen breaches the barriers protecting the brain. Here we describe a tool for investigating this process. This simple in vitro blood-brain-barrier (BBB) model, based on a human brain endothelial cell line grown on a permeable membrane, may be used to assay the BBB transmigration of C. neoformans or other neurotropic pathogens. © 2019 by John Wiley & Sons, Inc.

Candida albicans is an opportunistic human fungal pathogen that is able to cause both mucosal and systemic infections. It is also a frequent human commensal, where it is typically found inhabiting multiple niches including the gastrointestinal tract. One of the most remarkable features of C. albicans biology is its ability to undergo heritable and reversible switching between different phenotypic states, a phenomenon known as phenotypic switching. This is best exemplified by the white-opaque switch, in which cells undergo epigenetic transitions between two alternative cellular states. Here, we describe assays to quantify the frequency of switching between states, as well as methods to help identify cells in different phenotypic states. We also describe the use of environmental cues that can induce switching into either the white or opaque state. Finally, we introduce the use of mNeonGreen and mScarlet fluorescent proteins that have been optimized for use in C. albicans and which outperform commonly used fluorescent proteins for both fluorescence microscopy and flow cytometry. © 2019 by John Wiley & Sons, Inc.

Cryptosporidium is a genus of ubiquitous unicellular parasites belonging to the phylum Apicomplexa. Cryptosporidium species are the second largest cause of childhood diarrhea and are associated with increased morbidity. Accompanying this is the low availability of treatment and lack of vaccines. The major barrier to developing effective treatment is the lack of reliable in vitro culture methods. Recently, our lab has successfully cultivated C. parvum in the esophageal cancer–derived cell line COLO-680N, and has been able to maintain infection for several weeks. The success of this cell line was assessed with a combination of various techniques including fluorescent microscopy and qPCR. In addition, to tackle the issue of long-term oocyst production in vitro, a simple, low-cost bioreactor system using the COLO-680N cell line was established, which produced infectious oocysts for 4 months. This chapter provides details on the methodologies used to culture, maintain, and assess Cryptosporidium infection and propagation in COLO-680N. © 2019 by John Wiley & Sons, Inc.

Microsporidia are eukaryotic unicellular parasites that have been studied for more than 150 years. They are found throughout the world and are capable of infecting various invertebrate and vertebrate hosts. They can cause disease in both immune-compromised and immune-competent humans. In immune-compromised individuals, infections can be severe and often fatal. Microsporidia possess a unique, highly specialized invasion mechanism that involves a structure known as the polar tube as well as the spore wall. During spore germination, the polar tube rapidly discharges from the spore and deliver the sporoplasm into the host cell. Spores are the only stage of microsporidia that can survive outside of host cells. Since the first attempt to culture microsporidia in vitro in 1930s, their cultivation has served a critical role in the study and diagnosis of these parasites. In this chapter, we include methods on the cultivation, isolation, and cryopreservation of Encephalitozoon cuniculi, which can infect humans and provides a useful model for other microsporidia. These methods can also be utilized for the culture of Encephalitozoon hellem or Encephalitozoon intestinalis. © 2018 by John Wiley & Sons, Inc.

Trypanosoma congolense, together with T. vivax and T. brucei, causes African animal trypanosomiasis (AAT), or nagana, a livestock disease carried by bloodsucking tsetse flies in sub-Saharan Africa. These parasitic protists cycle between two hosts: mammal and tsetse fly. The environment offered by each host to the trypanosome is markedly different, and hence the metabolism of stages found in the mammal differs from that of insect stages. For research on new diagnostics and therapeutics, it is appropriate to use the mammalian life cycle stage, bloodstream forms. Insect stages such as procyclics are useful for studying differentiation and also serve as a convenient source of easily cultured, non-infective organisms. Here, we present protocols in current use in our laboratory for the in vitro culture of different life cycle stages of T. congolense—procyclics, epimastigotes, and bloodstream forms—together with methods for transfection enabling the organism to be genetically modified. © 2019 by John Wiley & Sons, Inc.