http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_1 The avian influenza (AI) virus is type A influenza isolated from and adapted to an avian host. Type A influenza belongs to the orthomyxovirdae virus family, is enveloped, and is pleiomorphic with a size ranging from 80–120 nm (reviewed in [1]). Type A influenza strains are classified by the serological subtypes of the primary viral surface proteins, the hemagglutinin (HA) and neuraminidase (NA). The HA has 16 subtypes (H1–H16) and contains neutralizing epitopes. Antibodies against the NA are not neutralizing, and there are nine neuraminidase or “N” subtypes. The “H” and N subtypes seem to be able to assort into any combination, and many of the 144 possible combinations have been found in natural reservoir species, although some combinations are more common than others. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_10 Immunohistochemical methods are commonly used for studying the pathogenesis of the avian influenza (AI) virus by allowing the identification of sites of replication of the virus in infected tissues and the correlation with the histopathological changes observed. In this chapter, the materials and methods for performing immunohistochemical detection of AI virus antigens in tissues are provided. The technique involves the following steps: heat-induced antigen retrieval; binding of a primary antibody to a virus type-specific antigen; antibody-antigen complex binding by a biotinylated secondary antibody; and binding of an enzyme-streptavidin conjugate. The enzyme is then visualized by application of the substrate chromogen solution to produce a colorimetric end product. Demonstration of AI virus antigen in tissues is based on chromogen deposition in the nucleus and/or cytoplasm of infected cells. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_11 Avian influenza (AI) viruses have been isolated from a wide diversity of free-living avian species representing several orders. Isolations are most frequently reported from aquatic birds in the Orders Anseriformes and Charadriiformes, which are believed to be the reservoirs for all AI viruses. Since their first recognition in the late 1800 s, AI viruses have been an important agent of disease in poultry and, occasionally, of nongallinaceous birds and humans. However, the recent highly pathogenic avian influenza (HPAI) H5N1 virus epidemics have increased the awareness of AI viruses and their potential implications among the scientific community, politicians, and the general public. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_12 Reverse genetics is the creation of a virus from a full-length cDNA copy of the viral genome, referred to as an infectious clone, and is the most powerful genetic tool in modern virology. The generation of influenza A viruses by reverse genetics has lagged mainly due to the inherent technical difficulties of providing a sufficient amount of all eight viral RNAs from cloned cDNA. A breakthrough was made in 1999 by utilizing the cellular enzyme RNA polymerase I for the synthesis of influenza viral RNAs. Although slightly different methods are being used in different laboratories for the rescue of the influenza virus, the basic concept of synthesizing viral RNA using RNA polymerase I remains the same. Coupled with in vitro mutagenesis, reverse genetics can be applied widely to accelerate progress in understanding the influenza virus life cycle, the generation of live-attenuated vaccines, and the use of influenza virus as vaccine and gene delivery vectors. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_13 The measurement of avian cellular immunity is critical to understanding the role and regulation of avian lymphocytes following avian influenza (AI) virus infection. Although the ability to measure avian T cell responses has steadily increased over the last few years, few studies have examined the role of cell-mediated immunity in avian species against the AI virus. Because of the structural and functional differences between mammalian and avian immune systems—including MHC architecture, different modes of somatic recombination for antibody production, and the absence of lymph nodes in birds—the extent to which birds and mammals regulate similar immune responses against the AI virus is currently under investigation. The increasing availability of monoclonal antibodies recognizing avian T cell-associated antigens as well as a number of inbred lines of chickens with genetically defined MHC haplotypes make this an important field of research for the future. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_14 Functional and molecular techniques have both been employed to measure the production of cytokines following influenza infection. Historically, the use of functional or antibody-based techniques was employed in mammalian immunology. In avian immunology, only a few commercial antibodies are available to measure avian cytokines. However, the determination of the genomic sequence of Gallus gallus species has made it possible to measure cytokine induction without monoclonal antibody- or functional-based tests, but rather based on molecular techniques. Although these tests do not measure functionally expressed cytokines, the lack of reagents to identify and quantify avian cytokines makes them critical to extend any measure of cytokine response. Measurement of cytokine induction, based on the design of primers and probes for RT-PCR or real-time RT-PCR for the cytokine mRNA, has become one of the more recent technologies reported to measure avian cytokines. It is important to note that small nucleotide polymorphisms between different lines of birds may result in substandard results when using published primer and probe sequences. This requires empirical testing to ensure adequate results. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_2 Successful detection of the avian influenza (AI) virus, viral antigen, nucleic acid, or antibody is dependent upon the collection of the appropriate sample type, the quality of the sample, and the proper storage and handling of the sample. The diagnostic tests to be performed should be considered prior to sample collection. Sera are acceptable samples for ELISA or agar gel precipitin tests, but not for real-time RT-PCR. Likewise, swabs and/or tissues are acceptable for real-time RT-PCR and virus isolation. The sample type will also depend on the type of birds that are being tested; oropharyngeal swabs should be collected from poultry, and cloacal swabs should be collected from waterfowl. This chapter will outline the collection of different specimen types and procedures for proper specimen handling. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_3 The efficient extraction and purification of viral RNA are critical for downstream molecular applications, whether it is the sensitive and specific detection of virus in clinical samples, virus gene cloning and expression, or quantification of the avian influenza (AI) virus by molecular methods from experimentally infected birds. Samples can generally be divided into two types: enriched (e.g., virus stocks) and clinical. Clinical type samples, which may be tissues or swab material, are the most difficult to process due to the complex sample composition and possibly low virus titers. In this chapter, two well-established procedures for the isolation of AI virus RNA from common clinical specimen types and enriched virus stocks for further molecular applications will be presented. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_4 Real-time RT-PCR (rRT-PCR) is a relatively new technology that has been used for avian influenza (AI) virus detection since the early 2000s for routine surveillance, during outbreaks, and for research. Some of the advantages of rRT-PCR are high sensitivity, high specificity, rapid time-to-result, scalability, cost, and quantitative nature. Furthermore, rRT-PCR can be used with numerous sample types, is less expensive than virus isolation in chicken embryos, and since infectious virus is inactivated early during processing, biosafety and biosecurity are also easier to maintain. This chapter will provide an overview of the USDA-validated rRT-PCR procedure for the detection of type A influenza. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_5 Serological methods, gene sequencing, and RT-PCR-based methods have all been used for the identification of influenza virus hemagglutinin (HA) subtypes. Compared to serological methods and gene sequencing, RT-PCR is fast, sensitive, and relatively inexpensive. However, since RT-PCR generally lacks the specificity of sequencing or serology, the most practical application of RT-PCR methods for subtype identification is either to target a few of the most important subtypes such as H5 and H7 or to use it in situations where a specific strain is being targeted, such as during an outbreak or with experimental samples. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_6 The avian influenza (AI) virus is usually isolated and propagated by inoculating either swab or tissue samples from infected birds into the chorioallantoic sac of embryonating chicken eggs. This is the accepted method, but occasionally an isolation may only be successful when inoculated either into the yolk sac or onto the chorioallantoic membrane of embryonating chicken eggs. Chorioallantoic fluid is harvested from eggs with dead or dying embryos and is tested for the presence of hemagglutinating antigen. If hemagglutination-positive, this indicates that the isolate may be the AI virus. The presence of the AI virus may be confirmed by either an agar gel immunodiffusion (AGID) assay, RT-PCR specific for AI virus, or a commercially available immunoassay kit specific for type A influenza. Instructions for AI virus primary isolation and propagation, preparing antigen for an AGID test, setting up an AGID test, and interpreting results are given. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_7 The hemagglutination (HA) assay is a tool used to screen cell culture or amnioallantoic fluid harvested from embryonating chicken eggs for hemagglutinating agents, such as type A influenza. The HA assay is not an identification assay, as other agents also have hemagglutinating properties. Live and inactivated viruses are detected by the HA test. Amplification by virus isolation in embryonating chicken eggs or cell culture is typically required before HA activity can be detected from a clinical sample. The test is, to some extent, quantitative [1 hemagglutinating unit (HAU) is equal to approximately 5–6 logs of virus]. It is inexpensive and relatively simple to conduct. Several factors (quality of chicken erythrocytes, laboratory temperature, laboratory equipment, technical expertise of the user) may contribute to slight differences in the interpretation of the test each time it is run. This chapter will describe the methods validated and used by the National Veterinary Services Laboratories (NVSL) for screening and identification of hemagglutinating viruses. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_8 The hemagglutination-inhibition (HI) assay is a classical laboratory procedure for the classification or subtyping of hemagglutinating viruses. For the avian influenza (AI) virus, the HI assay is used to identify the hemagglutinin (H) subtype of an unknown AI virus isolate or the HA subtype specificity of antibodies to AI virus. Since the HI assay is quantitative, it is frequently applied to evaluate the antigenic relationships between different AI virus isolates of the same subtype. The basis of the HI test is inhibition of hemagglutination with subtype-specific antibodies. The HI assay is a relatively inexpensive procedure utilizing standard laboratory equipment, is less technical than molecular tests, and is easily completed within several hours. However, when working with uncharacterized viruses or antibody subtypes, the library of reference reagents required for identifying antigentically distinct AI viruses and/or antibody specificities from multiple lineages of a single hemagglutinin subtype requires extensive laboratory support for the production and optimization of reagents. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-279-3_9 The neuraminidase-inhibition (NI) assay is a laboratory procedure for the identification of the neuraminidase (NA) glycoprotein subtype in influenza viruses or the NA subtype specificity of antibodies to influenza virus. A serological procedure for subtyping the NA glycoprotein is critical for the identification and classification of avian influenza (AI) viruses. The macro-procedure was first described in 1961 by D. Aminoff et al. [2] and was later modified to a microtiter plate procedure (micro-NI) by Van Deusen et al. [4]. The micro-NI procedure reduces the quantity of reagents required, permits the antigenic classification of many isolates simultaneously, and eliminates the spectrophotometric interpretation of results. Although the macro-NI has been shown to be more sensitive than the micro-NI, the micro-NI test is very suitable for testing sera for the presence of NA antibodies and has proven to be a practical and rapid method for virus classification. This chapter will provide an overview of the USDA-validated micro-NI procedure for the identification of subtype-specific NA in AIV and antibodies. 2008-02-08T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-468-1_1 Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen responsible for both hospital- and community-onset disease. Resistance to methicillin in S. aureus is mediated by PBP2a, a penicillin-binding protein with low affinity to β-lactams, encoded by the mecA gene. Accurate susceptibility testing of S. aureus isolates and screening of patients for colonization with MRSA are important tools to limit the spread of this organism. This review focuses on the clinical significance of MRSA infections and new approaches for the laboratory diagnosis and epidemiological typing of MRSA strains. 2007-09-27T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-468-1_10 As with many other bacterial species, the most commonly used method to assess staphylococcal biofilm formation in vitro is the microtiter plate assay. This assay is particularly useful for comparison of multiple strains including large-scale screens of mutant libraries. When such screens are applied to the coagulase-negative staphylococci in general, and Staphylococcus epidermidis in particular, they are relatively straightforward by comparison with microtiter plate assays used to assess biofilm formation in other bacterial species. However, in the case of clinical isolates of Staphylococcus aureus, including methicillin-resistant S. aureus, we have found it necessary to employ specific modifications including precoating of the wells of the microtiter plate with plasma proteins and supplementation of the medium with both salt and glucose. In this chapter, we describe the microtiter plate assay in the specific context of clinical isolates of S. aureus and the use of these modifications. A second in vitro method, which also is generally dependent on coating with plasma proteins and supplementation of the growth medium, is the use of flow cells. In this method, bacteria are allowed to attach to a surface and then monitored with respect to their ability to remain attached to the substrate and differentiate into mature biofilms under the constant pressure of fluid shear force. Although flow cells are not applicable to large-scale screens, we have found that they provide a more reproducible and accurate assessment of the capacity of S. aureus clinical isolates to form a biofilm. They also provide a means of analyzing structural differences in biofilm architecture and isolating bacteria and/or spent media for analysis of physiological and metabolic changes associated with the adaptive response to growth in a biofilm. While a primary focus of this chapter is on the use of in vitro assays to assess biofilm formation in clinical isolates of S. aureus, it is important to emphasize two additional considerations. First, it has become increasingly evident that biofilm formation in S. epiderimidis and S. aureus is not equivalent. Additionally, to date, most studies with S. aureus have been done with a very limited number of strains, almost all of which are derived from the NCTC strain designated 8325, and we have found that these strains are not representative of the most relevant clinical isolates. As with the specific elements of our flow cell system, we have written this chapter to reflect our focus on clinical isolates of S. aureus and the specific methods that we have found most reliable in that context. Second, as is often the case, in vitro methods do not necessarily reflect events that occur in vivo. Several in vivo methods to assess biofilm formation have been described, and these generally fall into one of two categories. The first focuses directly on staphylococcal diseases that are generally thought to include a biofilm component (e.g., endocarditis, osteomyelitis, septic arthritis). A discussion of these models is also beyond the scope of this chapter, but examples are easily found in the staphylococcal literature. The second approach uses some form of implanted device in an attempt to focus more directly on implant-associated biofilms. We use a model in which a small piece of Teflon catheter is implanted subcutaneously in mice and used as a substrate for colonization. We have the advantage of using bioluminescent derivatives of S. aureus clinical isolates and the IVIS® imaging system. However, because this system is not generally available, we restrict technical comments in this chapter to our use of an implanted catheter model evaluated by direct microbiological analysis of explanted catheters (2). 2007-09-27T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-468-1_11 Multiple drug resistance to antibiotics is a major public health problem. Many mechanisms may be involved in such resistance. Increasing data have shown that Staphylococcus aureus can invade different types of nonphagocytic cells, which, in turn, may contribute to evasion of the toxicity of certain antibiotics. The fibronectin-binding proteins are required for S. aureus to adhere to and internalize into the host cells. We have shown that a two-component signal transduction system, SaeRS, is essential for bacterial adhesion and invasion of the epithelial cells. 2007-09-27T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-468-1_12 This chapter explains computer techniques for comparing genes, proteins, or genomes of methicillin-resistant Staphylococcus aureus (MRSA). In the principle methodology for comparative genomics, first researchers obtain the data of DNA sequences and phenotypes for various strains of interest, and from those they infer what difference/similarity in sequences results in what difference/similarity in phenotypes. Usually, the obtained hypothesis provides guidance for the succeeding biological experiments, such as producing knockout strains or conducting transcriptome or proteome analysis, which examine the hypothesis. Even for physicians and experimental researchers, these computer-aided researches would be needed in order to understand the physiological characteristics and pathogenic abilities of the MRSA that they deal with in this “genome era.” This chapter involves no experiments and is confined to computer analysis. We explain methods for extracting the difference/similarity between sequences of nucleotide, amino acid, or even the whole genomes of bacteria. We also introduce how to compare the pathways between strains that possess different sets of genes. 2007-09-27T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-468-1_13 The microarray has shown tremendous potential for investigating gene expression profiles and expression levels in comparative biology; exploring the regulation mechanisms of gene expression; and evaluating target gene for developing new chemotherapeutic agents, vaccine, and diagnostic methods. In this chapter, we provide a detailed protocol for scientists who wish to investigate gene expression profiles by performing a microarray analysis, including different methods of RNA purification, decontamination, cDNA synthesis, fragmentation, and biotin labeling for hybridization using Affymetrix Staphylococcus aureus chips. 2007-09-27T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-468-1_14 Over the past decade numerous genomes of pathogenic bacteria were fully sequenced and annotated, while others are continuously being sequenced and published. To date, the sequences of > 440 bacterial genomes are publicly available for research purposes. These efforts in high-throughput sequencing parallel major improvements in methods permitting the study of whole transcriptome and proteome of bacteria. This provides a basis for a comprehensive understanding of the bacterial metabolism, adaptability to the environment, regulation, resistance pathways, and pathogenicity mechanisms of pathogens. Staphylococcus aureus is a Gram-positive human pathogen causing a wide variety of infections ranging from benign skin infections to life-threatening diseases. Furthermore, the spreading of multiresistance strains requiring the use of last-barrier drugs has resulted in the medical and scientific community focusing particularly on this pathogen. We describe here proteomic methods to prepare, identify, and analyze protein fractions, allowing the study of S. aureus on the organism level. Coupled with methods analyzing the whole bacterial transcriptome, this approach might contribute to the development of rapid diagnostic tests and to the identification of new drug targets. 2007-09-27T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-468-1_15 The role of the inanimate environment, including the air, in the transmission of methicillin-resistant Staphylococcus aureus (MRSA) infection is unclear; however, there are certain situations when evaluation of MRSA contamination of the environment is indicated. At this point, conventional culture methods are predominantly used, with molecular methods reserved for characterization of recovered isolates. A variety of methods are available for environmental sampling, and the objectives of sampling must be considered when choosing the appropriate technique. 2007-09-27T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-468-1_16 Methicillin-resistant Staphylococcus aureus (MRSA) has posed an immense problem for clinicians in the hospital setting for years, emerging as the most frequent nosocomial infection. To deal with this problem pathogen and others, infectious disease specialists have developed a variety of procedures for their control and prevention, involving options from preventative measures such as decolonization and isolation of MRSA-confirmed patients, to the more simple procedures of hand washing, expanding glove use, and reducing time in the hospital. With the realization that MRSA is now a community problem, there are expanded efforts toward more direct intervention, such as the use of anti-MRSA antibacterials and vaccines, in an attempt to reduce the overall burden of MRSA. 2007-09-27T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-468-1_17 We review data on the treatment of infections caused by drug-resistant Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA). In this review, we cover findings reported in the English language medical literature up to February 2006. Despite the emergence of resistant and multidrug resistant S. aureus, five effective drugs for which little resistance has been observed are in clinical use: vancomycin, quinupristin-dalfopristin, linezolid, tigecycline, and daptomycin. However, vancomycin is less effective for infections with MRSA isolates that have a high minimum inhibitory concentration in the susceptible range. Linezolid looks promising in the treatment of MRSA pneumonia and skin and soft-tissue infections (SSTIs). Daptomycin displays rapid bactericidal activity in vitro, and it has been shown to be noninferior to comparator agents in the treatment of SSTIs and bacteremia. Tigecycline was also noninferior to comparator drugs in the treatment of SSTIs. Clindamycin, trimethoprim-sulfamethoxazole, doxycycline, and minocycline are oral antistaphylococcal agents that may have utility in the treatment of SSTIs and osteomyelitis, but the clinical data for their efficacy is limited. There are four drugs with broad-spectrum activity against Gram-positive organisms at an advanced stage of clinical testing: ceptobiprole and three new glycopeptides with potent bactericidal activity, oritavancin, dalbavancin, and telavancin. Thus, there are currently many effective drugs to treat resistant S. aureus infections and many promising agents in the pipeline. Nevertheless, S. aureus remains a formidable adversary against which there are frequent treatment failures. The next goals are to determine the most appropriate indications and cost-effectiveness of each of these drugs in the treatment strategy against S. aureus. 2007-09-27T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-468-1_2 Staphylococcus aureus is a versatile pathogen associated with diverse clinical presentations. Only recently have the genetic factors underlying the virulence of this bacterial species become understood in a significant way. Methicillin-resistant S. aureus (MRSA) strains have been extremely important as nosocomial pathogens in health care facilities for more than three decades. Additionally, infections resulting from community-associated MRSA strains have emerged in the last decade and become a public health problem of global proportions. This changing epidemiology has spurred renewed interest in translating knowledge of the molecular determinants of virulence into rational prevention and control strategies. Four case histories are provided (three involving MRSA and one involving a methicillin-sensitive strain of S. aureus) that highlight the diversity of clinical presentations and relative virulence of S. aureus infections in humans. The molecular characterization of clonality and virulence gene profile is compared among the four cases. Significant genetic diversity exists among MRSA and sensitive strains of S. aureus. It is obvious that various combinations of virulence factors contribute to disease manifestations of infected patients. 2007-09-27T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-468-1_3 The widespread occurrence of methicillin-resistant Staphylococcus aureus (MRSA) or oxacillin-resistant MRSA is a major cause of concern worldwide. Although mainly located in hospital environments, these microorganisms have been reported to have the capacity to cause infections in the community. Resistance to methicillin implies resistance to all β-lactam antibiotics and, furthermore, MRSA isolates normally harbor resistance to other families of antibiotics such as co-trimoxazole and aminoglycosides. Prompt and accurate detection of MRSA isolates is therefore extremely important in clinical microbiology laboratories. In this chapter, we review the most common methods of susceptibility testing for MRSA. 2007-09-27T04:00:00Z