This project was conducted with funding from the Chemical, Biological, Radiological and Nuclear (CBRN) Research and Technology Initiative (CRTI) of the Canadian Food Inspection Agency, and the Alberta Prion Research Institute. The National Natural Science Foundation of China provided a scholarship (No. 30620120430) to W. Xu. The authors thank Brant Baker and Fred Van Herk, as well as Ruth Barbieri, Lisa Kalischuk-Tymensen, Andrew Olson, Lorna Selinger, Geoff Wallins and Homayoun Zahiroddini for their technical assistance.

Duplicate compost structures were constructed at the Lethbridge Research Centre (LRC) in Lethbridge, Alberta, Canada (Fig. 1). Large rectangular barley straw bales (260 x 120 x 80 cm; L x W x H) were used for the walls, and small bales (100 x 40 x 45 cm) for the floor. Large bales were oriented so walls were 120 cm thick, which maximized wall stability and heat retention. The small bales forming the floor were oriented with twine running horizontally to maximize absorbance in the event of a leak, yielding a floor 45 cm thick. Overall dimensions of the structures were 25 m x 5 m x 2.4 m; L x W x H). The construction site had a slope of approximately 1 , and the structures were oriented to encourage flow of leachate toward the sampling port that we inserted for experimental assessment. Moist feedlot manure was mixed and aerated by processing through a manure spreader and piling for 24 h prior to construction of the containment structures.
The compost bunkers were lined with heavy black/white plastic sheeting commonly used to cover silage piles. Sufficient surplus was left over the top in each direction to enable eventual fold-down and sealing of the compost bed within the bunker. To combat an ambient breeze, tires were used to weight the plastic in the bunker until the straw was loaded, but these were removed prior to the carcasses being placed. Once the leachate sampling port (necessary for experimental sampling only) was installed, loose barley straw was added into the bio-containment structure to form a base layer approximately 40 cm thick. This was accomplished by delivering a single round bale with a front end loader, and manually distributing the straw along the length of the bunker.
Sixteen feedlot cattle mortalities were placed onto the straw bed. These were cattle that had died within the previous 48 hours at nearby commercial feedlots, the majority having succumbed to bovine respiratory disease. The carcasses were aligned transversely within the composting bunker (Fig. 1), with a space of approximately 0.5 m between carcasses. To provide passive aeration, lengths of perforated flexible plastic drainage tubing (15 cm diameter) were laid between adjacent carcasses, embedded within the loose straw base. The ends of the tubing were directed vertically along the side walls extending beyond the top of the bunker, and held in place temporarily using tires extending past the upper surface of the walls.
Pre-conditioned feedlot manure was loaded into the bunkers to cover the carcasses to a final depth of 1.6 m. The manure was loaded over the side wall of the bunker, moving from one end of structure toward the other. The sample retrieval pyramids described below were positioned at their prescribed depths within the manure layer during this stage. Once the bunker was filled, the plastic sheeting was folded over the top of the piled manure and the ends of the perforated tubing were passed through it so that they were situated exterior to the wrap. The plastic was sealed with tape to establish a containment barrier around the straw, carcasses and manure. Eight aeration vents (50 x 50 x 15 cm) were placed on the top of each structure to promote passive aeration, and T-shaped connectors were attached to each end of the lengths of perforated tubing to promote air flow down into the tubes.
In this pilot study, experimental amendments to the compost structures were incorporated to enable investigation of pathogen inactivation and tissue degradation during the static composting process. Tissue and microbial samples pre-quantified by tissue weight or microbial enumeration were heat-sealed into nylon sample bags (5 x 9 cm; 50-&mu;m pore size), and packed into specialized pyramidal steel cages, designated as Baker Retrieval Pyramids (BRP)1. These pyramids were designed to enable their retrieval from the compost matrix at intervals during the composting process without seriously compromising containment or altering the dynamics of the composting process Within a BRP, eight bags (one each of eight sample types) were embedded in the same manure that was used to fill the bunker, arranged so that each bag was surrounded by manure and no bags were in direct contact with one another. Manure- and bag-filled BRPs were attached to lengths of logging chain and suspended at depths of 80 cm and 160 cm within the compost matrix. The chains were anchored to wooden poles spanning the bunkers at 1.5-m intervals (Fig. 1). T-type thermocouples placed in each BRP were also run along the chains to a data logger positioned externally to the compost structure. At specified intervals, BRPs were withdrawn vertically from the compost using a ratcheted come-along cable puller, and the plastic wrap was re-sealed. In the naturally arid climate of southern Alberta, little rainfall was received during this period, but the plastic wrap and domed shape of the composting material were effective for promoting lateral runoff to be absorbed by the straw bunker walls.

<ol>
	<li>Reuter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+temporal+collection+of+tissue+and+microbial+samples+from+static+composting+systems.">A simple method for temporal collection of tissue and microbial samples from static composting systems.</a> Canadian Biosystems Engineering. 50, 6.17-6.20 (2008).</li><li>Xu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biosecure+composting+system+for+disposal+of+cattle+carcasses+and+manure+following+infectious+disease+outbreak.">A biosecure composting system for disposal of cattle carcasses and manure following infectious disease outbreak.</a> J. Environ. Qual. 38, 437-450 (2009).</li></ol>

No conflicts of interest declared.

After 147 days of static composting, losses of total mass, dry matter (DM), organic matter, total carbon and total nitrogen were 23.7, 35.6, 52.9, 49.6, and 41.4%, respectively2. Within each structure, the volume of materials composted decreased from 118 to 71 m3. Decomposition of the bovine tissues monitored ranked as brain &gt; hoof &gt; bone. After only 7 days of composting, &gt;90% of brain tissue DM had decomposed, and 80% of hoof DM had decomposed within 56 d of composting. Complete loss of viability of Escherichia coli O157:H7 and Newcastle Disease was achieved within 14 days.
Intensive livestock production systems are particularly vulnerable to natural or intentional infectious disease outbreaks. Housing a large number of animals within a confined area gives rise to most infectious agents disseminating rapidly throughout the population. Containment is key to controlling any infectious disease outbreak, thus depopulation is used frequently as a means of preventing spread of the infectious agent to the larger livestock population. The depopulation scenario results in large numbers of livestock carcasses and contaminated manure requiring rapid disposal. Composting lends itself as a rapid-response disposal method for infected carcasses as well as manure and soil that may harbor infectious agents. We outline a composting procedure that can be conducted at the site of the disease outbreak, using materials readily available on-farm or from local farm-supply stores. In our study, infectious agents associated with beef cattle carcasses and manure were inactivated within 14 days of composting, an compost temperatures exceeded 55 &deg;C for more than one month. Production of leachate was extremely low, likely to due the absorbent nature of the loose straw base layer and our having optimized the DM content at initiation of composting. Total yields of leachate were less than 3 ppm of the initial compost mass (i.e., &lt;300 g per structure). Coliforms were detected in the leachate at up to 5.8 log10 CFU/mL at 14 days, but were not detectable after 101 days of composting. After 147 days, bovine carcasses were almost completely degraded with only a few long bones being recognizable. Bones were degraded further during the additional open-windrow composting cycle after the biocontained structures were opened, yielding final mature compost suitable for land application.
In conclusion, composting creates conditions that present substantial challenge to the survival of most pathogenic microorganisms. Free bacteria, protozoa and viruses are rapidly inactivated by the high temperature, alkalinity and high protease and nuclease activities within compost. This successful decomposition of mature feedlot cattle carcasses indicates that this static composting disposal procedure would be suitable for all common livestock. Care must be taken, however, to ensure that optimal carbon:nitrogen ratios and moisture levels are present in order for microbial kill conditions to be achieved. Pathogens inherently more heat-resistant, such as bacteria that form spores (e.g., anthrax), or those that are unusually recalcitrant, such as prions, may still remain infective after composting. Studies to elucidate the fate of these types of microorganisms during the composting process are currently underway in our laboratory.
<img src="/files/ftp_upload/1946/1946fig1.jpg" alt="Figure 1" /> Figure 1. Diagrammatic representation of the biosecure compost system comprising straw bale walls and floor, plastic sheeting enclosure, loose straw base, cattle carcasses, manure, perforated plastic ventilation tubing, and air vents, as well as experimental amendments (leachate port, sample retrieval pyramids and temperature sensors. (A) Transverse view (cross section). (B) Longitudinal view (side wall removed). All dimensions are in cm. From Xu et al. (2009)2

biocontained carcass composting for control of infectious disease outbreak in livestock

Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of animals housed within a confined area enables rapid dissemination of most infectious agents throughout a herd. Rapid containment is key to controlling any infectious disease outbreak, thus depopulation is often undertaken to prevent spread of a pathogen to the larger livestock population. In that circumstance, a large number of livestock carcasses and contaminated manure are generated that require rapid disposal.
Composting lends itself as a rapid-response disposal method for infected carcasses as well as manure and soil that may harbor infectious agents. We designed a bio-contained mortality composting procedure and tested its efficacy for bovine tissue degradation and microbial deactivation. We used materials available on-farm or purchasable from local farm supply stores in order that the system can be implemented at the site of a disease outbreak. In this study, temperatures exceeded 55&deg;C for more than one month and infectious agents implanted in beef cattle carcasses and manure were inactivated within 14 days of composting. After 147 days, carcasses were almost completely degraded. The few long bones remaining were further degraded with an additional composting cycle in open windrows and the final mature compost was suitable for land application. 
Duplicate compost structures (final dimensions 25 m x 5 m x 2.4 m; L x W x H) were constructed using barley straw bales and lined with heavy black silage plastic sheeting. Each was loaded with loose straw, carcasses and manure totaling ~95,000 kg. A 40-cm base layer of loose barley straw was placed in each bunker, onto which were placed 16 feedlot cattle mortalities (average weight 343 kg) aligned transversely at a spacing of approximately 0.5 m. For passive aeration, lengths of flexible, perforated plastic drainage tubing (15 cm diameter) were placed between adjacent carcasses, extending vertically along both inside walls, and with the ends passed though the plastic to the exterior. The carcasses were overlaid with moist aerated feedlot manure (~1.6 m deep) to the top of the bunker. Plastic was folded over the top and sealed with tape to establish a containment barrier and eight aeration vents (50 x 50 x 15 cm) were placed on the top of each structure to promote passive aeration. After 147 days, losses of volume and mass of composted materials averaged 39.8% and 23.7%, respectively, in each structure.

Watch this Scientific Journal Video about Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock at JoVE.com

Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

Using readily available materials, this biocontained composting system enables effective on-site disposal of large animal carcasses arising in the event of infectious disease outbreak. This procedure kills most infectious agents in carcasses and contaminated manure. Once infectious agent is confirmed non-viable, mature compost can be spread as fertilizer.

Intensive livestock production systems are particularly vulnerable to natural or intentional (bioterrorist) infectious disease outbreaks. Large numbers of ...

biocontained-carcass-composting-for-control-infectious-disease

Research

JoVE Journal

Immunology and Infection

10.8K Views.  Lethbridge Research Centre. Using readily available materials, this biocontained composting system enables effective on-site disposal of large animal carcasses arising in the event of infectious disease outbreak. This procedure kills most infectious agents in carcasses and contaminated manure. Once infectious agent is confirmed non-viable, mature compost can be spread as fertilizer.

Video: Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Biocontained Carcass Composting for Control of Infectious Disease Outbreak in Livestock

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Chapters in this video

Development of a Negative Selectable Marker for Entamoeba histolytica

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1^-/- Mice

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing