We thank Dr. Moon Nahm (University of Alabama Birmingham) for his invaluable assistance in establishing OPKA assays in our laboratory. This work was supported by National Institutes of Health Grant 1R01AI123383-01A1 to FYA.

1. Culture, Differentiation, and Validation of HL-60 Cells

<ol>
	<li>Prepare HL-60 cell culture media composed of 500 mL RPMI with L-glutamine and 50 mL heat-inactivated fetal bovine serum. Do not add antibiotics as this may affect the differentiation of the HL-60 cells.</li>
	<li>For propagation/maintenance of HL-60 cells, culture 5 x 106 cells in 10 mL of HL-60 cell culture media in 75 cm2 vented flasks at 37 &#176;C and 5% CO2. Passage cells every 3&#8722;4 days to maintain optimal cell concentrations. 
	NOTE: The cell concentration should not exceed 5 x 106/mL.</li>
	<li>Generate working stocks of HL-60 cells by aliquoting approximately 1 x 106 cells/mL in HL60 culture media with 10% dimethyl sulfoxide (DMSO) into 1 mL cryogenic tubes. 
	NOTE: Working stocks may be stored at -80 &#176;C. The master stock should be stored at -120 &#176;C.</li>
	<li>Differentiate HL-60 cells by culturing 1.5 x 107 cells in 15 mL of HL-60 cell culture media with 0.6%&#160;N,N-dimethylformamide (DMF) at 37 &#176;C and 5% CO2 in sterile filter-capped 75 cm2 flasks for 3 days prior to OPKA.</li>
	<li>Validate that the HL-60 cells have been successfully differentiated and are appropriate for use in the OPKA assay by testing viability and cell surface markers according to established flow cytometry protocols16,17,18,19. After differentiation, harvest HL-60 cells and stain approximately 1 x 104 cells with fluorescently-conjugated antibodies/stains for CD71, CD35, annexin V, and propidium iodide. 
	NOTE: Differentiated cells should be &#8805;65% viable, &#8805;55% CD35+, and &#8804;20% CD71+ as determined through established validation protocols14 (Figure 1).</li>
</ol>

2. Preparation of OPKA Buffers and Reagents

<ol>
	<li>Prepare 50 mL of sterile opsonization buffer B (OBB) by mixing 42.5 mL of sterile 1x phosphate-buffered saline (PBS) with Ca2+/Mg2+, 5 mL of heat-inactivated fetal bovine serum, and 2.5 mL of 0.1% sterile gelatin. Store at 4 &#176;C.</li>
	<li>Obtain baby rabbit complement and store at -80 &#176;C.</li>
	<li>Obtain or prepare bacterial culture plates (i.e., 15 x 100 mm2 5% sheep&#8217;s blood agar plates).</li>
</ol>

3. Preparation of Bacterial Stock Samples

<ol>
	<li>Obtain a stock of the bacterial strain(s) to be tested. 
	NOTE: For this protocol, serotype 3 Streptococcus pneumoniae (WU2, generously provided by Dr. Moon Nahm) is used.</li>
	<li>Grow the bacterial strain in an appropriate broth (i.e., Todd-Hewitt broth + 0.5% yeast extract for this WU2 strain) for approximately 2&#8722;4 h at 37 &#176;C. 
	NOTE: The optical density at 600 nm (OD600) of the culture should be between 0.6 and 0.8.</li>
	<li>Pellet the bacteria by centrifugation at 6,000 x g for 2 min and resuspend the cells in 10&#8722;30 mL of 15% glycerol in the appropriate broth. Aliquot the bacterial culture (500 &#181;L per aliquot) into sterile 1.5 mL centrifuge tubes and store at -80 &#176;C.</li>
	<li>Thaw out one vial of bacterial stock in a 37 &#176;C water bath. Pellet the bacterial cells and resuspend in 500 &#181;L of OBB under sterile conditions.</li>
	<li>Prepare different dilutions of the bacterial stock in OBB (i.e., 10 &#181;L of no dilution, 10 &#181;L of 1:10, 10 &#181;L of 1:100, etc.). Perform the OPKA assay (sections 4&#8211;6, including HL-60/complement co-culture) as described below using various dilutions of the untreated bacterial stock. Culture the plates overnight at 30 &#176;C (no CO2). 
	NOTE: The temperature 30 &#176;C is specific for WU2 to prevent overgrowth; other strains/serotypes may grow optimally at 37 &#176;C.</li>
	<li>Count the colonies for each dilution of untreated bacterial stock co-cultured with HL-60 cells and complement. Determine which dilution of bacteria yields the optimal number of countable colonies (approximately 80&#8722;120 CFUs for untreated bacteria co-cultured with HL-60 cells). Note this dilution for future OPKAs involving this bacterial stock.</li>
</ol>

4. Bacterial Treatment and Culture

<ol>
	<li>Thaw one tube of bacterial stock prepared in step 3.3. Pellet bacteria (6,000 x g for 2 min) and resuspend cell pellet in OBB at optimal dilution as determined in step 3.6.</li>
	<li>Pipette 10 &#181;L of resuspended bacterial dilution per well in a round-bottom 96-well cell culture plate.</li>
	<li>Add 20 &#181;L of appropriate antibody or drug treatment to each experimental well in duplicate. 
	NOTE: In this protocol, a serotype-specific antibody generated in mice is added as treatment X and a glycoside hydrolase enzyme known to degrade the serotype 3 polysaccharide capsule is added as treatment Y (Figure 2)4,20. For control wells, use 1x PBS or OBB, depending on the buffer used for treatment wells.</li>
	<li>Shake the sample plate at approximately 90 rpm for 1 h at room temperature. Adjust these conditions depending on the optimal temperature or shaking conditions of the treatments being tested.</li>
</ol>

5. HL-60 bacterial Co-culture

<ol>
	<li>Prepare HL-60 cells by harvesting the HL-60 differentiated cells that are treated with DMF three days prior (see step 1.4) into 15 mL conical tubes. Pellet the cells (500 x g, 3 min), discard the supernatant, and wash with at least 10 mL of 1x PBS.</li>
	<li>Pellet the washed cells (500 x g, 3 min), discard the supernatant, and resuspend the cells in OBB (start with 1 mL OBB and adjust for a final concentration of 1 x 107/mL after cell counting).</li>
	<li>Add baby rabbit complement (sterile, undiluted baby rabbit serum, age 3&#8722;4 weeks) at a 1:5 final volume. 
	NOTE: The final concentration of the HL-60-complement mixture should be 1 x 107/mL. If testing complement dependency, a second solution containing active HL-60 cells with heat-inactivated complement may be used (complement may be inactivated by incubating in a water bath at &#62;55 &#176;C for at least 30 min).</li>
	<li>After 1 h bacterial culture is complete (step 4.4), divide each sample (i.e., 10 &#181;L of each 30 &#181;L sample well into two new wells) into duplicate wells for two groups (i.e., use only 20 &#181;L of the original 30 &#181;L co-culture to account for pipetting error): one set will be co-cultured with HL-60-complement and one will include bacteria only. Add 50 &#181;L of the HL-60-complement mixture (from step 5.3) to each experimental set of wells (delegated +HL-60); add 50 &#181;L of OBB alone to the wells of bacteria only (delegated -HL-60). 
	NOTE: For this example, approximately 800 bacterial CFUs are used for the initial co-culture with 5 x 105/50 &#181;L HL-60 cells. If this multiplicity of infection is too high or too low as indicated by final colony numbers, adjust the initial bacterial dilution as opposed to the HL-60 cell count.</li>
	<li>Shake the 96-well plate at 37 &#176;C for 1 h (no CO2).</li>
</ol>

6. Sample Plating and Overnight Incubation

<ol>
	<li>Dilute each well 1:5 with OBB, so that each sample has a volume of at least 50 &#181;L.</li>
	<li>Pipette 50 &#181;L of each sample directly onto a designated area of a bacterial culture plate, ensuring adequate spacing between samples. For 15 x 100 mm2 round agar plates, pipet approximately 4 samples onto one plate.</li>
	<li>Cover and allow samples to dry for approximately 15 min at room temperature.</li>
	<li>Invert plates and culture overnight at 30 &#176;C (no CO2). Alternatively, culture plates in anaerobic jars to test whether anoxic conditions affect the bacterial growth or to control for morphology.</li>
	<li>After overnight culture, count the colonies in each designated sample area. Analyze data by comparing the number of live cells in each set to the corresponding control and/or samples that do not receive HL-60 cell co-culture (indicative of 100% cell survival, 0% cell killing).</li>
</ol>

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsular+polysaccharides+are+an+important+immune+evasion+mechanism+for+Staphylococcus+aureus.">Capsular polysaccharides are an important immune evasion mechanism for Staphylococcus aureus.</a> Human Vaccines and Immunotherapeutics. 9 (3), 480-487 (2013).</li><li>Ishibashi, K., Yamaguchi, O., Shiraiwa, Y., Ogihara, M., Shigeta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+therapy+of+Pseudomonas+aeruginosa+pyelonephritis+in+neutropenic+mice+with+human+antilipopolysaccharide+monoclonal+antibody+and+cefsulodin.">Combination therapy of Pseudomonas aeruginosa pyelonephritis in neutropenic mice with human antilipopolysaccharide monoclonal antibody and cefsulodin.</a> Journal of Urology. 155 (6), 2094-2097 (1996).</li><li>Middleton, D. R., Paschall, A. V., Duke, J. A., Avci, F. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+Hydrolysis+of+Pneumococcal+Capsular+Polysaccharide+Renders+the+Bacterium+Vulnerable+to+Host+Defense.">Enzymatic Hydrolysis of Pneumococcal Capsular Polysaccharide Renders the Bacterium Vulnerable to Host Defense.</a> Infection and Immunity. , (2018).</li><li>Baker, C. J., Noya, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+use+of+intravenous+immune+globulin+for+group+B+streptococcal+infection.">Potential use of intravenous immune globulin for group B streptococcal infection.</a> Reviews of Infectious Diseases. 12, 476-482 (1990).</li><li>Tian, H., Weber, S., Thorkildson, P., Kozel, T. R., Pirofski, L. 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The authors have nothing to disclose.

OPKAs serve essential roles in assessing antibody mediated immune responses induced by vaccinations6,8. The main significance of this simplified OPKA is the adaptability in the conditions to be tested (i.e., antibodies, enzyme treatments, etc.). In this sense, while this assay can be used to test the contribution of opsonins (i.e., antibodies) in phagocytosis, it can also be used to assess ways to overcome virulence factors (i.e., capsular polysaccharides) that n...

The opsonophagocytic killing assay (OPKA) is a critical tool for linking alterations in bacterial structure or function to subsequent changes in immune response and function. As such, it is frequently used as a complementary assay to determine immune-based efficacy of antibody treatments, vaccine candidates, enzyme optimization, etc. While in vivo assays are necessary to determine effective clearance or protection in a bacterial infection model, the OPKA can be used to assess immune contribution to bacterial cell death at the most basic components: bacteria, immune cells, and experimental treatments. Previous studies have shown that OPKAs can be modified and used for a variety of bacteria and serotypes, including Streptococcus pneumoniae1, Staphylococcus aureus2, Pseudomonas aeruginosa3. Furthermore, these optimized assays can be used to assess different experimental treatments, including the ability of an enzyme to make the bacterium more accessible to complement-mediated immune cells4 and antibody treatments to improve opsonization5. Classically, OPKA assay has been successfully used in basic and clinical research settings as a powerful indicator for protection induced by pathogen-specific antibodies6,7,8,9.
Different types of immune cells may be used for assessment of opsonophagocytic killing. One commonly used phagocytic population is the HL-60 human leukemic cell line. This cell line can be kept as inactivated promyelocytes in culture; however, they can be differentiated into various activated states via different drug treatments10,11. Treatment of HL60 with N,N-dimethylformamide differentiates the cell line into activated neutrophils with strong phagocytic activity11. While HL-60 cells have been optimized and are frequently used for these phagocytosis assays10, other primary polymorphonuclear leukocytes can be used as the immune arm of the experiment12.
Additionally, these assays can be simplified13 or multiplexed14 to look at multiple antibiotic-resistant strains of the bacteria to be tested. The multiplexed method has been made more feasible through the development of software that can efficiently count bacterial colony forming units (CFUs) per spot on an agar plate15. Here, we describe a streamlined method using one bacterial strain, HL-60 cells, baby rabbit complement, and blood agar plates. With this method, multiple treatments can be assessed quickly to address specific research questions on how the innate immune response to bacterial infection can be modulated.

<table border="0" cellpadding="0" cellspacing="0" style="width:636px;" width="637">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Annexin V (APC conjugated)</td>
			<td style="width:147px;">BioLegend</td>
			<td style="width:119px;">640919</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">anti-CD35, human (PE conjugated)</td>
			<td style="width:147px;">BioLegend</td>
			<td style="width:119px;">333405</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">anti-CD71, human (PE conjugated)</td>
			<td style="width:147px;">BioLegend</td>
			<td style="width:119px;">334105</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:204px;">bacterial strain to be used (ie, Streptococcus pneumoniae, WU2)</td>
			<td style="width:147px;">Bacterial Respiratory Reference Laboratory (Dr. Moon Nahm)&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">blood agar plates</td>
			<td>Hardy Diagnostic</td>
			<td>A10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">Fetal Clone serum</td>
			<td>HyClone</td>
			<td>SH30080.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">glycerol</td>
			<td>Sigma</td>
			<td>G9012-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">HL-60 cells</td>
			<td>ATCC</td>
			<td>CCL-240</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">IgG Isotype Control (PE conjugated)</td>
			<td style="width:147px;">BioLegend</td>
			<td style="width:119px;">400907</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:204px;">N,N-dimethylformamide (DMF)</td>
			<td>Fisher Chemical</td>
			<td>UN2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">propidium iodide</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:119px;">P4864</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:204px;">RPMI media with L-glutamine</td>
			<td>Corning</td>
			<td>10-040-CV</td>
			<td></td>
		</tr>
	</tbody>
</table>

opsonophagocytic killing assay to assess immunological responses against bacterial pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

Validation of HL-60 differentiation should be performed before starting the OPKA. This can be accomplished using flow cytometry to determine the extracellular expression of CD11b, CD35, CD71, and annexin V (Figure 1). Propidium iodide can also be used as a viability marker. After being treated with DMF for 3 days, expression of CD35 should be increased (&#8805;55% of all cells) and expression of CD71 should be decreased (&#8804;20% of all cells). The percentage of annexin V+ and propidium io...

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Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

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Immunology and Infection

12.0K Views.  University of Georgia. This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

Video: Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

Enzyme-linked Immunospot Assay (ELISPOT):  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Adaptive immunity is an important component to clearance of intracellular pathogens. The ability to detect and quantify these responses in humans is an important diagnostic tool. The enzyme-linked immunospot assay (ELISPOT) is gaining popularity for its ability to identify cellular immune responses against microbial antigens, including immunosuppressed populations such as those with HIV infection, transplantation, and steroid use. This assay has the capacity to quantify the immune responses against specific microbial antigens, as well as distinguish if these responses are Th1 or Th2 in character. ELISPOT is not limited to the site of inflammation. It is versatile in its ability to assess for immune responses within peripheral blood, as well as sites of active involvement such as bronchoalveolar lavage, cerebral spinal fluid, and ascites. Detection of immune responses against a single or multiple antigens is possible, as well as specific epitopes within microbial proteins. This assay facilitates detection of immune responses over time, as well as distinctions in antigens recognized by host T cells. Dual color ELISPOT assays are available for detection of simultaneous expression of two cytokines. Recent applications for this technique include diagnosis of extrapulmonary tuberculosis, as well as investigation of the contribution of infectious antigens to autoimmune diseases.

Enzyme-linked Immunospot Assay ELISPOT:  Quantification of Th-1 Cellular Immune Responses Against Microbial Antigens

Identification of microbial targets of adaptive immunity in idiopathic diseases can be accomplished by the use of the enzyme-linked immunospot assay.

Adaptive immunity is an important component to clearance of intracellular pathogens.  The ability to detect and quantify these responses in humans is an ...

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating histoplasmosis caused by the fungus Histoplasma capsulatum and 2) to examine the role of virulence factors as target for therapy. To generate mAbs, mice are immunized, the immune responses are assessed using a solid phase ELISA system developed in our laboratory, and the best responder mice are selected for isolation of splenocytes for fusion with hybridoma cells. C57BL/6 mice have been extensively used to study H. capsulatum pathogenesis and provide the best model for obtaining the data required. In order to assess the role of the mAbs in infection, mice are intraperitoneally administered with either mAb to H. capsulatum or isotype matched control mAb and then infected by either intravenous (i.v.), intraperitoneal (i.p.), or intranasal (i.n.) routes. In the scientific literature, efficacy of mAbs for fungal infections in mice relies on mortality as an end point, in conjunction with colony formin units (CFU) assessments at earlier time points. Survival (time to death) studies are necessary as they best represent human disease. Thus, efficacy of our intervention would not adequately be established without survival curves. This is also true for establishing efficacy of vaccine or testing of mutants for virulence. With histoplasmosis, the mice often go from being energetic to dead over several hours. The capacity of an intervention such as the administration of a mAb may initially protect an animal from disease, but the disease can relapse which would not be realized in short CFU experiments. In addition to survival and fungal burden assays, we examine the inflammatory responses to infection (histology, cellular recruitment, cytokine responses). For survival/time to death experiments, the mice are infected and monitored at least twice daily for signs of morbidity. To assess fungal burden, histopathology, and cytokine responses, the mice are euthanized at various times after infection. Animal experiments are performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine.

Passive Administration of Monoclonal Antibodies Against H. capsulatum and Others Fungal Pathogens

C57BL/6 mice have been used to study Hc pathogenesis and provide the best model. We are exploring the potential benefits of humoral immunity against this fungus and generated several mAbs [to histone H2B and a heat shock protein 60kDa] that we tested for their protective efficacy after intraperitoneal administration.

The purpose of the use of this methodology is 1) to advance our capacity to protect individuals with antibody or vaccine for preventing or treating ...

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions 1. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage 2, 3. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle 4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection 7. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization 8. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella typhimurium and Mycobacterium marinum. Micro-injection of bacteria and multi-color fluorescence imaging are essential techniques involved in the application of zebrafish embryo infection models.

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen ...

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

Application of Long-term cultured Interferon-&#947; Enzyme-linked Immunospot Assay for Assessing Effector and Memory T Cell Responses in Cattle

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is controlled up to 95 % of the T cells generated during the expansion phase are eliminated (i.e., contraction phase) and memory T cells remain, sometimes for a lifetime. In humans, two functionally distinct subsets of memory T cells have been described based on the expression of lymph node homing receptors. Central memory T cells express C-C chemokine receptor 7 and CD45RO and are mainly located in T-cell areas of secondary lymphoid organs. Effector memory T cells express CD45RO, lack CCR7 and display receptors associated with lymphocyte homing to peripheral or inflamed tissues. Effector T cells do not express either CCR7 or CD45RO but upon encounter with antigen produce effector cytokines, such as interferon-&#947;. Interferon-&#947; release assays are used for the diagnosis of bovine and human tuberculosis and detect primarily effector and effector memory T cell responses. Central memory T cell responses by CD4+ T cells to vaccination, on the other hand, may be used to predict vaccine efficacy, as demonstrated with simian immunodeficiency virus infection of non-human primates, tuberculosis in mice, and malaria in humans. Several studies with mice and humans as well as unpublished data on cattle, have demonstrated that interferon-&#947; ELISPOT assays measure central memory T cell responses. With this assay, peripheral blood mononuclear cells are cultured in decreasing concentration of antigen for 10 to 14 days (long-term culture), allowing effector responses to peak and wane; facilitating central memory T cells to differentiate and expand within the culture.

Long-term cultured interferon-&#947; enzyme-linked immunospot assay is used as a measure of central memory responses and correlates with protective anti-mycobacterial vaccine responses. With this assay, peripheral blood mononuclear cells are stimulated with mycobacterial antigens and interleukin-2 for 14 days, enabling differentiation and expansion of central memory T cells.

Effector and memory T cells are generated through developmental programing of na&#239;ve cells following antigen recognition. If the infection is ...

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

A High-throughput Shigella-specific Bactericidal Assay

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing activity by assessing the ability of antibodies in serum to bind to bacteria and activate complement. This complement activation results in the lysis and killing of the target bacteria. These assays are valuable because they go beyond quantifying antibody production to elucidate the biological functions that these antibodies have, allowing researchers to study the role that antibodies may play in preventing infection. SBAs have been used to study immune responses for many human pathogens, but there is no widely accepted methodology for Shigella at present. Historically, SBAs have been very labor-intensive, requiring many time-consuming steps to accurately quantify surviving bacteria. This protocol describes a simple, robust, and high-throughput assay that measures functional antibodies specific for Shigella in serum in vitro. The method described here offers many advantages over traditional SBAs, including the use of frozen bacterial stocks, 96 well assay plates, a micro-culture system, and automated colony-counting. All of these modifications make this assay less labor-intensive and more high-throughput. This protocol is simpler and faster to perform than traditional SBAs while still using simple technologies and readily available reagents. The protocol has been successfully applied in multiple independent laboratories and the assay is robust and reproducible. The assay can be used to assess immune responses in pre-clinical as well as clinical studies. Quantifying shigellacidal antibody titers both before and after antigen exposure (either by immunization or infection) allows for a broader understanding of how functional antibody responses are generated and their contribution to protective immunity. The development of this standardized, well-characterized assay may greatly facilitate Shigella vaccine design.

A High-throughput Shigella-specific Bactericidal Assay

Here we present a protocol to measure Shigellacidal activity of antibodies in serum. Serum is mixed with bacteria and exogenous complement, incubated, and the reaction mixture is plated on agar plates. Viable bacteria form colonies which are counted, using an automated colony enumerator, and used to determine the bactericidal titer.

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...