J. Chua, N.A. Fisher, D. DeShazer and A.M. Friedlander designed the procedures described in the manuscript. J. Chua, N.A. Fisher, S.D. Falcinelli and D. DeShazer performed the experiments. J. Chua wrote the manuscript.
The authors thank Joshua J. W. Roan, Nora D. Doyle, Nicholas R. Carter and Steven A. Tobery for excellent technical assistance and David P. Fetterer and Steven J. Kern for statistical analysis.
The work was supported by the Defense Threat Reduction Agency Proposal #CBCALL12-THRB1-1-0270 to A.M.F and #CBS.MEDBIO.02.10.RD.034 to D.D.
Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.
The content of this publication does not necessarily reflect the views or policies of the Department of Defense, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

1. Preparations for Maintaining a Hissing Cockroach Colony
<ol>
	<li>Prepare cages for the hissing cockroaches to live in. Apply a thin layer of petroleum jelly, approximately 20 to 30 mm in width, to the circumference of the inner walls near the top of the cage to prevent the hissing cockroaches from climbing out of the cage and escaping. 
	NOTE: Hissing cockroaches can be housed in a variety of containers that have a large floor space, are of sufficient height, and have lids. Use mouse cages (~43 cm x 23 cm x 20 cm). For cages destined for 37 &#176;C, do not apply petroleum jelly.</li>
	<li>Include a cardboard egg carton placed upside down inside the cage to provide a hiding place for the naturally shy insects. Do not substitute the cardboard egg carton with one made of polystyrene foam to prevent ingestion of plastic.</li>
	<li>Do not provide bedding for easier clean-up and to increase the visibility of newly hatched nymphs.</li>
	<li>Obtain dry dog food which contains a composition of ~20% crude protein. Coarsely grind the dog food with a food processor or blender and store at 4 &#176;C.</li>
	<li>Obtain several shallow dishes and aquarium stones or a sponge for food and water. 
	NOTE: Petri dishes are recommeded.</li>
</ol>
2. Hissing Cockroach Care and Breeding
<ol>
	<li>Obtain outbred Madagascar hissing cockroaches (~5 cm) from a commercial breeder. The species used in this protocol is Gromphadorhina laevigata. Unpack the shipping box containing the hissing cockroaches immediately upon receipt.</li>
	<li>Follow appropriate institutional guidelines for the use of personal protective equipment in handling animals. 
	NOTE: Use thick disposable gloves for handling cockroaches as the tarsal claws of a hissing cockroach are sharp.</li>
	<li>Transfer up to 75 large hissing cockroaches (&#62;3 cm) per large cage. Transfer the newly hatched and smaller nymphs (&#60;3 cm) to a separate cage. 
	NOTE: Keeping significantly more than 75 large hissing cockroaches per mouse cage could lead to deteriorating health of the colony and could also result in deaths.</li>
	<li>Handle a newly molted cockroach, which is off-white in color, gently and avoid squeezing it. Alternatively, allow the exoskeleton to darken and harden before handling. 
	NOTE: It is not necessary to remove the mites, Gromphadorholaelaps schaeferi, that often accompany hissing cockroaches upon receipt from breeders. The beneficial mites keep the hissing cockroaches clean and are harmless to humans.</li>
	<li>Feed hissing cockroaches with ground dry dog food in a shallow dish once or twice a week. Provide enough dog food to ensure that the hissing cockroaches have sufficient food until the next feeding. Discard any food that has become wet and moldy.</li>
	<li>In addition to dog food, provide cut-up fruits and vegetables, such as apples, potatoes, or lettuce, in a shallow dish. Discard moldy or rotten food.</li>
	<li>Provide drinking water once or twice a week in a shallow dish but do not overfill the dish to prevent spillage in the cage. Place either small aquarium stones or a sponge in the dish to provide a landing pad for small nymphs. Use reverse osmosis water, if available.</li>
	<li>Keep hissing cockroaches in the dark at temperatures ranging from 21 &#176;C to 30 &#176;C. Keep large hissing cockroaches destined for experimentation at a lower temperature (~21 &#176;C) for ~2 months to decrease breeding and pregnancy. In contrast, keep nymphs at higher temperatures (28 &#176;C to 30 &#176;C) to hasten growth. 
	NOTE: Keep cages at room temperature (21 &#176;C) in a dark cabinet.</li>
	<li>Provide humidity by including a separate pan of water if hissing cockroaches are kept in an incubator.</li>
	<li>Clean the cage by scooping out dry feces regularly or by transferring hissing cockroaches to a clean cage every 2 to 3 weeks. Keep the bottom of the cage dry. Clean the cage immediately if excess water is allowed to accumulate and mix with the excrement at the bottom of the cage.</li>
</ol>
3. Cockroach Preparation for Experimentation
<ol>
	<li>Transfer the appropriate number of hissing cockroaches in a cage to a 37 &#176;C humidified incubator 1 to 3 weeks prior to an experiment for acclimation. Include a control group for injection. This acclimation period is critical for avoiding temperature shock during experimentation.</li>
	<li>Do not apply petroleum jelly to cages destined for 37 &#176;C because the higher temperature melts the jelly.</li>
	<li>Check the hissing cockroaches every 1 to 2 days to replace food and water and clean the cage.</li>
	<li>Obtain clear disposable plastic food containers with lids for grouping the hissing cockroaches during experimentation. For ventilation, punch holes in the lid or the side of the container with a nail and hammer. 
	NOTE: Use screw cap containers over snap cap containers in the biosafety level 3 setting. Snap cap containers can be reinforced with tape, if necessary.</li>
	<li>On the day of injection, distribute 6 to 12 acclimatized hissing cockroaches into groups per container, ensuring equal distribution by sex and body mass.</li>
	<li>Determine the sex of individual hissing cockroaches. Determine the sex by the prominence of horns found on males and the lack thereof on females.</li>
	<li>Weigh the individual hissing cockroaches. To facilitate the weighing of a hissing cockroach on the balance, enclose it within two weigh boats that have been tared. 
	NOTE: For consistency between experiments, use hissing cockroaches with a weight of 4 to 8 g.&#160;However, no difference in survival after infection have been found between small (1.5 to 2 g) and large (6 to 8 g) hissing cockroaches. For drug studies, use hissing cockroaches with a weight of ~5 g to obtain a more consistent drug concentration per body mass.</li>
	<li>In lieu of a water dish, include high water content fruit or vegetables, such as a slice of apple or potato. Discard rotten food. Do not provide additional water to keep the container dry.</li>
	<li>Return hissing cockroaches to 37 &#176;C until injections.</li>
</ol>
4. Bacterial Culture and Preparations
NOTE: The bacterial species used in this protocol are B. mallei, B. pseudomallei, and B. thailandensis. All manipulations with B. mallei and B. pseudomallei must be performed in Class II or Class III biological safety cabinets located in a biosafety level (BSL) 3 laboratory. Perform manipulations with B. thailandensis in similar biological safety cabinets located either in a BSL2 or BSL3 laboratory. Follow institutional standard operating procedure for BSL3 work. Follow institutional guidelines for use of personal protective equipment when handling bacteria.
<ol>
	<li>Prepare a master plate of Burkholderia at least 3 days prior to infection. Use Luria-Bertani (Lennox) (LB) agar for B. pseudomallei or B. thailandensis and use LB agar supplemented with 4% glycerol for B. mallei. Streak bacteria from a 25% glycerol stock stored at -80 &#176;C. 
	NOTE: Always streak the master plate directly from the frozen glycerol stock; avoid serial passage of the bacteria from plate to plate as this may cause reduced virulence.</li>
	<li>Inoculate 10 to 20 mL LB broth with several colonies of B. pseudomallei or B. thailandensis from the master plate. Similarly inoculate LB broth supplemented with 4% glycerol for B. mallei.</li>
	<li>Shake the broth culture at 175 to 250 rpm at 37 &#176;C for ~18 h.</li>
	<li>Centrifuge 2 to 3 mL of culture at 5,000 x g for 10 min.</li>
	<li>Discard the supernatant and resuspend the bacterial pellet in sterile phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 1.8 mM KH2PO4, pH 7.4).</li>
	<li>Dilute the bacteria with PBS to achieve an optical density (OD) of 0.5, measured at an absorbance at 600 nm in a spectrophotometer.</li>
	<li>Serially dilute the bacteria ten-fold in PBS from the starting OD of 0.5. Use these suspensions for infection.</li>
	<li>Determine the colony forming units (CFU) of the inoculum by serial dilution and plating of 100 &#181;L aliquots on agar media. Incubate plates at 37 &#176;C for 24 to 48 h.</li>
</ol>
5. Drug Preparations
<ol>
	<li>Determine the appropriate amount of drug to be given per body mass. The CLQ dosage given to the hissing cockroach was based on the standard dosage for mammals, which is 50 mg/kg/day.</li>
	<li>Resuspend or dilute the drug of interest in vehicle. For example, dilute CLQ in PBS at a concentration of 12 mg/mL. This concentration provides 300 &#181;g of drug to a ~6 g hissing cockroach in a 25 &#181;L injected volume.</li>
	<li>If necessary, sterilize the drug solution by passing it through a 0.22 &#181;m syringe filter.</li>
	<li>Store the drug solution at 4 &#176;C until use. Warm the drug solution to at least 21 &#176;C (room temperature) prior to injections.</li>
</ol>
6. Assembly of the Injector
<ol>
	<li>Set the pointer to the desired volume (25 &#181;L) by rotating the adjustment screw on a repetitive pipette.</li>
	<li>Push the release bar of the repetitive pipette inward and pull the pusher outward. The repetitive pipette is now ready to accommodate a loaded syringe. 
	NOTE: Calibrate the volume ejected by the repetitive pipette by measuring the amount of water ejected on a balance.</li>
	<li>Fill a 1 mL syringe with suspension containing either bacteria or drug.</li>
	<li>Attach syringe to a sterile 26 or 27 G x &#189; inch (or shorter) needle.</li>
	<li>Tap the syringe to float the air bubbles to the top and expel the bubbles and some suspension into a container filled with 10% bleach.</li>
	<li>Snap the syringe onto the syringe clip of the repetitive pipette with the needle bevel facing up.</li>
	<li>Pull the release bar outward and press the dispenser button firmly to perform blank injections into a container filled with 10% bleach until the syringe plunger is against the pusher.</li>
	<li>Perform 1 or 2 additional blank injections to ensure liquid is being ejected out of the syringe. The repetitive pipette is ready for injections.</li>
</ol>
7. Cockroach Injections
<ol>
	<li>Perform all cockroach injections in a Class II or Class III biological safety cabinet in a BSL2 or BSL3 setting using institutional recommended personal protective equipment.</li>
	<li>Clean work surfaces in the safety cabinet that may come in contact with the hissing cockroaches. Use 10% bleach followed by 70% ethanol to remove residual bleach. Allow the surface to air dry.</li>
	<li>With one hand, grip the hissing cockroach by its side. Immobilize a hissing cockroach such that it is unable to recoil during injection. Bend the hissing cockroach slightly so that the cutaneous membranes between the abdominal terga are exposed.</li>
	<li>With the other hand, hold the repetitive pipette such that the needle is at a 0&#176; to 30&#176; angle from the dorso-ventral midline of the hissing cockroach. Puncture the cutaneous membrane adjacent to the 3rd, 4th, or 5th tergum from the posterior end. 
	NOTE: Entry of the needle into the hissing cockroach at the angle indicated ensures that the needle does not go through and exit out of the hissing cockroach. The injection site ensures that the ejected material is contained within the abdominal cavity filled with hemolymph. Practice holding the hissing cockroaches and injecting with water before undertaking injections with live bacteria or drug.</li>
	<li>Push the dispense button firmly to inject the volume. Gently withdraw the needle at the same angle as used for entry.</li>
	<li>Place the injected hissing cockroach in a separate container to distinguish it from the hissing cockroaches that have not yet been injected. Wipe off any hemolymph that may have oozed out from the injection site.</li>
	<li>Continue injecting other hissing cockroaches within a group using the same syringe and needle. Stop injecting before the syringe plunger reaches the bottom of the barrel to prevent incomplete dosing in the last injection.</li>
	<li>For multiple injections in a single hissing cockroach, such as those with bacteria and drug, inject at different sides of a tergum on the dorsal side of the hissing cockroaches. Alternatively, perform multiple injections by injecting at different terga (3rd to 5th).</li>
	<li>Ensure that the lids of the containers are securely fastened. Reinforce the lids with tape, if necessary.</li>
	<li>Incubate hissing cockroaches in a humidified 37 &#176;C incubator.</li>
</ol>
8. Recording Hissing Cockroach Morbidity and Mortality
<ol>
	<li>Perform all hissing cockroach examinations in a Class II or Class III biological safety cabinet with institutional recommended personal protective equipment. Clean the surfaces in the safety cabinet with 10% bleach followed by 70% ethanol. Allow the work area to air dry.</li>
	<li>Score hissing cockroaches once or twice daily over a period of 1 to 2 weeks using the morbidity scoring table (Table 1).</li>
	<li>Remove dead hissing cockroaches from the container and record the number of survivors.</li>
	<li>Transfer remaining live hissing cockroaches to a clean container if excess moisture has accumulated at the bottom of the container. Alternatively, use disposable paper towels to wipe off excess moisture.</li>
	<li>Replace rotten food daily and return hissing cockroaches to the humidified 37 &#176;C incubator.</li>
	<li>At the end of study, place remaining survivors in a biohazard bag and freeze at -80 &#176;C to euthanize.</li>
	<li>Statistically analyze the data14 to determine LD50 and by Kaplan-Meier and Log-Rank analysis for survival. As with all insect experimentation, some deaths may occur in the control group. These deaths are attributed to the natural mortality of insects 15. For a more detailed discussion on how to account for deaths in the control group, see reference15.</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+hemocytes+and+their+role+in+immunity.">Insect hemocytes and their role in immunity.</a> Insect Biochem Mol Biol. 32 (10), 1295-1309 (2002).</li><li>Bergin, D., Reeves, E. P., Renwick, J., Wientjes, F. B., Kavanagh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superoxide+production+in+Galleria+mellonella+hemocytes:+identification+of+proteins+homologous+to+the+NADPH+oxidase+complex+of+human+neutrophils.">Superoxide production in Galleria mellonella hemocytes: identification of proteins homologous to the NADPH oxidase complex of human neutrophils.</a> Infect Immun. 73 (7), 4161-4170 (2005).</li><li>Browne, N., Heelan, M., Kavanagh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+the+structural+and+functional+similarities+of+insect+hemocytes+and+mammalian+phagocytes.">An analysis of the structural and functional similarities of insect hemocytes and mammalian phagocytes.</a> Virulence. 4 (7), 597-603 (2013).</li><li>Lemaitre, B., Hoffmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+defense+of+Drosophila+melanogaster.">The host defense of Drosophila melanogaster.</a> Annu Rev Immunol. 25, 697-743 (2007).</li><li>Mulder, P. 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C., Fraser, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sound+production+in+the+cockroach,+Gromphadorhina+portentosa:+Evidence+for+communication+by+hissing.">Sound production in the cockroach, Gromphadorhina portentosa: Evidence for communication by hissing.</a> Behav Ecol Sociobiol. 6 (4), 305-314 (1980).</li><li>Chua, J., 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=pH+Alkalinization+by+Chloroquine+Suppresses+Pathogenic+Burkholderia+Type+6+Secretion+System+1+and+Multinucleated+Giant+Cells.">pH Alkalinization by Chloroquine Suppresses Pathogenic Burkholderia Type 6 Secretion System 1 and Multinucleated Giant Cells.</a> Infect Immun. 85 (1), e0058616 (2017).</li><li>Fisher, N. A., Ribot, W. J., Applefeld, W., DeShazer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Madagascar+hissing+cockroach+as+a+novel+surrogate+host+for+Burkholderia+pseudomallei,+B.+mallei+and+B.+thailandensis.">The Madagascar hissing cockroach as a novel surrogate host for Burkholderia pseudomallei, B. mallei and B. thailandensis.</a> BMC Microbiol. 12, 117 (2012).</li><li>Haraga, A., West, T. E., Brittnacher, M. J., Skerrett, S. J., Miller, S. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+and+Galleria+mellonella+infection+models+reflect+the+virulence+of+naturally+occurring+isolates+of+B.+pseudomallei,+B.+thailandensis+and+B.+oklahomensis.">Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis.</a> BMC Microbiol. 11 (1), 11 (2011).</li><li>Pilatova, M., Dionne, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burkholderia+thailandensis+is+virulent+in+Drosophila+melanogaster.">Burkholderia thailandensis is virulent in Drosophila melanogaster.</a> PLoS One. 7 (11), e49745 (2012).</li><li>Ramarao, N., Nielsen-Leroux, C., Lereclus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+insect+Galleria+mellonella+as+a+powerful+infection+model+to+investigate+bacterial+pathogenesis.">The insect Galleria mellonella as a powerful infection model to investigate bacterial pathogenesis.</a> J Vis Exp. (70), e4392 (2012).</li><li>Eklund, B. 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The authors have nothing to disclose.

Optimal experimental conditions begin with a healthy hissing cockroach colony, which requires a minimal but consistent time commitment. Although hissing cockroaches can go for a relatively long period of time (~weeks) without food and water, weekly or bi-weekly cage maintenance must be provided. This includes checking the food and water supply and ensuring that the cage is dry. Maintaining dry living conditions is especially important during acclimation and incubation at higher temperatures; we find that more hissing coc...

The use of insects as alternative non-mammalian animal models to study bacterial pathogenesis and innate host defense has been gaining momentum in recent years. Logistically, this is due to their relatively inexpensive cost and the ease in obtaining, handling, and caring for insects compared to mammals. There is also no regulatory policy governing the use of insects in research; it is not subject to the purview or restrictions set forth by any animal use committee or government agency. Insects as surrogate animal models are particularly amenable to comprehensive screening studies for virulence factors, host-pathogen interactions, and assessments of anti-microbial drug efficacy. Their use can reduce the number of mammals used for research thereby overcoming some of the ethical dilemmas inherent to the conduct of animal experimentation 1,2.
Insects may serve as surrogate hosts because there is a high degree of commonality between the innate immune systems of insects and mammals 1,3. Both insect plasmatocytes and mammalian macrophages phagocytose microorganisms 4. The insect counterpart to the neutrophil is the hemocyte 5,6. Intracellular oxidative burst pathways in insect and mammalian cells are similar; reactive oxygen species in both are produced by orthologous p47phox and p67phox proteins 5. The signaling cascades downstream of Toll receptors in insects and Toll-like receptors and Interleukin-1 in mammals are also remarkably similar; both result in production of antimicrobial peptides, such as defensins 7. Thus, insects can be utilized to study general innate immune mechanisms that are shared by metazoans.
An insect called the Madagascar hissing cockroach from the genus Gromphadorhina, is one of the largest cockroach species that exists, typically reaching 5 to 8 cm at maturity. It is native only to the island of Madagascar and is characterized by the hissing sound it makes - a sound that is produced when the hissing cockroach expels air through respiratory openings called spiracles 8. The distinctive hiss serves as a form of social communication among hissing cockroaches for courtship and aggression 9 and can be heard when a male is disturbed in its habitat. The Madagascar hissing cockroach is slow moving compared to the American cockroach and other urban pest species. It is easy to care for and breed; a pregnant hissing cockroach can produce 20 to 30 offspring at a time. A baby hissing cockroach, called a nymph, reaches sexual maturity in 5 months after undergoing 6 molts and can live up to 5 years both in the wild and in captivity 8.
We have utilized the Madagascar hissing cockroach as a surrogate host for infection with the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis 10,11. The virulence of these pathogens in hissing cockroaches was compared to their virulence in the benchmark animal model for Burkholderia, the Syrian hamster. We found that the 50% lethal dose (LD50) of B. pseudomallei and B. mallei was similar in both models 11. Interestingly, B. thailandensis, although avirulent in the rodent model, is lethal in the hissing cockroach 11. This difference with respect to B. thailandensis infection underscores the utility of the hissing cockroach model; B. thailandensis attenuating mutants can be more readily resolved in the hissing cockroach than in rodent models. Furthermore, as B. thailandensis is often used as the model organism for B. pseudomallei and B. mallei 10,12,13, identifying attenuating mutations in it could lead to similar targets in its more virulent relatives.
Despite the difference in virulence of B. thailandensis in the hissing cockroach versus the Syrian hamster, mutations in critical virulence factors, such as those in the type 6 secretion system-1 (T6SS-1), which are attenuating in B. mallei and B. pseudomallei, are similarly attenuating for B. thailandensis 11. The hissing cockroach model is further validated in that individual T6SS mutants (T6SS-2 to T6SS-6) in B. pseudomallei, which have no bearing on virulence in Syrian hamsters, remain virulent in the hissing cockroaches 11. Thus, the hissing cockroach is a viable surrogate animal model for the three Burkholderia species. We recently utilized the hissing cockroach as a surrogate animal model to examine the efficacy of the anti-malarial drug chloroquine (CLQ) against Burkholderia infection 10 and its toxicity.
Here, we describe the rearing and care of the Madagascar hissing cockroach and provide details on how to infect this insect with three Burkholderia species. Furthermore, we illustrate that the hissing cockroach is a viable surrogate model to study virulence and drug efficacy in Burkholderia infections and that it likely can also serve as a surrogate host for other bacterial pathogens in similar studies.

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	<tbody>
		<tr>
			<td>Madagascar hissing cockroach 
			&#160;&#160; 
			&#160; 
			&#160; 
			&#160;</td>
			<td>Carolina Biological Supply Co, Burlington, NC&#160;</td>
			<td>143668</td>
			<td></td>
		</tr>
		<tr>
			<td>Kibbles n Bits, any flavor</td>
			<td>Big Heart Pet Brands, San Francisco, CA</td>
			<td>UPC #079100519378</td>
			<td></td>
		</tr>
		<tr>
			<td>Snap on disposable plastic containers or equivalent</td>
			<td>Rubbermaid, Huntersville, NC</td>
			<td>UPC #FG7F71RETCHIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Screw on disposable plastic containers or equivalent</td>
			<td>Rubbermaid, Huntersville, NC</td>
			<td>UPC #FG7J0000TCHIL</td>
			<td></td>
		</tr>
		<tr>
			<td>Tridak STEPPER series repetitive pipette</td>
			<td>Dymax Corporation 
			www.dymax.com</td>
			<td>T15469</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe (1 mL)&#160;</td>
			<td>Becton Dickinson, Franklin Lakes, NJ</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle (26 or 27G x 1/2)</td>
			<td>Becton Dickinson, Franklin Lakes, NJ</td>
			<td>305109, 305111</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroquine diphosphate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>C6628</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Gibco/ Thermo Fisher Scientific, Gaithersburg, MD</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Luria- Bertani (Lennox)</td>
			<td>Becton Dickinson, Sparks, MD</td>
			<td>240230</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar&#160;</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A1296</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G6279</td>
			<td></td>
		</tr>
	</tbody>
</table>

the madagascar hissing cockroach as an alternative non-mammalian animal model to investigate virulence, pathogenesis, and drug efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

This section illustrates the results that were obtained when Madagascar hissing cockroaches were infected with B. mallei, B. pseudomallei, or B. thailandensis; the results show that this insect is a tractable animal model for different species of Burkholderia in studying virulence, drug toxicity, and drug efficacy against bacterial infection. More hissing cockroaches survived in groups that were infected with the attenuated mutants (&#916;hcp1) than in ...

Watch this Scientific Journal Video about The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy at JoVE.com

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

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Research

JoVE Journal

Immunology and Infection

10.9K Views.  United States Army Medical Research Institute of Infectious Diseases. We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

Video: The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Using Phylogenetic Analysis to Investigate Eukaryotic Gene Origin

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms&#39; long evolutionary history or scarcity of fossilization.
In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. Two phylogenetic trees constructed using theoretically distinct methods have consistently&#160;suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene.&#160;It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.

A method of constructing a phylogenetic tree based on sequence homology of SWEETs from eukaryotes and SemiSWEETs from prokaryotes is described. Phylogenetic analysis is a useful tool for explaining the evolutionary relatedness between homologous proteins or genes from different organism groups.

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...