Name: the madagascar hissing cockroach as an alternative non-mammalian animal model to investigate virulence, pathogenesis, and drug efficacy
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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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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 cockroaches die and at a faster rate at higher temperatures when containers were not cleaned daily.
The key to consistent dosing or inoculation of the hissing cockroach is to press the dispenser button on the repetitive pipette firmly. We recommend practicing this technique, loading of the syringe onto the repetitive pipette, and performing blank injections. The most time-consuming step of the procedure for an operator new to the technique is holding or immobilizing the hissing cockroach during injection. Therefore, we also highly recommend practicing the technique of holding and injecting multiple hissing cockroaches before tackling a more ambitious project. This can be achieved by maintaining a small group of hissing cockroaches that is used exclusively for injection practices. Although we have found that injection can be performed quickly when holding the hissing cockroach on its side, other techniques for holding hissing cockroaches (e.g. immobilizing a hissing cockroach on a smooth curved surface; perching the hissing cockroach on the middle finger while the index finger and thumb immobilize it) may be preferred and should be explored by different operators.
The use of the hissing cockroach model affords several advantages over other insect models (e.g. the wax worm larva Galleria mellonella and the fruit fly Drosophila melanogaster) that have been previously used as animal models with Burkholderia infection 16,17,18. For example, the experimental window for a hissing cockroach ranges from months to years allowing flexibility to the researchers, whereas that for a wax worm larva is only five days 19,20. For a wax worm larva, the five day period also coincides with cocoon encasement; removal of cocoons is a labor intensive process that may cause physical trauma to the larvae 20. More importantly, a B. thailandensis T6SS-1 mutant that is attenuated in both the Syrian hamster and the hissing cockroach 11, was virulent in Galleria, suggesting that Galleria is not a good model for the study of some mutants such as T6SS in B. thailandensis (data not shown).
The use of the hissing cockroach presents several advantages over the fruit fly. The hissing cockroach is large and of a substantial body mass with a tough exoskeleton that allows it to be easily handled during injections. In contrast, the fruit fly is small and requires specialized equipment for inoculation. Also, whereas the hissing cockroach naturally lives in temperatures that are similar to or exceed human body temperature, the optimal temperature for the fruit fly is between 22 to 28 &#176;C. This makes the fruit fly of limited use in the context of studying processes that are dependent on human body temperature (such as multi-nucleated giant cell formation in Burkholderia 10).
Some disadvantages to the use of hissing cockroaches do exist. The genetics of the hissing cockroach are not as well studied as those of Drosophila or even Galleria. The hissing cockroach also has a substantial &#34;ick&#34; or gross factor. However, the hissing cockroach remains an attractive and viable surrogate host for Burkholderia by providing clear advantages to its use in research that are unique to the species. As we have illustrated that the Madagascar hissing cockroach is a tractable surrogate host for Burkholderia, it very likely can also serve as a surrogate host for other bacterial pathogens and we are currently utilizing it in such studies.

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.

<table><tbody><tr><td>Madagascar hissing cockroach&lt;br/&gt; &amp;nbsp;&amp;nbsp;&lt;br/&gt; &amp;nbsp;&lt;br/&gt; &amp;nbsp;&lt;br/&gt; &amp;nbsp;</td><td>Carolina Biological Supply Co, Burlington, NC&amp;nbsp;</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&lt;br/&gt; www.dymax.com</td><td>T15469</td><td></td></tr><tr><td>Syringe (1 mL)&amp;nbsp;</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&amp;nbsp;</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 groups that were infected with wildtype B. pseudomallei K96243, parental B. mallei SR1, or B. thailandensis DW503 (Figure 1). Conversely, infection with virulent mutants (&#916;hcp2 or &#916;hcp3) killed the hissing cockroaches similarly to the wildtype B. pseudomallei (Figure 1). Infection with the mammalian avirulent Burkholderia species, B. thailandensis E264, and its aminoglycoside sensitive derivative DW503, show that the hissing cockroach model is particularly suitable for elucidating mutations in B. thailandensis that lead to attenuation (Figure 2). Thus, it is a more fitting animal model for B. thailandensis studies than rodent models. Increasing concentrations or multiple injections of CLQ did not kill the hissing cockroaches; this illustrates that drug toxicity can also be tested in the hissing cockroach model (Figure 3). Further, the efficacy of CLQ against B. thailandensis infection is shown in Figure 4. Important aspects of hissing cockroach care and infection are shown in Figure 5. Table 1 can be used to score the morbidity of hissing cockroaches during experiments.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56491/56491fig1.jpg" /> 
Figure 1: Hissing cockroach survival after injection with virulent and attenuated Burkholderia. Eight hissing cockroaches per group were injected with 25 &#181;L of bacterial suspension. Hissing cockroaches were checked for survival once a day for 5 days. (A) Hissing cockroaches were injected with parental B. mallei SR1 (open square) or the &#916;hcp1 mutant (closed square) at 100 CFUs. (B) Hissing cockroaches were injected with wild type B. pseudomallei K96243 (open square), &#916;hcp1 (closed square), &#916;hcp2 (open triangle), or &#916;hcp3 (open circle) mutant at 10 CFUs. (C) Hissing cockroaches were injected with parental B. thailandensis DW503 (open square) or &#916;hcp1 mutant (closed square) at 100 CFUs. Figure originally published in reference 11. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56491/56491fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56491/56491fig2.jpg" /> 
Figure 2: Hissing cockroach survival after injection of increasing concentrations of B. thailandensis for LD50 determination. Eight hissing cockroaches per group were injected with wildtype B. thailandensis E264 (A) or the aminoglycoside sensitive derivative DW503 (B) and survival was scored for 7 days. The LD50 is 3 CFUs for E264 and 6 CFUs for DW503. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56491/56491fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56491/56491fig3.jpg" /> 
Figure 3: Hissing cockroach survival after injection with chloroquine. Five hissing cockroaches per group were injected once (A) or twice on two consecutive days (B) with 250 (diamond), 500 (square), or 1,000 &#181;g (triangle) CLQ or PBS (circle) and survival was scored for 7 days. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56491/56491fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56491/56491fig4.jpg" /> 
Figure 4: Hissing cockroach survival after infection with B. thailandensis and treatment with chloroquine. 10 to 12 hissing cockroaches per group were infected with B. thailandensis DW503 and not treated (square), infected with B. thailandensis DW503 and treated with CLQ (triangle), treated with CLQ alone (diamond), or were uninfected and untreated (circle). Survival was recorded for 7 days. The survival curve, a composite of 4 separate experiments, is expressed as a percentage equal to the total number of survivors divided by the total number of hissing cockroaches for each treatment on the days indicated. The CFU inoculum given ranged from 10 to 20 LD50. Figure originally published in reference 10. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56491/56491fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56491/56491fig5.jpg" /> 
Figure 5: Images related to the hissing cockroach model. (A) A female hissing cockroach lacks protrusions on its head. (B) A male hissing cockroach can be identified by the presence of horns. (C) A hissing cockroach must molt out of its exoskeleton to grow. The emerging insect is white in color but gradually darkens as the new exoskeleton hardens. (D) Hissing cockroaches may be housed in a snap cap plastic container with ventilation holes during an experiment. (E) Under BSL3 conditions, hissing cockroaches are housed in screw cap plastic containers. (F) A large mouse cage is used to house a hissing cockroach colony. It should contain food, water and a cardboard egg carton for hiding. (G) Hissing cockroaches are injected with a 1 mL syringe attached to a repetitive pipette. (H) A hissing cockroach is inoculated by injection through the cutaneous membrane between the abdominal terga. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56491/56491fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1</td>
			<td>Live, Normal</td>
			<td>-actively mobile 
			-able to grasp and hold onto fingers when hissing cockroach is picked up</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Live, Lethargic</td>
			<td>-immobile but crawls when prodded</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Live, Moribund</td>
			<td>-immobile with legs tucked in 
			-does not move when prodded 
			-antennae and/ or legs move when prodded</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Dead</td>
			<td>-immobile with legs tucked in 
			-antennae do not move when prodded 
			-legs do not move when prodded</td>
		</tr>
	</tbody>
</table>
Table 1: Hissing cockroach morbidity scoring system. The overall score for a group of hissing cockroaches is based on the hissing cockroach with the highest score in the group.

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 ...

the-madagascar-hissing-cockroach-as-an-alternative-non-mammalian

Universidad San Sebastian

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

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Genetics

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

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 ...

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic fungal pathogen of humans, is the fourth most common cause of nosocomial infections which results in ~40% mortality. However, other C. albicans infections are less severe and rarely lethal and include vulvovaginal candidiasis, impacting ~75% of women, as well as oropharyngeal candidiasis, predominantly impacting infants, AIDS patients and cancer patients. While murine models are most frequently used to study C. albicans pathogenesis, these models predominantly assess host survival and are costly, time consuming, and limited in replication. Therefore, several mini-model systems, including Drosophila melanogaster, Danio rerio, Galleria mellonella, and Caenorhabditis elegans, have been developed to study C. albicans. These mini-models are well-suited for screening mutant libraries or diverse genetic backgrounds of C. albicans. Here we describe two approaches to study C. albicans infection using C. elegans. The first is a fecundity assay which measures host reproduction and monitors survival of individual hosts. The second is a lineage expansion assay which measures how C. albicans infection affects host population growth over multiple generations. Together, these assays provide a simple, cost-effective way to quickly assess C. albicans virulence.

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

Fungal opportunist pathogens can cause life-threatening as well as minor infections, but non-lethal phenotypes are frequently ignored when studying virulence. Therefore, we developed a nematode model that monitors both the survival and reproduction aspects of host to investigate fungal virulence.

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic ...

Applications of RNA Interference in American Cockroach

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous characteristics. Altogether with well-annotated genome sequences, these advantages have made American cockroach, Periplaneta americana, an important hemimetabolous insect model. Limited by the shortage of knockout strategy, effective RNA interference (RNAi)-based gene knockdown becomes an indispensable technique in functional gene research of P. americana. The present protocol describes the RNAi operation techniques in P. americana. The protocol includes (1) selection of the P. americana at proper developmental stages, (2) preparation for the injection setting, (3) dsRNA injection, and (4) gene knockdown efficiency detection. RNAi is a powerful reverse genetic tool in P. americana. The majority of P. americana tissues are sensitive to extracellular dsRNA. Its simplicity allows researchers to quickly obtain dysfunctional phenotypes under one or multiple targeting dsRNA injections, enabling researchers to better use the P. americana for developmental and metamorphic studies.

The present protocol describes step-by-step guidelines for the RNAi operation techniques in P. americana.

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous ...

Developmental Biology

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Infecting Mice with Malassezia spp. to Study the Fungus-Host Interaction

Animal models are crucial for infectious disease research. They provide an important basis for analyzing the full spectrum of interactions that occur between microbes and their host in vivo in a tissue-specific manner. Pathogenic fungi are increasingly recognized as a serious threat for humans and exploiting such infection models have greatly improved our understanding of fungal pathogenicity. Species of the genus Malassezia are the most abundant fungi of the human skin microbiota and they are also associated with the development of severe inflammatory skin disorders such as seborrheic dermatitis and atopic dermatitis. However, a causative link between Malassezia and disease pathogenesis remains unknown, a fact that can be attributed to the poor knowledge of the complex crosstalk of Malassezia with the skin immune system. This protocol describes the establishment of an experimental mouse model that allows studying the interaction of Malassezia with the mammalian skin in vivo. It outlines the method for cultivating Malassezia spp. under laboratory conditions, how to infect the murine skin with Malassezia spp. and how to assess the outcome of infection by means of the skin inflammation and fungal burden analyses. The model described here works in fully immunocompetent animals and does not rely on immune suppressive or antibiotic pretreatment of the animals. It is furthermore adaptable to virtually all genetically modified mouse strains and can be combined with other skin disease models. These features make this infection model a very powerful tool for studying in detail the innate and adaptive immune response of the host against Malassezia in the skin in vivo.

Infecting Mice with Malassezia spp. to Study the Fungus-Host Interaction

This protocol outlines the establishment of a mouse model for studying Malassezia-host interactions in the skin. It describes the cultivation of Malassezia in vitro, the infection of the murine skin with Malassezia, and the subsequent analysis of the inflammation and the fungal burden in the skin tissue.

Animal models are crucial for infectious disease research. They provide an important basis for analyzing the full spectrum of interactions that occur ...

Drosophila melanogaster Larva Injection Protocol

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly and raise ethical issues. Unconventional models are notoriously cheap, easy to handle and culture, and do not take much space. They are genetically amenable and possess complete genome sequences, and their use presents no ethical considerations. The fruit fly Drosophila melanogaster, for instance, has provided great insights into a variety of behavior, development, metabolism, and immunity research. More specifically, D. melanogaster adult flies and larvae possess several innate defense reactions that are shared with vertebrate animals. The mechanisms regulating immune responses have been mostly revealed through genetic and molecular studies in the D. melanogaster model. Here a novel larval injection technique is provided, which will further promote investigations of innate immune processes in D. melanogaster larvae and explore the pathogenesis of a wide range of microbial infections.

Drosophila melanogaster Larva Injection Protocol

Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

The use of unconventional models to study innate immunity and pathogen virulence provides a valuable alternative to mammalian models, which can be costly ...

Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment and maintenance of gnotobiotic American cockroaches (Periplaneta americana). This approach includes built-in sterility checks for ongoing quality control. Gnotobiotic insects are defined here as cockroaches that still contain their vertically transmitted endosymbiont (Blattabacterium) but lack other microbes that normally reside on their surface and in their digestive tract. For this protocol, egg cases (oothecae) are removed from a (nonsterile) stock colony and surface sterilized. Once collected and sterilized, the oothecae are incubated at 30 &#176;C for approximately 4&#8722;6 weeks on brain-heart infusion (BHI) agar until they hatch or are removed due to contamination. Hatched nymphs are transferred to an Erlenmeyer flask containing a BHI floor, sterile water, and sterile rat food. To ensure that the nymphs are not housing microbes that are unable to grow on BHI in the given conditions, an additional quality control measure uses restriction fragment-length polymorphism (RFLP) to test for nonendosymbiotic microbes. Gnotobiotic nymphs generated using this approach can be inoculated with simple or complex microbial communities and used as a tool in gut microbiome studies.

Establishment and Maintenance of Gnotobiotic American Cockroaches Periplaneta americana

This protocol is used in establishing and maintaining gnotobiotic American cockroaches (Periplaneta americana) by surface sterilizing the egg cases (oothecae) prior to hatching. These gnotobiotic insects contain their vertically transmitted Blattabacterium endosymbionts but have axenic guts.

Gnotobiotic animals are a powerful tool for the study of controls on microbiome structure and function. Presented here is a protocol for the establishment ...

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

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Establishment and Maintenance of Gnotobiotic American Cockroaches (Periplaneta americana)