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

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...

<ol>
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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. I. <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+as+a+model+system+for+the+study+of+the+virulence-associated+type+III+secretion+system+of+Burkholderia+pseudomallei.">Burkholderia thailandensis as a model system for the study of the virulence-associated type III secretion system of Burkholderia pseudomallei.</a> Infect Immun. 76 (11), 5402-5411 (2008).</li><li>West, T. E., Frevert, C. W., Liggitt, H. D., Skerrett, S. 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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. E., 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=The+orange+spotted+cockroach+(Blaptica+dubia,+Serville+1839)+is+a+permissive+experimental+host+for+Francisella+tularensis.">The orange spotted cockroach (Blaptica dubia, Serville 1839) is a permissive experimental host for Francisella tularensis.</a> PeerJ Preprints. 4, e1524v1522 (2016).</li></ol>

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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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