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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Preparations for Maintaining a Hissing Cockroach Colony

1. 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 °C, do not apply petroleum jelly.
2. Include a cardboard egg carton placed upside down inside the cage to ....

Results

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 (Δhcp1) than in group.......

Discussion

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.......

Materials

List of materials used in this article

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