Sporulation and Germination of Paenibacillus larvae Cells.

Endospores are metabolically dormant cells formed by a variety of Gram-positive bacteria within the phylum Firmicutes in response to nutrient limiting or otherwise unfavorable growth conditions. American foulbrood disease (AFB) is a serious disease of honeybees that is caused by the introduction of Paenibacillus larvae endospores into a honeybee colony. Progression to fulminant disease and eventual collapse of the colony requires multiple rounds of endospore germination, vegetative replication, endospore formation, and subsequent spread within the colony. This unit includes protocols for the in vitro sporulation and germination of P. larvae to assist investigators in the study of these processes. © 2018 by John Wiley & Sons, Inc.

Growth and Laboratory Maintenance of Paenibacillus larvae.

Paenibacillus larvae is a Gram-positive, spore-forming bacterium and the causative agent of American foulbrood disease (AFB), a highly contagious, fatal disease affecting managed honeybee (Apis mellifera) colonies. As the etiological agent of American foulbrood disease, P. larvae is the most economically significant bacterial pathogen infecting honeybees. This unit includes protocols for the in vitro growth and laboratory maintenance of P. larvae. © 2018 by John Wiley & Sons, Inc.

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.

The orange spotted cockroach (Blaptica dubia, Serville 1839) is a permissive experimental host for Francisella tularensis.

Francisella tularensis is a zoonotic bacterial pathogen that causes severe disease in a wide range of host animals, including humans. Well-developed murine models of F. tularensis pathogenesis are available, but they do not meet the needs of all investigators. However, researchers are increasingly turning to insect host systems as a cost-effective alternative that allows greater increased experimental throughput without the regulatory requirements associated with the use of mammals in biomedical research. Unfortunately, the utility of previously-described insect hosts is limited because of temperature restriction, short lifespans, and concerns about the immunological status of insects mass-produced for other purposes. Here, we present a novel host species, the orange spotted (OS) cockroach (Blaptica dubia), that overcomes these limitations and is readily infected by F. tularensis. Intrahemocoel inoculation was accomplished using standard laboratory equipment and lethality was directly proportional to the number of bacteria injected. Progression of infection differed in insects housed at low and high temperatures and F. tularensis mutants lacking key virulence components were attenuated in OS cockroaches. Finally, antibiotics were delivered to infected OS cockroaches by systemic injection and controlled feeding; in the latter case, protection correlated with oral bioavailability in mammals. Collectively, these results demonstrate that this new host system provides investigators with a new tool capable of interrogating F. tularensis virulence and immune evasion in situations where mammalian models are not available or appropriate, such as undirected screens of large mutant libraries.

The Madagascar hissing cockroach as a novel surrogate host for Burkholderia pseudomallei, B. mallei and B. thailandensis.

BACKGROUND: Burkholderia pseudomallei and Burkholderia mallei are gram-negative pathogens responsible for the diseases melioidosis and glanders, respectively. Both species cause disease in humans and animals and have been designated as category B select agents by the Centers for Disease Control and Prevention (CDC). Burkholderia thailandensis is a closely related bacterium that is generally considered avirulent for humans. While it can cause disease in rodents, the B. thailandensis 50% lethal dose (LD50) is typically ≥ 104-fold higher than the B. pseudomallei and B. mallei LD50 in mammalian models of infection. Here we describe an alternative to mammalian hosts in the study of virulence and host-pathogen interactions of these Burkholderia species. RESULTS: Madagascar hissing cockroaches (MH cockroaches) possess a number of qualities that make them desirable for use as a surrogate host, including ease of breeding, ease of handling, a competent innate immune system, and the ability to survive at 37°C. MH cockroaches were highly susceptible to infection with B. pseudomallei, B. mallei and B. thailandensis and the LD50 was <10 colony-forming units (cfu) for all three species. In comparison, the LD50 for Escherichia coli in MH cockroaches was >105 cfu. B. pseudomallei, B. mallei, and B. thailandensis cluster 1 type VI secretion system (T6SS-1) mutants were all attenuated in MH cockroaches, which is consistent with previous virulence studies conducted in rodents. B. pseudomallei mutants deficient in the other five T6SS gene clusters, T6SS-2 through T6SS-6, were virulent in both MH cockroaches and hamsters. Hemocytes obtained from MH cockroaches infected with B. pseudomallei harbored numerous intracellular bacteria, suggesting that this facultative intracellular pathogen can survive and replicate inside of MH cockroach phagocytic cells. The hemolymph extracted from these MH cockroaches also contained multinuclear giant cells (MNGCs) with intracellular B. pseudomallei, which indicates that infected hemocytes can fuse while flowing through the insect's open circulatory system in vivo. CONCLUSIONS: The results demonstrate that MH cockroaches are an attractive alternative to mammals to study host-pathogen interactions and may allow the identification of new Burkholderia virulence determinants. The importance of T6SS-1 as a virulence factor in MH cockroaches and rodents suggests that the primary role of this secretion system is to target evasion of the innate immune system.