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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 LD50in 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.
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
370
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.
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
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