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

Abstract

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.

Figure 1

A representative juvenile Madagascar hissing cockroach used as a surrogate host forB. pseudomallei,B. mallei, andB. thailandensisinfection studies. The black arrows show the locations where bacteria were inoculated into the dorsal abdominal section of the MH cockroach, between the third and the fifth terga from the posterior.

Figure 2

B. pseudomalleiis virulent for the MH cockroach and T6SS-1 mutants are attenuated. Groups of eight MH cockroaches were challenged by the intra-abdominal route of infection and MH cockroach deaths were monitored for 5 days at 37°C. (A) 10¹ cfu. (B) 10² cfu. (C) 10³ cfu. (D) 10⁴ cfu. (E) 10⁵ cfu. Bp, K96243; Bp Δhcp1, DDS1498A; Bp ΔvgrG1-5’, DDS1503-1A; Bp ΔvgrG1-3’, DDS1503-2A.

Figure 3

B. pseudomalleiT6SS-2, T6SS-3, T6SS-4, T6SS-5, and T6SS-6 mutants are virulent in the MH cockroach. (A) 10¹ cfu. (B) 10² cfu. (C) 10³ cfu. (D) 10⁴ cfu. (E) 10⁵ cfu. Bp, K96243; Bp Δhcp2, DDS0518A; Bp Δhcp3, DDS2098A; Bp Δhcp4, DDS0171A; Bp Δhcp5, DDS0099A; Bp Δhcp6, DDL3105A.

Figure 4

B. malleiandB. thailandensisare virulent for the MH cockroach and their T6SS-1 mutants are attenuated. (A) 10¹ cfu. (B) 10² cfu. (C) 10³ cfu. Bm, SR1; Bm Δhcp1, DDA0742; Bt, DW503; Bt Δhcp1, DDII0868.

Figure 5

B. pseudomalleimultiplies inside MH cockroach hemocytes. Panel A is a representative micrograph of hemolymph obtained from a MH cockroach infected with B. pseudomallei K96243 and stained with DAPI. The white arrows show hemocytes that harbor intracellular B. pseudomallei. The white scale bar is 100 μm. Panels B and C show a higher magnification of a B. pseudomallei-infected hemocyte using bright field microscopy (B) and stained with DAPI and a Burkholderia-specific rabbit polyclonal antibody (C). The secondary antibody used, Alexa Fluor 588 goat anti-rabbit IgG, stained B. pseudomallei green. The magnified inset in C shows individual bacilli within the hemocyte cytosol and the white arrows show extracellular bacteria in the hemolymph. The white scale bars in B and C are 20 μm. The results are representative images from eight MH cockroaches infected with ~ 10³ cfu of B. pseudomallei K96243.