Comparative experimental subcutaneous glanders and melioidosis in the common marmoset (Callithrix jacchus)

Abstract

Glanders and melioidosis are caused by two distinct Burkholderia species and have generally been considered to have similar disease progression. While both of these pathogens are HHS/CDC Tier 1 agents, natural infection with both these pathogens is primarily through skin inoculation. The common marmoset (Callithrix jacchus) was used to compare disease following experimental subcutaneous challenge. Acute, lethal disease was observed in marmosets following challenge with between 26 and 1.2 × 10(8) cfu Burkholderia pseudomallei within 22-85 h. The reproducibility and progression of the disease were assessed following a challenge of 1 × 10(2) cfu of B. pseudomallei. Melioidosis was characterised by high levels of bacteraemia, focal microgranuloma progressing to non-necrotic multifocal solid lesions in the livers and spleens and multi-organ failure. Lethal disease was observed in 93% of animals challenged with Burkholderia mallei, occurring between 5 and 10.6 days. Following challenge with 1 × 10(2) cfu of B. mallei, glanders was characterised with lymphatic spread of the bacteria and non-necrotic, multifocal solid lesions progressing to a multifocal lesion with severe necrosis and pneumonia. The experimental results confirmed that the disease pathology and presentation is strikingly different between the two pathogens. The marmoset provides a model of the human syndrome for both diseases facilitating the development of medical countermeasures.

Keywords: Burkholderia; animal model; histology.

Figure 1

Comparative virulence of different doses of bacteria administered by the subcutaneous route to marmosets. (a) Burkholderia pseudomallei (P < 0.001, general linear model with covariate), (b) Burkholderia mallei (P = 0.01, general linear model with covariate).

Figure 2

Core body temperature of marmosets postchallenge with either 2.97 × 10² ± 56 cfu of B pseudomallei or 3.65 × 10² ± 105 cfu of Burkholderia mallei by the subcutaneous route.

Figure 3

Bacterial load in various marmoset tissues at specific times postchallenge by the subcutaneous route. (a) Burkholderia pseudomallei, mean challenge dose 1.85 × 10² ± 15 cfu. Statistically significant changes in the liver (P = 0.004), spleen (P = 0.003), kidney (P = 0.002), lung (P = 0.003), brain (P = 0.002) and blood (P = 0.001) were observed with time, with most of the significance occurring between t = 12 and t = 48 or for terminal animals (Independent sample Kruskal–Wallis Test). (b) Burkholderia mallei, mean challenge dose 1.79 × 10² ± 14 cfu. Statistically significant changes in the liver (P = 0.004), spleen (P = 0.003), kidney (P = 0.002), lung (P = 0.002), brain (P = 0.002) and blood (P = 0.001) were observed with time, with most of the significance occurring between t = 24 and t = 144 or for terminal animals (Independent sample Kruskal–Wallis Test).

Figure 4

Representative H&E and IHC stained tissue sections from the spleen of a marmoset humanely euthanised at 48 h p.c. challenged with 1.85 × 10² ± 57 cfu of Burkholderia pseudomallei by the subcutaneous route. (a) H & E showing multifocal lesion with severe necrosis (++++), (b) Burkholderia pseudomallei antigen IHC showing abundant bacteria associated with the lesion (++++), (c) IHC staining showing a moderate number of macrophages associated with the lesion (+++), (d) IHC staining showing very few T cells associated with the lesion (+), (e) IHC staining showing very few B cells associated with the lesion (+), (f) IHC staining showing very few inducible Nitric oxide synthase (iNOS) producing cells associated with the lesion (+).

Figure 5

Representative H&E and IHC stained tissue sections from the inoculation site of a marmoset humanely euthanised at 36 h p.c. challenged with 1.85 × 10² ± 57 cfu of Burkholderia pseudomallei by the subcutaneous route. (a) H & E showing multifocal lesion with severe necrosis (++++), (b) B. pseudomallei antigen IHC showing abundant bacteria associated with the lesion (++++), (c) IHC staining showing a small number of macrophages associated with the lesion (++), (d) IHC staining showing very few T cells associated with the lesion (+), (e) IHC staining showing no B cells associated with the lesion, (f) IHC staining showing very few inducible Nitric oxide synthase (iNOS) producing cells associated with the lesion (+).

Figure 6

Key blood parameter data from marmosets challenged with 1.85 × 10² ± 57 cfu of Burkholderia pseudomallei by the subcutaneous route at specific times postchallenge. (a) Liver function enzymes, ALT (Alanine transaminase), AST (Aspartate aminotransferase), ALKP (Alkaline phosphatase), (b) AMYL (amylase), LDH (lactate dehydrogenase), (c) White blood cell distribution, WBC (white blood cell count × 10⁹/l), %NEU (per cent neutrophils), %LYM (Per cent lymphocytes), %MONO (per cent monocyte). Each time point represents data from four different animals humanely euthanised at that time, therefore connecting lines are only included to demonstrate the trend of the data sets. Error bars represent the standard error of the mean (SEM).

Figure 7

Representative H&E and IHC stained tissue sections from the liver of a marmoset humanely euthanised at 144 h p.c. challenged with 1.79 × 10² ± 20 cfu of Burkholderia mallei by the subcutaneous route. (a) H & E showing non-necrotic multifocal solid lesions (++), (b) Burkholderia mallei antigen IHC showing a small number of bacteria associated with the lesion (++), (c) IHC staining showing a small number macrophages associated with the lesion (++), (d) IHC staining showing very few T cells associated with the lesion (+), (e) IHC staining showing no B cells associated with the lesion, (f) IHC staining showing a small number inducible Nitric oxide synthase (iNOS) producing cells associated with the lesion (++).

Figure 8

Representative H&E and IHC stained tissue sections from the lungs of a marmoset humanely euthanised at 144 h p.c. challenged with 1.79 × 10² ± 20 cfu of Burkholderia mallei by the subcutaneous route. (a) H & E showing multifocal lesion with severe necrosis (++++), (b) B. mallei antigen IHC showing abundant bacteria associated with the lesion (++++), (c) IHC staining showing abundant macrophages associated with the lesion (++++), (d) IHC staining showing a small number of T cells associated with the lesion (++), (e) IHC staining showing very few B cells associated with the lesion (+), (f) IHC staining showing a small number inducible Nitric oxide synthase (iNOS) producing cells associated with the lesion (++).

Figure 9

Key disease features from marmosets challenged with 1.79 × 10² ± 20 cfu of Burkholderia mallei by the subcutaneous route at specific times postchallenge. (a) Splenic weight with time, (b) Liver function enzymes, ALT (Alanine transaminase), AST (Aspartate aminotransferase), ALKP (Alkaline phosphatase), (c) Platelet and clotting factors, PLT (Platelet, K/μl), PT (Prothrombin Times, s), aPPT (activated partial thromboplastin time, s), (d) White blood cell distribution, WBC (white blood cell count × 10⁹/l), %NEU (per cent neutrophils), %LYM (per cent lymphocytes). Each time point represents data from four different animals humanely euthanised at that time, therefore connecting lines are only included to demonstrate the trend of the data sets. Error bars represent the standard error of the mean (SEM).