Pathogenesis of percutaneous infection of goats with Burkholderia pseudomallei: clinical, pathologic, and immunological responses in chronic melioidosis

Abstract

Melioidosis is a severe suppurative to granulomatous infection caused by Burkholderia pseudomallei. The disease is endemic to South-East Asia and Northern Australasia and is also of interest as a potential biological weapon. Natural infection can occur by percutaneous inoculation, inhalation or ingestion, but the relative importance of each route is unknown. Experimental infection models using mice have shown inhalation to be the most lethal route of exposure, but few studies have examined the pathogenesis of percutaneous infection despite its presumptive importance in natural disease. Caprine models are useful in the study of melioidosis because goats are susceptible to natural infection by B. pseudomallei, display similar epizootiology/epidemiology to that of humans within the endemic range and develop similar pathologic lesions. Percutaneous inoculation with 10(4) CFU of B. pseudomallei produced disease in all experimental animals with rapid dissemination to the lungs, spleen and kidneys. Initial fever was brief, but temperatures did not return to pre-infection levels until day 18, concurrent with a dramatic lymphocytosis and the transition to chronic disease. Distribution and appearance of gross pathologic and radiographic lesions in goats were similar to caprine aerosol infection and to reported human disease. The similarities seen despite different routes of infection suggest that host or bacterial factors may be more important than the route of infection in disease pathogenesis. The nature of melioidosis in goats makes it amenable for modelling additional risk factors to produce acute clinical disease, which is important to the study of human melioidosis.

Keywords: Burkholderia pseudomallei; goat; melioidosis; model; pathogenesis.

Figure 1

Mean temperatures and selected representative goats. Peaks in temperature were typically seen on days 1 and 2, although later peaks are possible as seen in BpG49 on day 4. Day 18 was the first day that the mean temperature was not significantly different from day 0 (*P = 0.14). The peak in BpG57 on day 28 highlights the chronic active nature of disease at later time points. Mean (•), BpG48 (), BpG49 (), BpG56 (), BpG57 (□).

Figure 2

Mean granulocyte count, lymphocyte count and temperature. Granulocytes () showed an initial peak on day 2 and then on day 18 before a significant decrease on day 32 and a return to pre-infection levels on day 39. Lymphocytes () first increase over pre-infection levels on day 11, with a significant increase out of the normal range on day 18, which is associated with a significant decrease in temperature () to pre-infection levels. Lymphocytes significantly decrease to pre-infection levels on day 32. (‡) Different than previous measure and pre-infection, (†) different than previous measure but no different than pre-infection, (*) different than pre-infection, (ø) not different than previous measure or pre-infection, significance P < 0.05.

Figure 3

Thoracic and extirpated lung radiographs of goats infected percutaneously with B. pseudomallei. (a) BpG47, day 7, right lateral thorax, moderate bronchointerstitial infiltrates and small ill-defined nodular infiltrates in the dorsocaudal and cranioventral lung lobes (arrows). (b) BpG59, day 14, left lateral thorax, moderate-severe bronchointerstitial infiltrates and numerous nodules (5–11 mm) throughout the pulmonary parenchyma (arrows). (c) BpG54, day 21, left lateral thorax, moderate diffuse bronchointerstitial infiltrates, patchy alveolar infiltrates in the mid-caudal lung lobes, bronchoalveolar infiltrates in cranioventral lung lobes (arrow) and numerous nodular opacities (6–16 mm) throughout the pulmonary parenchyma. (d) BpG59, day 42, progression of the bronchointerstitial infiltrates with several poorly defined opacities/alveolar infiltrates within the lungs, although nodules are less defined than on day 14. (e) BpG54, day 21, extirpated lungs, numerous nodules (thin arrow) and several focal areas of consolidation (arrow) throughout the parenchyma with a dense bronchial pattern diffusely. (f) BpG59, day 42, extirpated lungs, severe diffuse bronchointerstitial pattern with a large 2 cm cavitated nodule (inset) in the right caudal lung lobe (arrow) with several other nodules in the remaining lungs.

Figure 4

Gross lesions of caprine melioidosis. (a) Lung, numerous, multifocal, discrete to coalescing abscesses/pyogranulomas associated with extensive consolidation and haemorrhage; (b) lung, typical subpleural targetoid pyogranulomas with tan purulent centres and consolidated hyperaemic rims that are rarely associated with fibrous pleural adhesions; (c) spleen, multiple, large pyogranulomas with adhesions of the splenic capsule to the peritoneum; (d) kidney, multifocally extensive and coalescing pyogranulomas spanning from cortex to medulla.

Figure 5

Extrapulmonary histologic lesions of percutaneous caprine melioidosis. (a) Skin, multifocally extensive neutrophilic infiltrates (N) expand the deep dermis surrounding vessels and adnexa with subepidermal clefting (C); (b) prescapular LN, capsulitis (C) and early subcapsular abscess (A) involving the subjacent lymphoid follicle; (c) heart, lymphoplasmacytic interstitial myocarditis (arrow) infiltrating along tissue planes; (d) spleen, upper panel: neural microabscesses (M) in a larger splenic nerve, lower panel: mild suppurative perineuritis (arrow) and minimal numbers of neutrophils infiltrating (arrow head) a smaller splenic nerve; (e) liver, vasocentric portal microabscess (M) obliterating the lower pole of a portal vein (V); (f) kidney, suppurative pyelonephritis with degenerate neutrophils (N) filling the lumen, infiltrating the pelvic lining forming a mucosal abscess (arrow), and invading the suburothelial stroma; (g) adrenal, microabscesses (arrows) with lytic necrosis are scattered within the zonae fasciculata and reticularis; and (h) mesenteric LN, phagocytically active macrophages with abundant intrahistiocytic (white arrow) and extracellular (black arrow) coccobacilli. Haematoxylin and eosin staining.

Figure 6

Respiratory lesions of percutaneous caprine melioidosis. (a) Suppurative tracheitis showing neutrophil transcytosis (arrows) and submucosal expansion by a thick band of lymphoplasmacytes (L); (b) suppurative bronchiolitis with focal necrosis of the bronchiolar wall (arrow) and luminal aggregates of neutrophils; (c) early vasocentric abscess formation, neutrophils (N) exiting vessel (V) and filling perivascular alveolar and interalveolar spaces; (d) microscopic pulmonary haemorrhage centreed on interlobular vessel (V), haemorrhage (H) to the left of interlobular septum (S) and oedema (E) to the right; (e) a fibrinocellular thrombus (T) occludes the lumen of a small vein; (f) small arteriole, typical lesion of leukocytoclastic vasculitis with neutrophil infiltration (arrow) into the wall and medial hyperplasia, evidenced by a mitotic figure (arrowhead); (g) chronic active bronchointerstitial pneumonia showing septal fibrosis (F) alongside alveolar oedema (E), and suppurative inflammation (arrow); and (h) pulmonary pyogranuloma encased in a thick fibrous capsule (C), an outer mantle of lymphocytes (L) and an inner core of macrophages (M), including giant cells (arrow), which have effected near complete resolution of the liquefactive centre via phagocytosis of neutrophils (N) and cellular debris. Haematoxylin and eosin staining.

Figure 7

Cytokine production in goats infected with B. pseudomallei. Responses of (a) TGF-β1 (log pg/ml), (b) IFNγ (log ng/ml) and (c) CCL2 (log ng/ml) in aerosol () and percutaneous () groups. Significant elevations were only present for CCL2 in aerosol-infected goats and IFNγ in percutaneously infected goats. TGF-β1 shows an initial decrease followed by increases through day 21 before returning to pre-infection levels on day 42. (‡) Different than previous measure and pre-infection, (†) different than previous measure but no different than pre-infection, (*) different than pre-infection, (ø) not different than previous measure or pre-infection, significance P < 0.05.