Figure 1

Figure 1. Postmortem photographs of an American quarter horse in study of multisystemic disease and septicemia caused by presumptive Burkholderia pseudomallei, Florida, USA. A) The submandibular lymph node was enlarged...

In 2006, an 8-year-old quarter horse gelding with a left retropharyngeal abscess was seen at the University of Florida Veterinary Medical Center (Gainesville, Florida, USA). Clinical examination and radiographs revealed a well-circumscribed 20 × 10 × 18-cm round soft tissue mass caudal to the ramus of the mandible with displacement of the left guttural pouch. Other examination findings included diffuse interstitial pneumonia, multiple cutaneous ulcers on the dorsal midline, anterior uveitis in the right eye, enlarged mesenteric lymph nodes, and an aneurysm of the right renal artery. Because of the animal’s declining clinical condition, it was humanely euthanized. At necropsy examination, gross findings included a large, soft, round abscess in the retropharyngeal space, compressing the guttural pouch with a draining tract into the epidermis (Figure 1, panel A). The mesenteric lymph nodes were diffusely enlarged, ranging from 2.0 to 6.0 cm in diameter, with purulent material and hemorrhage (Figure 1, panel B). Other gross findings included diffuse interstitial pneumonia with multifocal 2.0 × 2.0 × 2.0-cm peribronchiolar abscesses (Figure 1 panel C). Small 1.0 × 1.0 × 1.0-cm randomly scattered areas of necrosis were multifocally scattered in the liver (Figure 1, panel D); anterior uveitis was present in the right eye.

Figure 2

Figure 2. Photomicrography of tissues from study of multisystemic disease and septicemia caused by presumptive Burkholderia pseudomalleiin American quarter horse, Florida, USA. Photomicrographs show hematoxylin and eosin staining of submandibular...

Tissue specimens were fixed in 10% neutral buffered formalin, processed routinely, and embedded in paraffin. Paraffin-embedded sections were cut 4 μm thick and examined after staining with hematoxylin and eosin. We performed histochemical Gram stain to screen for bacteria. Histologic sections from the affected submandibular lymph node (Figure 2, panel A), lung (Figure 2, panel B), and mesentery revealed multifocal to coalescing abscesses and pyogranulomas containing necrotic cellular debris and degenerative neutrophils mixed with hemosiderophages (Figure 2, panels C–E). Numerous macrophages and neutrophils contained intracytoplasmic 1–2 µm gram-negative bacilli (Figure 2, panel F).

Postmortem aerobic culture of the retropharyngeal and mesenteric lymph nodes revealed pure B. cepacia complex growth. A fatty acid analysis using gas chromatography of the bacterial isolate supported this finding. B. cepacia complex is a heterogeneous group of bacteria from the Burkholderia genus that typically causes opportunistic infections in immunocompromised hosts. Disease outbreaks often occur in the hospital setting through contaminated medical devices or in patients with chronic respiratory diseases, such as cystic fibrosis (10).

The clinical manifestations of this case were unusual for B. cepacia complex and more consistent with the clinical course of acute, highly pathogenic Burkholderia species. We suspect that the organism was misidentified. The precise methodology of the original diagnosis was not reported in the case documents and is unknown to the authors of this report. Current biochemical systems in historically nonendemic areas often mistakenly identify B. pseudomallei strains as members of B. cepacia complex (11). Our team concluded that further investigation was necessary. Unfortunately, because of DNA fragmentation during routine tissue processing for histopathology, attempts at isolating bacterial DNA from the formalin-fixed paraffin-embedded tissue samples were unsuccessful. A limitation of this study is the inability to extract bacterial DNA from the tissue block, preventing definitive identification of B. pseudomallei.

Figure 3

Figure 3. Immunohistochemical examination of tissues from American quarter horse with multisystemic disease and septicemia caused by presumptive Burkholderia pseudomallei, Florida, USA. Photomicrographs show immunohistochemistry for monoclonal antibodies 3A2 (A),...

B. pseudomallei employs a network of polysaccharides, including capsular polysaccharides (CPS) and lipopolysaccharides (LPS), to enhance virulence and immune evasion (12,13). A panel of monoclonal antibodies targeting B. pseudomallei and B. mallei CPS (4C4), typical LPS O-Ag serotype A (4C7), atypical LPS O-Ag serotype B and its variant B2 (3A2, 5B4), and B. mallei LPS O-Ag (3D11) were used (13). The secondary antibody was biotinylated goat anti-mouse IgG (1:200 dilution). Intrahistiocytic bacilli were immunopositive for 4C4, 3A2, and 5B4 and immunonegative for 4C7 and 3D11 (Figure 3). We tested 19 different strains of B. cepacia complex and demonstrated no cross-reactivity to 4C4, 4C7, 3A2, and 5B4 on Western blot analysis (Table). Previous studies have shown B. pseudomallei cross-reactivity by some B. cepacia complex strains to B. pseudomallei–like CPS-specific antibodies (4C4) (14) and B. mallei to monoclonal antibodies to typical type A LPS (4C7); however, cross-reactivity of either species to B. pseudomallei atypical type B LPS (3A2 and 5B4) has not been reported, and cross-reactivity was not noted in our experiments (15). The immunohistochemistry and Western blot results suggest infection with B. pseudomallei with atypical O-Ag type B or B2.

This case report describes the histomorphology and immunohistochemical identification of an atypical serotype of B. pseudomallei in a horse with clinical signs consistent with melioidosis. Although type A is the most common LPS O-Ag type of B. pseudomallei, accounting for most infections, types B and B2 are found more frequently in Australia (13). The route of exposure and travel history of this horse is unknown. On the basis of the immunohistochemistry results in this case, we conclude that the initial culture and biochemical analysis misidentified B. cepacia complex. Western blot assay analysis of purified LPS from numerous bacteria in the B. cepacia complex failed to highlight any of the mentioned monoclonal antibodies. This case underscores the potential pathological effects of B. pseudomallei in horses and other animals in the United States, emphasizing the need for increased awareness and understanding of its emergence as a potential pathogen in diverse species.