Natural Burkholderia mallei Infection in Dromedary, Bahrain

We confirm a natural infection of dromedaries with glanders. Multilocus variable number tandem repeat analysis of a Burkholderia mallei strain isolated from a diseased dromedary in Bahrain revealed close genetic proximity to strain Dubai 7, which caused an outbreak of glanders in horses in the United Arab Emirates in 2004.


The Study
On a small private farm, 2 of 7 horses had positive serologic reactions and showed typical clinical signs of glanders. On the same premises, 6 dromedaries were kept several meters away from the sick horses in a separate enclosure. Three dromedaries that showed clinical signs of glanders, including severe mucopurulent discharge from both nostrils (Figure 1, panel A), fever, emaciation, and fatigue, died. One of these dromedaries underwent necropsy. Serum samples from this dromedary tested positive for glanders with both the OIE-acknowledged complement fi xation test (titer 10+++) and with the Central Veterinary Research Laboratory-developed in-house competitive ELISA (7) with an inhibition of 57%.
An EDTA blood sample was incubated for 11 days in a blood culture system (Oxoid, Cambridge, UK) until it became positive. This fl uid was then cultured on sheep blood agar at 37°C for 72 h. The isolate stained poorly gram-negative, was rod shaped, and tested oxidase positive. Suspected B. mallei colonies were analyzed with the API 20 NE-test (bioMérieux, Marcy l'Etoile, France) and were positive for nitrate, glucose assimilation, arginine dehydrolase (after 4 days of incubation), N-acetyl glucosamine, and potassium gluconate. The API 20 NEtest identifi ed the colonies as B. mallei because the same API ID number (1140504) occurred as in the previously isolated Dubai 7 strain (1).
During necropsy, typical glanderous lesions in the lung, choanae, and nasal septae were observed. Golf ballsized reddish-gray nodules resembling tubercles with a central gray necrotic zone were detected in the lungs. In the choanae and nasal septae, stellate scars, ulcers, and honeycomb necrotic patches covered with yellow pus (Figure 1, panel B) were seen. Glanderous lesions were absent from other organs.
The presence of B. mallei in lung and choanae specimens was examined by using standard culturing techniques as described by Wittig et al. (1). For bacterial growth, sheep blood agar plates were incubated at 37°C for 72 h. B. mallei was directly isolated from the pus, which had accumulated in the choanae, but not from nasal and eye swabs and not from the lung lesions. However, the tissue samples were stored at -20°C for >20 days before incubation.
Multilocus variable number tandem repeat analysis based on 23 different loci (10) was used for further subtyping through sequencing of the variable number tandem repeat regions (online Appendix Table, www.cdc.gov/EID/ content/17/7/1277-appT.htm). Phylogenetic analysis of these data was performed as described by Hornstra et al. (10) and compared with existing B. mallei strains ( Figure  2). In this analysis, the strain (THSK2) isolated from the dromedary clustered with B. mallei strain Dubai 7 ( Figure  2) that had been isolated from a horse in the United Arab Emirates (11).

Conclusions
Old World camels, the dromedary (C. dromedarius), and the Bactrian camel (C. bactrianus) are susceptible to B. mallei (glanders) and B. pseudomallei (melioidosis) infection (12,13). However, reports of B. mallei infection in dromedaries have described artifi cial infections (4,5). We report natural B. mallei infection in a dromedary that occurred during a glanders outbreak in horses.
Clinical signs as well as gross pathologic and microscopic lesions of the diseased dromedary were similar to changes seen in equids. These changes were dominated by severe mucopurulent nasal discharge, nodules and ulcers with pus in the choanae and nasal septae, and granulomas in the lungs that resembled tubercle lesions (pseudo tubercles).
B. mallei was isolated from venous blood, indicating septicemia. The pathogen was also directly isolated from the pus, which had accumulated in the choanae, but not from nasal and eye swabs and, unexpectedly not from the lung lesions. A possible explanation for the failure to isolate B. mallei from the nasal swabs was the heavy growth of various other contaminating bacteria because no selective culture medium exists for B. mallei. It could also be explained by storage of the samples at -20°C for >20 days, which probably destroyed the bacteria.
The genetic relatedness of the strain isolated from the dromedary to the strain isolated in 2004 from horses in the United Arab Emirates suggests that this strain might be endemic to this region. It also appears to be genetically distinct from a recent outbreak in Pakistan, demonstrating the persistence of multiple strains on a larger geographic scale. Isolation of this pathogen from both camels and horses poses new challenges to the international trade of equids from and to countries where camels are raised.