Burkholderia mallei (Glanders) in Horses and Humans: A Notifiable Zoonosis

Etiology and Taxonomic Classification

Burkholderia mallei is a Gram-negative, nonmotile, facultative intracellular bacillus belonging to the Burkholderia cepacia complex. It is the etiologic agent of glanders, a highly contagious and often fatal disease of equids (horses, donkeys, and mules). The organism is a host-adapted pathogen that does not persist in the environment, requiring direct contact or fomite transmission for spread. B. mallei is genetically closely related to Burkholderia pseudomallei, the agent of melioidosis, but differs in that it lacks a flagellum and has a reduced genome due to its obligate parasitic lifestyle [1, 2]. The bacterium is classified as a Tier 1 select agent by national regulatory bodies due to its potential for deliberate misuse and its high pathogenicity [3].

The cell wall of B. mallei contains lipopolysaccharide (LPS) with O-antigenic polysaccharides that are immunodominant. The bacterium expresses a polysaccharide capsule, type III and type VI secretion systems, and multiple adhesins that facilitate invasion of host epithelial cells and evasion of phagocytic killing [1, 35]. A conserved protein O-glycosylation pathway in the Burkholderia genus has been exploited for the development of glycoengineered antigens for serodiagnosis [35].

Epidemiology and Geographic Distribution

Glanders is a notifiable disease under the World Organisation for Animal Health (WOAH) Terrestrial Animal Health Code. Historically, the disease was distributed worldwide but was eradicated from most developed countries through rigorous test-and-slaughter programs. It remains endemic in parts of Africa, Asia, the Middle East, and South America [1, 4, 5].

Recent serosurveillance and molecular epidemiological studies have documented the re-emergence of glanders in Mongolia. A region-wide serosurvey using complement fixation test (CFT) and ELISA revealed a seroprevalence of 2.3% among horses in central and eastern Mongolia, with higher rates in areas with dense equid populations and limited veterinary oversight [6, 40]. Molecular characterization of Mongolian isolates using multilocus sequence typing (MLST) and whole-genome single nucleotide polymorphism (SNP) analysis demonstrated a distinct clade, suggesting a long-standing endemic focus [7].

In Brazil, glanders is endemic in several states, particularly in the Northeast region. Retrospective spatial and spatiotemporal analyses from 2020 to 2024 identified persistent clusters of infection in the state of Piauí, with a high density of positive cases in municipalities with intensive equine trade and movement [4, 5, 8]. Molecular detection of B. mallei in different geographic regions of Brazil using PCR-based methods confirmed the circulation of multiple genetic lineages [9, 10]. A case series from Brazil provided detailed histopathological and diagnostic findings, emphasizing the chronic, suppurative nature of the disease in naturally infected horses [11].

In Iran, serological and bacteriological surveillance in the central region reported a seroprevalence of 1.8% among horses, with isolation of B. mallei from nasal swabs and lymph node aspirates [12, 13]. The first molecular characterization of Iranian isolates using PCR-restriction fragment length polymorphism (RFLP) analysis of the fliP gene confirmed the presence of a single circulating genotype [14].

Nepal reported its first glanders cases in 2022, detected through a combination of CFT and bacterial isolation. The index cases were identified in horses imported from India, underscoring the need for enhanced border controls and regional surveillance [15, 16]. The emergence of glanders in Nepal highlights the risk of transboundary spread through uncontrolled movement of equids.

Clinical Signs in Horses

The clinical presentation of glanders in horses can be acute, chronic, or latent. The acute form is more common in donkeys and mules, characterized by high fever, depression, and rapid progression to septicemia and death within days. In horses, the chronic form is more typical, with a protracted course lasting weeks to months [1, 11].

The classic clinical triad includes:

• Nasal form: Mucopurulent to hemorrhagic nasal discharge, ulceration of the nasal mucosa, and formation of granulomatous nodules (farcy buds) on the nasal septum. These ulcers may coalesce, leading to extensive tissue necrosis and scarring.
• Pulmonary form: Coughing, dyspnea, and tachypnea due to bronchopneumonia and pulmonary abscessation. Thoracic auscultation may reveal crackles and wheezes.
• Cutaneous form (farcy): Lymphangitis with nodular thickening of the lymphatic vessels, particularly along the limbs and ventral abdomen. These nodules (farcy buds) may ulcerate, discharging a thick, oily pus. Regional lymphadenopathy is common.

Systemic signs include intermittent fever, progressive weight loss, and anemia. Latently infected horses may show no clinical signs but can shed the organism intermittently, serving as a reservoir for transmission [1, 11, 38].

Pathology and Pathogenesis

B. mallei enters the host through the respiratory tract, oral mucosa, or broken skin. The bacterium adheres to and invades respiratory epithelial cells and alveolar macrophages, surviving within phagosomes by inhibiting phagolysosomal fusion. The type III secretion system (T3SS) injects effector proteins that disrupt host cell signaling, promoting intracellular survival and dissemination [1].

Gross pathological findings in chronic glanders include:

• Nasal cavity: Ulcerative rhinitis with raised, granulomatous nodules on the nasal septum and turbinates. Histologically, these lesions consist of a central necrotic core surrounded by epithelioid macrophages, multinucleated giant cells, and a fibrous capsule.
• Lungs: Multiple, well-encapsulated abscesses (1-5 cm in diameter) scattered throughout the lung parenchyma. These abscesses contain thick, caseous pus and are surrounded by a zone of fibroplasia. Histopathology reveals pyogranulomatous pneumonia with central liquefactive necrosis.
• Lymph nodes: Enlarged, caseous lymphadenitis of the submandibular, retropharyngeal, and mediastinal lymph nodes. Cut section shows a characteristic "starburst" pattern of necrosis and fibrosis.
• Skin and lymphatics: Nodular lymphangitis with thrombophlebitis and perivascular granulomatous inflammation. Immunohistochemical staining using an anti-BpaB antibody has been shown to specifically label B. mallei antigens within these lesions, providing a valuable diagnostic tool for formalin-fixed tissues [39].

Immunoglobulin and cytokine profiling in naturally infected equids has demonstrated a predominant Th1-type response, with elevated levels of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in the acute phase. Chronic infection is associated with a mixed Th1/Th2 response, with increased IgG1 and IgG4/7 subclasses [17, 18].

Zoonotic Transmission and Human Disease

Glanders is a zoonotic disease, with transmission to humans occurring through direct contact with infected equids or their exudates. Occupational exposure is the primary risk factor, affecting veterinarians, farriers, slaughterhouse workers, and laboratory personnel [1, 41]. The bacterium can enter the human body through abrasions in the skin, inhalation of aerosols, or contamination of mucous membranes.

Human glanders presents in three main forms: localized cutaneous infection, pulmonary infection, and septicemic disease. Cutaneous infection typically follows inoculation through a skin wound, resulting in a nodular lymphangitis with regional lymphadenopathy. Pulmonary infection, acquired through inhalation, manifests as pneumonia with abscess formation, as documented in a case report from China [19]. Septicemic glanders is rapidly fatal if untreated, with high fever, hypotension, and multiorgan failure [20, 21].

A serological survey of humans exposed to B. mallei-infected equids in India found that 6.7% of occupational contacts had detectable antibodies, indicating subclinical infection is possible [41]. This finding underscores the importance of personal protective equipment (PPE) and biosafety measures for personnel handling infected animals.

Diagnostic Approaches

Diagnosis of glanders requires a combination of clinical examination, serological testing, bacterial isolation, and molecular methods. Due to the zoonotic risk and the notifiable status of the disease, all diagnostic procedures should be performed in biosafety level 3 (BSL-3) facilities.

Serological Methods

The complement fixation test (CFT) remains the prescribed test for international trade under WOAH guidelines. However, CFT has limitations, including low sensitivity in chronic cases and prozone effects. Several ELISA-based methods have been developed as alternatives [34].

A competitive ELISA (cELISA) using a monoclonal antibody against B. mallei LPS has shown high sensitivity (98.2%) and specificity (99.5%) in validation studies [3]. A commercial glanders ELISA has been validated as an alternative to CFT for international trade of equidae, with a diagnostic sensitivity of 96.7% and specificity of 98.9% [34].

Protein microarray-guided development has led to a highly sensitive and specific dipstick assay for glanders serodiagnostics. This assay uses recombinant antigens (BimA, BopA, and TssB) that are recognized by antibodies from infected horses, providing a rapid, field-deployable test [22].

A chemiluminescent western blot assay has been developed and validated for glanders serodetection. This method uses whole-cell lysate of B. mallei and detects antibodies against multiple protein bands, offering higher specificity than CFT in cross-reactivity studies with B. pseudomallei [23].

Immunoblotting assays based on the whole-cell proteome have been developed for serodiagnosis, demonstrating utility in differentiating glanders from melioidosis [33, 45].

A microsphere-based immunoassay (Luminex technology) has been developed for the serological detection of glanders in equids. This multiplex assay allows simultaneous detection of antibodies against multiple B. mallei antigens, improving throughput and reducing sample volume [42].

Bacterial Isolation and Identification

Isolation of B. mallei from clinical specimens (nasal swabs, pus from lymph nodes, or tissue biopsies) is the gold standard for diagnosis. The bacterium grows on blood agar, MacConkey agar, and selective media (e.g., Ashdown's medium) after 48-72 hours of incubation at 37°C. Colonies are small, smooth, and mucoid, with a characteristic "earthy" odor. Biochemical identification is based on oxidase positivity, nitrate reduction, and inability to ferment lactose [1, 12].

Molecular Diagnostics

Several PCR-based methods have been developed for the detection of B. mallei DNA. A comparative analysis of PCR primer sets targeting the fliP, Burkholderia intracellular motility A (bimA), and B. mallei-specific insertion sequences found that the fliP gene target provided the highest analytical sensitivity (10 fg of genomic DNA) [24].

Real-time quantitative PCR (qPCR) assays targeting the B. mallei-specific Bma gene cluster have been validated for use in equine tissue samples, with a limit of detection of 1-10 colony-forming units per reaction [25]. A multiplex real-time PCR kit for the differential diagnosis of glanders and melioidosis has been developed and assessed for laboratory practice [44].

A novel ready-to-use loop-mediated isothermal amplification (LAMP) method has been developed for the detection of B. mallei and B. pseudomallei. This method uses a set of six primers targeting the groEL gene and can be performed at a constant temperature of 65°C, with results visualized by color change within 30 minutes. The LAMP assay has a sensitivity of 100% and specificity of 98.5% when tested against a panel of 50 B. mallei isolates [26].

A recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR) visualization system has been constructed for the rapid detection of B. mallei. This method targets the Bma gene cluster and uses Cas12a nuclease for signal amplification. The RPA-CRISPR assay has a limit of detection of 1 copy/μL and can be completed in 45 minutes without the need for thermal cycling equipment [27].

Diagnostic Workflow

The following Mermaid diagram illustrates a recommended diagnostic workflow for glanders in horses.

flowchart TD
    A[Clinical suspicion: nasal discharge, lymphadenopathy, skin nodules], > B{Serological screening}
    B, > C[CFT or cELISA]
    C, >|Positive| D[Confirmatory testing]
    C, >|Negative| E[No evidence of infection; monitor]
    D, > F[Immunoblot or chemiluminescent western blot]
    F, >|Positive| G[Collect samples: nasal swab, pus, tissue]
    F, >|Negative| H[False positive; rule out cross-reaction]
    G, > I[Bacterial culture on selective media]
    I, >|Growth| J[Biochemical identification + MALDI-TOF]
    I, >|No growth| K[PCR or LAMP on clinical sample]
    J, > L[Confirm by PCR: fliP or Bma target]
    K, >|Positive| M[Report as confirmed case]
    K, >|Negative| N[Consider latent infection; retest in 30 days]
    L, > M
    M, > O[Notify veterinary authorities]
    O, > P[Quarantine, euthanasia, disinfection]

Treatment and Antimicrobial Susceptibility

Treatment of glanders in horses is generally not recommended due to the zoonotic risk and the potential for latent carriers. In most endemic countries, test-and-slaughter policies are enforced. However, in rare cases where treatment is attempted (e.g., valuable breeding stock in non-endemic regions), antimicrobial therapy must be guided by susceptibility testing.

B. mallei is intrinsically resistant to many beta-lactam antibiotics due to the production of beta-lactamases. The organism is susceptible to doxycycline, ciprofloxacin, imipenem, and trimethoprim-sulfamethoxazole. Combination therapy with two or more agents is recommended to prevent the emergence of resistance [1, 36].

In vitro antibacterial activity of sulbactam-durlobactam against pathogenic Burkholderia species has been demonstrated, with minimum inhibitory concentrations (MIC90) of 2-4 μg/mL for B. mallei. This beta-lactam/beta-lactamase inhibitor combination shows promise as a therapeutic option [36].

Control and Prevention

Control of glanders relies on early detection, strict quarantine, and elimination of infected animals. Key measures include:

• Surveillance: Regular serological testing of equids in endemic areas using CFT or ELISA. Systematic monitoring of infected herds by serial CFT, bacterial isolation, and PCR is essential for identifying new cases and assessing the effectiveness of control measures [38].
• Movement control: Restriction of equine movement from endemic to non-endemic regions. Border controls and pre-export testing are critical for preventing transboundary spread [15, 16].
• Biosecurity: Isolation of suspect animals, use of dedicated equipment, and disinfection of contaminated premises. B. mallei is susceptible to common disinfectants, including 1% sodium hypochlorite, 70% ethanol, and 2% glutaraldehyde.
• Euthanasia: Infected animals should be humanely euthanized to eliminate the source of infection. Carcasses must be disposed of by incineration or deep burial.
• Personal protective equipment: Personnel handling suspect animals must wear gloves, goggles, and respiratory protection to prevent zoonotic transmission [41].

No licensed vaccine is currently available for glanders. Research efforts have focused on identifying protective antigens, including the type VI secretion system proteins and LPS. A glycoengineered antigen exploiting the conserved protein O-glycosylation pathway in Burkholderia has been developed for serodiagnosis but has not yet been translated into a vaccine [35].

Molecular Epidemiology and Strain Typing

Molecular typing of B. mallei isolates has provided insights into the global epidemiology of glanders. MLST and whole-genome SNP analysis have revealed a clonal population structure with limited genetic diversity. Isolates from India (2015-2016) were found to belong to a single sequence type (ST1), with minor SNP variations allowing for strain tracing [28, 31].

In Brazil, molecular characterization of isolates from 2014-2017 using MLST and variable-number tandem repeat (VNTR) analysis identified two major clonal complexes, CC1 and CC2, with distinct geographic distributions [10]. The 16S rDNA and internal transcribed spacer (ITS) sequence diversity of B. mallei isolates from India (2013-2019) was low, with >99.8% sequence identity among strains, confirming the high genetic conservation of this pathogen [29].

A genetic variant of B. mallei detected in Kuwait was found to have a deletion in the fliP gene, which could lead to false-negative results in PCR assays targeting this locus. This finding highlights the need for multiple molecular targets in diagnostic algorithms [37].

Conclusion

Glanders remains a significant threat to equine health and a persistent zoonotic risk in endemic regions. The disease is notifiable due to its high pathogenicity, potential for rapid spread, and the absence of effective vaccines. Advances in molecular diagnostics, including LAMP, RPA-CRISPR, and multiplex immunoassays, have improved the speed and accuracy of detection. However, control efforts are hampered by the lack of a licensed vaccine, the existence of latent carriers, and the challenges of transboundary surveillance. Continued investment in serosurveillance, molecular epidemiology, and biosecurity measures is essential for the eventual eradication of this ancient zoonosis.

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