Patient Presenting with Abscess Unresponsive to Treatment and Progressive to Osteomyelitis: A Rare Cause Burkholderia mallei.

Glanders is a rare zoonotic disease caused by Burkholderia mallei (B. mallei). B. mallei can cause pneumonia, abscesses, osteomyelitis in severe cases, sepsis, and even death in humans. In this report, we present a 15-year-old male patient living in a rural area who was diagnosed with glanders. The patient, who did not have any previous disease, was followed up with a diagnosis of pneumonia in the hospital, where he was admitted with complaints of cough and abdominal pain and presented to us with pain, redness, and swelling in his leg. Magnetic resonance imaging of the lower extremity revealed osteomyelitis in the fourth and fifth metatarsals of the right foot. B. mallei growth was detected in the abscess culture. Meropenem treatment was started. The patient's symptoms regressed with treatment. The patient was discharged with oral ciprofloxacin for B. mallei eradication. Glanders are usually transmitted through direct contact with infected animals, especially single-hoofed animals such as horses, or through inhalation of aerosols containing B. mallei. It is a rare disease-causing pneumonia and abscesses and can be life-threatening in severe cases. Diagnosis of glanders is difficult because the initial symptoms are non-specific. Isolation of B. mallei in culture is the gold standard for diagnosing the disease. There is no clear recommendation for treating glanders and imipenem; meropenem ceftazidime can be used based on antibiotic susceptibility tests.

Isolation, serological and molecular methods in screening of Burkholderia mallei in East Azerbaijan province, Iran.

Glanders caused by Burkholderia mallei is one of the most dangerous zoonotic diseases in solipeds. Clinical diagnosis of this disease in its early stages in horses, is difficult. This study investigated serological and molecular identification of B. mallei in East Azerbaijan province. In the third and fourth quarters of 2020, throughout 2021, and in the first and second quarters of 2022, the complement fixation test (CFT) was performed on 350 horses. The malleination was used to confirm the positive CFT cases. Blood samples were taken for culture and for preparing serums to perform the enzyme-linked immunosorbent assay (ELISA). Deep eye discharge, nostril, cutaneous ulcers and lymph fluid swabs were cultured, and polymerase chain reaction (PCR) was carried out. Eleven horses were CFT-positive. Based on the malleination on the 11 horses, six were affected by glanders, five were not affected (false positive), and one horse was CFT-negative despite exhibiting clinical signs. It was positive by malleination, ELISA and PCR. A total number of seven positive cases of glanders were diagnosed. The B. mallei could not be isolated, but the Burkholderia cepacia complex was isolated in one case. Except for three cases, the results of the CFT, mallein and ELISA tests were consistent. The amount of confidence interval was 95.00%. It is suggested that ELISA could be used as a complement to CFT in screening and, if positive results are observed in one of the tests, the entire herd must be examined more accurately using the mallein and western blot confirmatory tests.

First molecular characterization of Burkholderia mallei strains isolated from horses in Mongolia.

Glanders, a highly contagious and often fatal disease affecting equids, is caused by Burkholderia mallei. Although sporadic cases of equine glanders have recently been documented in Mongolia, genome sequencing and molecular studies of the bacteria within this region are lacking. This study provided the first molecular characterization of B. mallei isolated from four native Mongolian horses from two different provinces in 2019 and 2022 by applying whole-genome sequencing with two SNP types (previously developed genotyping with 15 SNP markers that provide global coverage of the B. mallei population and the core genome coding SNP typing developed in this study). The Mongolian isolates were located within the L3B1 cluster, which was previously associated with the V-120 strain from Russia. Within the L3B1 cluster shared by neighboring countries, they were in a unique subbranch. In this study, specific SNP markers unique to the Mongolian strains were identified to track these strains using a high-resolution melting analysis (HRMA). This study revealed the unique phylogenetic background of Mongolian strains isolated from the eastern part of Mongolia. HRMA specific to the Mongolian subbranch may contribute to the molecular epidemiological monitoring of glanders in Mongolia and surrounding countries.

Instances of Biowarfare in World War I (1914-1918).

During World War I (WWI), also referred to as 'The Great War,' Germany implemented a pioneering biowarfare program as part of a broader military strategy to undermine Allied forces by targeting their logistical and supply capabilities. This initiative, unprecedented in its systematic and strategic application, utilized a variety of pathogens, primarily targeting animal populations, to disrupt support systems without contravening international laws, specifically the 1907 Hague Convention. The operations, shrouded in secrecy and largely led by the German General Staff, included sophisticated sabotage actions against both enemy and neutral states. The allegations and usage of bioweapons increased the interest of the Great Powers in further developing their own biowarfare program.

Tracking the footsteps of Burkholderia mallei: determination of the molecular differences and potential resistance genes.

Background/aim: Chemical biological radiological nuclear threats are at an important point in the agenda of world health today, as they can cause mass deaths. B. mallei attracts attention as a potential biological warfare agent due to its features such as multidrug resistance, a rapid transmission mechanism via aerosol, the absence of a complete treatment protocol for the infection it causes, and the absence of an approved vaccine for protection against the bacteria. B. mallei suspect samples must be studied by experienced personnel in biosafety level III laboratories. B mallei is a difficult and troublesome pathogen to diagnose and many unknowns about B. mallei today. Therefore, the aim of the study was to determine the molecular differences and potential resistance genes of B mallei strains. Materials and methods: Determination of the molecular differences and potential resistance genes of B mallei strains with new bioinformatics approaches by comparatively examining the data of 29 B mallei strains, 10 of which were isolated from Türkiye, on the genome list of the National Biotechnology Information Center (NCBI). Results: According to the genome annotations of the origins, the origin containing the highest number of CDS which is 5172 was found as the 11th strain obtained in Türkiye in 1949. The origin with the highest number of pseudogenes was determined as 23,344 (China 7) origin. Two hundred and eighty-five pseudogenes found in this strain were obtained from a knee effusion in Myanmar. According to chromosome 2 data, B. mallei strain was determined as the most similar strain to ATCC 23344, line 11 with NCTC 10229 strain, and SAVP1 strain was determined as the least similar strain. When the antimicrobial resistance gene markers of the isolates included in the study were examined, amrA and amrB, qacG ade, Burkholderia pseudomallei Omp38 were found to be carrying. Conclusion: In terms of public health, it was thought that the data obtained as a result of our study about B mallei, which is defined as a biological weapon, is very valuable for creating treatment protocols to be applied to possible epidemics in the future. In addition, the available genetic epidemiological data of these strains belonging to a category that is dangerous to work with in a laboratory environment were reviewed.

Knowledge, awareness and perception about equine glanders among veterinarians and medical professionals in India.

Glanders is a highly infectious and notifiable disease of equines that occurs due to Burkholderia mallei. In India, glanders re-emerged in 2006 and thereafter regular outbreaks have been reported in various states (n = 14). Frequent and prolonged contact with equids with glanders may transmit B. mallei infection to humans. This study was designed to learn more about the Knowledge, Awareness and Perception (KAP) of veterinarians, para veterinarians, and physicians about equine glanders, which will help in enhancing the nation-wide glanders eradication programme. A total of 165 respondent's from 11 Indian states and one union territory were surveyed. Most of the respondents (n = 160) were from equine glanders affected or endemic states. Knowledge gap analysis revealed that 40.3 and 22% of the participants were not aware of government regulations and the transmission of glanders, respectively. These are major concerns given the wide spread occurrence of disease in the country. Awareness test on glanders revealed that 65(39.4%) participants would collect biological samples for laboratory confirmation, 67(40.6%) would inform the concerned authorities and 106 (64.2%) replied that they would eliminate the glanders infected equines. Analysis of perception towards equine glanders showed that majority of the participants (n = 113, 68.4%) observed that equine keepers were reluctant to disclose the clinical symptoms of B. mallei infection. Furthermore, non-co-operation and unwillingness by superiors (33.9%), financial (31%), administrative (28.4%), and technical limitations (27.8%) were major constraints under the perception analysis. This study reveals that veterinarians need to be educated on governmental policies and guidelines on equine glanders with regular training and awareness programs. Intersectoral co-ordination to investigate human glanders is also needed.

Burkholderia pseudomallei Complex Subunit and Glycoconjugate Vaccines and Their Potential to Elicit Cross-Protection to Burkholderia cepacia Complex.

Burkholderia are a group of Gram-negative bacteria that can cause a variety of diseases in at-risk populations. B. pseudomallei and B. mallei, the etiological agents of melioidosis and glanders, respectively, are the two clinically relevant members of the B. pseudomallei complex (Bpc). The development of vaccines against Bpc species has been accelerated in recent years, resulting in numerous promising subunits and glycoconjugate vaccines incorporating a variety of antigens. However, a second group of pathogenic Burkholderia species exists known as the Burkholderia cepacia complex (Bcc), a group of opportunistic bacteria which tend to affect individuals with weakened immunity or cystic fibrosis. To date, there have been few attempts to develop vaccines to Bcc species. Therefore, the primary goal of this review is to provide a broad overview of the various subunit antigens that have been tested in Bpc species, their protective efficacy, study limitations, and known or suspected mechanisms of protection. Then, we assess the reviewed Bpc antigens for their amino acid sequence conservation to homologous proteins found in Bcc species. We propose that protective Bpc antigens with a high degree of Bpc-to-Bcc sequence conservation could serve as components of a pan-Burkholderia vaccine capable of protecting against both disease-causing groups.

Development and validation of a chemiluminescent western blot assay for glanders (Burkholderia mallei) serodetection.

Glanders, caused by Burkholderia mallei, is a zoonotic disease of equids. Serologic testing for glanders is required by disease-free countries before international movement of equids. The World Organisation for Animal Health Terrestrial Manual recommends the complement fixation test (CFT) for clearance of individual animals for movement, but the CFT is prone to false-positive results. A colorimetric western blot (WB) assay was developed and validated to resolve false-positive CFT results; however, that assay is relatively time-consuming, and the interpretation is subjective. We present here a procedurally similar chemiluminescent WB assay that performs comparably to the validated colorimetric WB assay and offers noticeable benefits of decreased time-to-result and greater ease of interpretation.

Assessment of the Virulence of the Burkholderia mallei Strain BAC 86/19 in BALB/c Mice.

Burkholderia mallei is an aerobic, Gram-negative, non-motile bacillus. As an obligate mammalian pathogen, it primarily affects solipeds. Although rarely transmitted to humans, the disease it causes, glanders, is classified as a zoonosis. The bacterium was officially eradicated in Brazil in 1969; however, it reemerged after three decades. This study aims to assess the virulence of a specific B. mallei strain, isolated in Brazil, in BALB/c mice through intranasal infection. The strain, B. mallei BAC 86/19, was obtained from the tracheal secretion of a young mare displaying positive serology but no clinical signs of glanders. Post-mortem examinations revealed macroscopic lesions consistent with the disease, however. In mice, the LD50 was determined to be approximately 1.59 × 105 colony-forming units (CFU)/animal. Mice exposed to either 0.1 × LD50 or 1 × LD50 displayed transient weight loss, which resolved after three or five days, respectively. B. mallei persisted within the liver and lung for five days post-infection and in the spleen for seven days. These findings underscore the detectable virulence of the Brazilian B. mallei BAC 86/19 strain in mice, which are relatively resilient hosts. This research points to the importance of the continued investigation of the virulence mechanisms and potential countermeasures associated with B. mallei infections, including their Brazilian isolates.

Glanders Diagnosis in an Asymptomatic Mare from Brazil: Insights from Serology, Microbiological Culture, Mass Spectrometry, and Genome Sequencing.

This manuscript elucidates the occurrence of glanders in an asymptomatic mare from Brazil presenting positive Burkholderia mallei antibody titers. The diagnosis was established through a multi-pronged approach encompassing microbiological culture, mass spectrometry, and genome sequencing. The outbreak occurred in 2019 in Tatuí, São Paulo, Brazil, and the infected mare, despite displaying no clinical symptoms, had multiple miliary lesions in the liver, as well as intense catarrhal discharge in the trachea. Samples were collected from various organs and subjected to bacterial isolation, molecular detection, and identification. The strain was identified as B. mallei using PCR and confirmed by MALDI-TOF mass spectrometry. Whole-genome sequencing revealed a genome size of 5.51 Mb with a GC content of 65.8%, 5871 genes (including 4 rRNA and 53 tRNA genes), and 5583 coding DNA sequences (CDSs). Additionally, 227 predicted pseudogenes were detected. In silico analysis of different genomic loci that allow for differentiation with Burkholderia pseudomallei confirmed the identity of the isolate as B. mallei, in addition to the characteristic genome size. The BAC 86/19 strain was identified as lineage 3, sublineage 2, which includes other strains from Brazil, India, and Iran. The genome sequencing of this strain provides valuable information that can be used to better understand the pathogen and its epidemiology, as well as to develop diagnostic tools for glanders.

Glanders (Burkholderia mallei infection) in an Iranian man: A case report.

Background: Glanders is a rare disease that has been eradicated in many countries but may be difficult to diagnose due to its nonspecific symptoms. This disease, which can be highly fatal if left untreated, is caused by a bacterium called Burkholderia mallei. Humans can get the disease through contact with infected animals, such as horses. Over time, various treatment strategies have been proposed for this disease, and attempts haveeven been made to develop a vaccine, but thus far, no effective vaccine has been developed to prevent it. Case presentation: In this article, we describeacase of Glanders disease in KamkarArabnia Hospital in Qom, Iran. The patient was a 22-year-old man with headache, fever, chills, diarrhea, and vomiting of blood, and was admitted to the infectious ward in isolation. Conclusion: The lack of definitive diagnostic symptoms and the rarity of this disease make it difficult to diagnose, and one should be careful in dealing with its symptoms. Also, paying attention to the patient's medical history and travel history to endemic areas, can lead to timely diagnosis and treatment.

Molecular detection of Burkholderia mallei in different geographic regions of Brazil.

Glanders is a contagious disease of equids caused by the Gram-negative bacterium Burkholderia mallei. In Brazil, the disease is considered to be reemerging and has been expanding, with records of equids with positive serology in most of the federative units. However, there are few reports describing the genotypic detection of the agent. This study demonstrated the detection of B. mallei by species-specific PCR directly from tissues or from bacterial cultures, followed by amplicon sequencing in equids (equines, mules, and asinines) with positive serology for glanders in all five geographic regions of Brazil. The molecular evidence of B. mallei infection in serologically positive equids in this study expands the possibility of strain isolation and the conduction of epidemiological characterizations based on molecular information. The microbiological detection of B. mallei in cultures from nasal and palate swabs, even in equids without clinical manifestations, raises the possibility of environmental elimination of the agent.

Epidemiological Situation of Glanders in the State of Pará, Brazil.

Glanders is an anthropozoonosis caused by the bacteria Burkholderia mallei, affecting mainly equids. It has been eradicated in North America, Australia, and Western Europe, but continues to occur sporadically in countries in Asia, Africa, the Middle East, and South America. Its notification is mandatory by the World Organization for Animal Health. After 30 years, the disease reappeared in Brazil in 1999 and, thereafter, 1,413 outbreaks have been reported. However, the epidemiological situation of the disease in the country is not adequately known. Thus, 2718 animals from 654 properties in the state of Pará were randomly selected by sampling and examined using a serial protocol with Complement Fixation and Western Blot serological tests. The prevalence of properties infected with glanders in the state was estimated at 1.68% [0.84; 3.33] and of seropositive animals at 0.50% [0.27; 0.94]. The introduction of animals was individualized as a risk factor for disease introduction in the properties (OR = 5.9 [1.4; 25.5]). Despite the low prevalence of infected properties and seropositive animals, the state must review actions to fight the disease, considering that the strategies implemented have not affected the endemic balance of the disease. This process must involve all public and private agents interested in the topic.

A Case Report of Burkholderia mallei Infection Leading to Pneumonia.

BACKGROUND: Glanders is a rare zoonotic disease caused by Burkholderia mallei. Humans can be infected by B. mallei, which causes cutaneous lymphadenitis and pneumonia, leading to sepsis and death in severe cases. CASE PRESENTATION: We report a case of a 60-year-old male who was diagnosed with glanders. The patient who had a history of diabetes presented with cough, expectoration, and fever. Computed tomography (CT) imaging showed B. mallei infection in the right upper lobe of the lung with mediastinal lymph node involvement and the lingual segment of the left lung. Moreover, the posterior basal segment of the lower lobe of both lungs had inflammation. Subsequently, B. mallei infection was confirmed by lymph node biopsy and bronchoalveolar lavage multiplex PCR-based targeted gene sequencing. After meropenem treatment, the patient was discharged, and CT imaging showed reduced absorption of pulmonary inflammatory lesions. CONCLUSIONS: Glanders is a rare disease that can cause skin infection, lymphadenitis, and pneumonia, and in severe cases, it can be life-threatening. The diagnosis of this disease mainly relies on microbiological culture and pathological biopsy. Diagnosis is also facilitated by multiplex PCRbased targeted gene sequencing. Glanders is treated with cephalosporins, carbapenems, and other sensitive antibiotics.

Protein Microarray-Guided Development of a Highly Sensitive and Specific Dipstick Assay for Glanders Serodiagnostics.

Burkholderia mallei, the causative agent of glanders, is a clonal descendant of Burkholderia pseudomallei, the causative agent of melioidosis, which has lost its environmental reservoir and has a restricted host range. Despite limitations in terms of sensitivity and specificity, complement fixation is still the official diagnostic test for glanders. Therefore, new tools are needed for diagnostics and to study the B. mallei epidemiology. We recently developed a highly sensitive serodiagnostic microarray test for human melioidosis based on the multiplex detection of B. pseudomallei proteins. In this study, we modified our array tests by using anti-horse IgG conjugate and tested sera from B. mallei-infected horses (n = 30), negative controls (n = 39), and horses infected with other pathogens (n = 14). Our array results show a sensitivity of 96.7% (confidence interval [CI] 85.5 to 99.6%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The reactivity pattern of the positive sera on our array test allowed us to identify a set of 12 highly reactive proteins of interest for glanders diagnosis. The B. mallei variants of the three best protein candidates were selected for the development of a novel dipstick assay. Our point-of-care test detected glanders cases in less than 15 min with a sensitivity of 90.0% (CI, 75.7 to 97.1%) and a specificity of 100.0% (CI, 95.4 to 100.0%). The microarray and dipstick can easily be adopted for the diagnosis of both B. mallei and B. pseudomallei infections in different animals. Future studies will show whether multiplex serological testing has the potential to differentiate between these pathogens.

Identification of Burkholderia mallei Isolates with Polymerase Chain Reaction-Restriction Fragment Length Polymorphism.

Burkholderia mallei is the main cause of glanders as a dangerous contagious zoonosis disease that is mostly observed in single-hoofed animals, especially horses. Modern molecular techniques have been recently employed to improve epidemiology for identifying and searching for strains of this bacterium at different times and locations. Due to the unknown number of circulating strains and lack of preventive methods, glanders is still observed in the form of epidemics. The present study aimed to evaluate six field isolates plus two laboratory strains of Borkolderia mallei and Burkholderia pseudomallei using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. All the isolates and strains were microbially cultured in the glycerol nutrient and glycerol agar media. The individually grown colonies of the bacterium were used in the biochemical tests. The DNA of isolates was extracted by boiling, and the PCR-RFLP test was conducted on their genome. Finally, the bacterium was injected into guinea pigs to induce the Straus reaction. The biochemical assays (or bioassays) confirmed the isolates as Burkholderia mallei. The PCR-RFLP assay demonstrated a product for Burkholderia mallei with a length of 650 bp. Nevertheless, 250 and 400 bp were produced for Burkholderia pseudomallei. The swollen scrotum pointed to the occurrence of the Straus reaction. The PCR-RFLP is a proper differential diagnosis technique for B. mallei; moreover, it is a suitable method for differentiating between Burkholderia mallei and Burkholderia pseudomallei. This technique can detect Burkholderia mallei in a short time with high precision and sensitivity.

Efficacy of finafloxacin in a murine model of inhalational glanders.

Burkholderia mallei, the causative agent of glanders, is principally a disease of equines, although it can also infect humans and is categorized by the U.S. Centers for Disease Control and Prevention as a category B biological agent. Human cases of glanders are rare and thus there is limited information on treatment. It is therefore recommended that cases are treated with the same therapies as used for melioidosis, which for prophylaxis, is co-trimoxazole (trimethoprim/sulfamethoxazole) or co-amoxiclav (amoxicillin/clavulanic acid). In this study, the fluoroquinolone finafloxacin was compared to co-trimoxazole as a post-exposure prophylactic in a murine model of inhalational glanders. BALB/c mice were exposed to an aerosol of B. mallei followed by treatment with co-trimoxazole or finafloxacin initiated at 24 h post-challenge and continued for 14 days. Survival at the end of the study was 55% or 70% for mice treated with finafloxacin or co-trimoxazole, respectively, however, this difference was not significant. However, finafloxacin was more effective than co-trimoxazole in controlling bacterial load within tissues and demonstrating clearance in the liver, lung and spleen following 14 days of therapy. In summary, finafloxacin should be considered as a promising alternative treatment following exposure to B. mallei.

Current status of glanders in Brazil: recent advances and challenges.

Glanders is an infectious disease that causes serious damage to the equine production chain in countries where it occurs endemically and poses a risk to public health. This study aimed to conduct an integrative review of the advances in the knowledge of glanders in Brazil over the last three decades since its re-emergence. Documentary research was conducted for the period between the years 2000 and 2022. SCOPUS and PUBMED databases were used to search for scientific articles, dissertations, and thesis, in addition to the Brazilian Digital Library of Thesis and Dissertations (BDTD). A total of 41 documents were retrieved, including 12 dissertations, five theses, and 24 scientific articles. The Federal Rural University of Pernambuco (UFRPE) group provided a noteworthy amount of material on this topic (25 documents, four theses, three dissertations, and 18 scientific articles). During this period, ten dissertations, one thesis, and six scientific articles published by other groups in other states of the Federation were also identified. It was concluded that there was a significant number of scientific publications with relevant data on the clinical, epidemiological, pathological, microbiological, serological, and molecular characteristics of glanders disease in Brazil. Additionally, training of human resources regarding this disease led to an increase in the nucleation of research groups, especially in the northeast region of Brazil. Despite significant advances, new research groups and specific funding are still needed for the development of more accurate diagnostic methods, immunizing production, training of veterinarians to recognize the disease, and more robust programs to control and eradicate the disease in Brazil.

Sequencing-Based Genotyping of Pakistani Burkholderia mallei Strains: A Useful Way for Investigating Glanders Outbreaks.

Burkholderia (B.) mallei is a host-adapted equine pathogen that causes glanders, a re-emerging zoonotic disease, which is endemic in Pakistan and other developing countries and seriously impacts the global equine movement. Due to globalization, the geographical restriction of diseases vanishes and the lack of awareness of and experience with eradicated diseases in industrialized countries also promotes the re-introduction of infections in these regions. Owing to the high equine population, the Pakistani province Punjab is a potential hotspot where several glanders outbreaks have been seen over last two decades. For determining the genomic diversity of B. mallei in this and other equine-populated prefectures, the genomes of 19 B. mallei strains isolated between 1999 and 2020 in different locations were sequenced and their genotypes were determined. Particularly, for genetically highly homogenous pathogens like B. mallei genotyping techniques require a high discriminatory power for enabling differentiation on the strain level. Thus, core-genome single nucleotide polymorphism (cgSNP) analysis was applied for distinguishing the highly similar strains. Furthermore, a whole-genome sequence-based core genome multi locus sequence typing (cgMLST) scheme, specific to B. mallei, was developed and additionally applied to the data. It was found that B. mallei genotypes in Pakistan persisted over time and space and genotype clusters preferred connection with a time point rather than the place of isolation, probably due to frequent equine movement, which promotes the spread of glanders. The cgMLST approach proved to work in accord with SNP typing and may help to investigate future glanders outbreaks.