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.

The structural basis for deubiquitination by the fingerless USP-type effector TssM.

Intracellular bacteria are threatened by ubiquitin-mediated autophagy, whenever the bacterial surface or enclosing membrane structures become targets of host ubiquitin ligases. As a countermeasure, many intracellular pathogens encode deubiquitinase (DUB) effectors to keep their surfaces free of ubiquitin. Most bacterial DUBs belong to the OTU or CE-clan families. The betaproteobacteria Burkholderia pseudomallei and Burkholderia mallei, causative agents of melioidosis and glanders, respectively, encode the TssM effector, the only known bacterial DUB belonging to the USP class. TssM is much shorter than typical eukaryotic USP enzymes and lacks the canonical ubiquitin-recognition region. By solving the crystal structures of isolated TssM and its complex with ubiquitin, we found that TssM lacks the entire "Fingers" subdomain of the USP fold. Instead, the TssM family has evolved the functionally analog "Littlefinger" loop, which is located towards the end of the USP domain and recognizes different ubiquitin interfaces than those used by USPs. The structures revealed the presence of an N-terminal immunoglobulin-fold domain, which is able to form a strand-exchange dimer and might mediate TssM localization to the bacterial surface.

Spread analysis of glanders in the state of Piauí, northeastern Brazil.

Brazil is strategic in controlling neglected zoonoses, such as glanders, in its territory. Among the Brazilian states, Piauí is a strategic state for the spread of the disease in the country. The present study aimed to evaluate the spatial and temporal distribution of official cases of glanders in Piauí between 2015 and 2022. The glanders cases were located in the municipalities of the north and central-north mesoregions, mainly in Campo Maior, Teresina and Altos. The highest incidence risk (IR) occurred in of Altos (IR = 257.9), Sussuapara (IR = 158.4), and Teresina (IR = 157.7). A primary cluster was formed with a relative risk of 14.88 between 2019 and 2022, encompassing 34 municipalities in the north and central-north regions. In Piauí, glanders is well localized, with the potential for spread across borders. This is the first study demonstrating the distribution of reported cases of glanders in the state of Piauí.

Serological and Bacteriological Surveillance of Glanders Among Horses in Central Region of Iran.

Glanders is the oldest and very contagious disease among horses caused by Burkholderia mallei. The disease occurs as a chronic form in horses. Hence, because of the prolonged shedding, numerous horses can potentially get infected by one horse with glanders. Glanders is endemic in Iran and this causes occasional occurrence in horse population of the country. The aim of this study was to determine the incidence of B.mallei infection in horses in two central provinces of Iran. A total of 517 serum samples were collected from stable horses in Tehran and Alborz provinces. Among the studied horses, seven presented fever, anorexia, dyspnea, subcutaneous abscesses, nasal and cutaneous discharges, emaciation, and lymphadenopathy. Nasal and ocular discharges and subcutaneous abscesses were sampled for bacterial culture and PCR. The sera were examined by means of complement fixation test (CFT) and indirect enzyme-linked immunosorbent assay (iELISA). Seropositive cases were further examined by Mallein test. The results derived from the present study indicated that only 1.35% of the studied horses were positive in CFT, iELISA and Mallein test, of which only in 42.85% B.mallei was successfully cultured on blood agar and glycerinated nutrient media and confirmed by PCR. Periodic serological tests along with quarantine can benefit reduction of the occurrence of the disease in horses in Iran.

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.

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.

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.

Pathogenic bacteria remodel central metabolic enzyme to build a cyclopropanol warhead.

Bacteria of the Burkholderia pseudomallei (BP) group pose a global health threat, causing the infectious diseases melioidosis, a common cause of pneumonia and sepsis, and glanders, a contagious zoonosis. A trait of BP bacteria is a conserved gene cluster coding for the biosynthesis of polyketides (malleicyprols) with a reactive cyclopropanol unit that is critical for virulence. Enzymes building this warhead represent ideal targets for antivirulence strategies but the biochemical basis of cyclopropanol formation is unknown. Here we describe the formation of the malleicyprol warhead. We show that BurG, an unusual NAD+-dependent member of the ketol-acid reductoisomerase family, constructs the strained cyclopropanol ring. Biochemical assays and a suite of eight crystal structures of native and mutated BurG with bound analogues and inhibitors provide snapshots of each step of the complex reaction mechanism, involving a concealed oxidoreduction and a C-S bond cleavage. Our findings illustrate a remarkable case of neofunctionalisation, where a biocatalyst from central metabolism has been evolutionarily repurposed for warhead production in pathogens.

First glanders cases detected in Nepal underscore the need for surveillance and border controls.

BACKGROUND: Glanders is a transmissible zoonotic disease caused by Burkholderia mallei that infects equids and humans. No glanders cases in equids were reported so far in Nepal. CASE PRESENTATION: Following suspected glanders in animals with clinical signs in different regions in Nepal, serum samples were tested by CFT, ELISA and Luminex® tests. Two horses and a mule tested positive for glanders by all tests, while two other equids only tested positive by ELISA and Luminex®. Analysis of swabs and pus samples by a PCR system targeting B. mallei confirmed the presence of the bacterium in the samples collected from the 3 equids that yielded positive results in all serological tests. Genotyping of the three PCR positive samples with a SNP-based method identified a genotype closely related to the B. mallei strains circulating in India. CONCLUSION: Confirmation of glanders cases underscores the need of implementing a surveillance program in Nepal and a strict control of the animal movement across the borders.

Glanders and brucellosis in equids from the Amazon region, Brazil.

Glanders and brucellosis are zoonotic infectious diseases that affect equids in several countries worldwide. On Marajó Island (Amazon region of Brazil), Marajoara and Puruca horses, which are well adapted to the climatic and territorial adversities of the region, play a fundamental role in the local economy and in the sociocultural lives of the population. However, these animals have undergone a drastic reduction in number, markedly due to precarious veterinary care, unknown causes of morbidity and mortality, and disordered crossing with other breeds introduced to the island. Thus, this study aimed to investigate the occurrence of glanders and brucellosis in equids on a property located in the municipality of Soure, Marajó Island (Brazil). Serum samples were collected from 388 animals (357 horses and 31 mules), maintained in an extensive breeding system, in a property that was also extensively breeding buffaloes, goats, and sheep, with contact among species. The sera were tested for glanders using an indirect ELISA (ELISAi), and the results were confirmed by immunoblotting. The diagnosis of brucellosis was made using the Rose Bengal test (RBT) and confirmed through the Serum Agglutination test (SAT) and 2-mercaptoethanol test. In the case of glanders, 2.31% (9/388) of animals were positive in ELISAi test, of which eight had results confirmed by immunoblotting, representing 2.06% seropositivity in the entire herd. For brucellosis, serum samples from 6.7% (26/388) horses were reactive in the RBT, of which 4.12% (18/388) had a titer ≥50 and 2.06% (8/388) had a titer ≥100 in the SAT. This is the first study to report the occurrence of glanders and equine brucellosis in the municipality of Soure/Marajó Island. Monitoring the occurrence of such diseases is extremely important since they affect the herds economically and zootechnically, in addition to their high zoonotic potential. The number of animals sampled in this study, as well as the way they are raised and managed, is representative of the total equid population of the island. These results, combined with previous studies on buffaloes, indicate that these diseases are endemic in the Marajo Island.

Molecular characterization of Burkholderia mallei strains isolated from horses in Brazil (2014-2017).

Glanders is an infectious zoonosis caused by Burkholderia (B.) mallei that mainly affects equids. The objective of this work was to provide additional knowledge on the diversity of the strains circulating in Brazil. Six Burkholderia mallei isolates obtained during necropsies of glanderous horses between 2014 and 2017 in two different states (Pernambuco and Alagoas) were analyzed by polymerase chain reaction-high-resolution melting (PCR-HRM). While four strains (9902 RSC, BM_campo 1, BM_campo 3 and UFAL2) clustered in the L3B2 branch, which already includes the Brazilian 16-2438_BM#8 strain, two strains (BM_campo 2.1 and BM_campo 2.2) clustered within the L3B3sB3 branch, which mostly includes older isolates, from Europe and the Middle East. Whole genome sequencing of two of these strains (UFAL2 and BM_campo 2.1), belonging to different branches, confirmed the HRM typing results and refined the links between the strains, including the description of the L3B3Sb3Gp1SbGp1 genotype, never reported so far for contemporary strains. These results suggest different glanders introduction events in Brazil, including a potential link with strains of European origin, related to colonization or trade.

Characterization of immunoglobulin and cytokine responses in Burkholderia mallei infected equids.

Burkholderia mallei causes a highly fatal infectious disease in equines known as glanders. It is one of the OIE listed notifiable diseases, which entails strict control policy measures once B. mallei infection is confirmed in the susceptible hosts. Humans, especially equine handlers, veterinary professionals and laboratory workers are at greater risk to acquire the B. mallei infection directly through prolonged contact with glanderous equines, and indirectly through unprotected handling of B. mallei contaminated materials. Further, natural resistance of B. mallei to multiple antibiotics, aerosol transmission, lack of effective vaccine and treatment make this organism a potential agent of biological warfare. Results of experimental B. mallei infection in mouse and non-human primates and immunization with live attenuated B. mallei strains demonstrated that activation of early innate and adaptive immune responses play a critical role in controlling B. mallei infection. However, the immune response elicited by the primary hosts (equids) B. mallei infection is poorly understood. Therefore, we aimed to investigate immune responses in glanders affected horses (n = 23) and mules (n = 1). In this study, chronically infected equids showed strong humoral responses (IgM, IgG and IgA) specific to B. mallei type 6 secretory proteins such as Hcp1, TssA and TssB. The infected equids also elicited robust cellular responses characterized by significantly elevated levels of IFN-γ, TNF-α, IL-12, IL-17 and IL-6 in PBMCs. In addition, stimulation of equine PBMCs by Hcp1 resulted in the further elevation of these cytokines. Thus, the present study indicated that antibody response and T helper cell (Th) type 1-associated cytokines were the salient features of chronic B. mallei infection in horses. The immune responses also suggest further evaluation of these proteins as potential vaccine candidates.

16S rDNA and ITS Sequence Diversity of Burkholderia mallei Isolated from Glanders-Affected Horses and Mules in India (2013-2019).

Glanders is a highly contagious and fatal infection of equids caused by the bacteria known as Burkholderia mallei. It is one of the notifiable equine diseases and is still present in Asia, South America and Africa. In India, glanders re-emerged in 2006, and thereafter, increasing numbers of cases were reported in different regions of the country. Between 2013 and 2019, 39 B. mallei were isolated from glanders-affected horses (n = 30) and mules (n = 9) from seven states of India such as Uttar Pradesh, Haryana, Delhi, Himachal Pradesh, Gujarat, Maharashtra and Tamil Nadu. In this study, the phylogenetic relationships of these isolates were assessed by sequence analysis of 16S rDNA gene and ITS region. Purified PCR-amplified products of 16S rDNA gene and ITS region were sequenced, aligned and phylogenetic trees were constructed using MEGA 11 software. Additionally, B. mallei 16S rDNA (n = 36) and ITS (n = 18) sequences available in the GenBank were also included for analysis to determine the diversity of older B. mallei isolates with recent Indian isolates. Both the phylogeny showed that the majority of the recent isolates from India are closely related to each other, but are genetically diverse from older isolates that originated from India. Nucleotide substitutions were also observed in a single and double position in 12 recent and two old Indian isolates. The study also indicates that similar B. mallei strains were responsible for glanders outbreaks in different states (Uttar Pradesh- Himachal Pradesh and Uttar Pradesh- Haryana) and this is due to the migration of infected animals from one state to another state. This study implies that 16S rDNA and ITS region may be used for molecular characterization of B. mallei associated with glanders in resource-limited settings.

Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei, a Tier 1 select agent.

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/µL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.

Molecular epidemiology of Burkholderia mallei isolates from India (2015-2016): New SNP markers for strain tracing.

Glanders, caused by a bacterium called B. mallei, is primarily an infectious horse and human disease. Although its incidence is rare in developed countries, it is nonetheless prevalent in several geographical areas of the world. There is a lack of cost-effective, rapid and specific molecular typing tools for epidemiological tracing of glanders cases. We previously reported an SNP-based typing method that categorizes global B. mallei strains into three lineages (L1 to L3), as well as additional branches, sub-branches and groups. However, further discrimination of the Indian and Pakistani isolates within the L2B2sB2 sub-branch was not possible due to the lack of sufficient epidemiological markers. In this study, 10 B. mallei strains isolated from four states in India during 2015-2016 were whole genome sequenced; SNP analysis further confirmed their position in the L2B2sB2 branch. To better track the strains, four new markers targeting Indian or Pakistani strains, and specifically targeting sub-groups within the Indian strains, were identified. The new SNP markers were tested and validated on the 10 Indian isolates included in this study as well as on 6 contemporary B. mallei Pakistani strains. These rapid and discriminating typing tools will contribute to the epidemiological monitoring of B. mallei infections, particularly in South Asia and the Middle East, endemic regions of the disease.

Validation of three qPCR for the detection of Burkholderia mallei in equine tissue samples.

Burkholderia mallei is the causative agent of glanders, a zoonosis listed by the World Organization for Animal Health as of mandatory notification. In this work, a comparison of three qPCR protocols was made, two of them based on articles by other authors and one standardized in house, this last one aiming at a genomic region that does not exist in other species of the Burkholderia genus. All qPCRs showed high efficiency and good repeatability. However, reactions with Cq between 36 and 40 were considered suspicious and unreliable, requiring greater clinical criteria to analyze the results.

Molecular Typing of Burkholderia mallei Isolates from Equids with Glanders, India.

We collected 10 Burkholderia mallei isolates from equids in 9 districts in India during glanders outbreaks in 2013-2016. Multilocus variable-number tandem-repeat analysis showed 7 outbreak area-related genotypes. The study highlights the utility of this analysis for epidemiologically tracing of specific B. mallei isolates during outbreaks.