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.

Activation of Toll-Like Receptors by Live Gram-Negative Bacterial Pathogens Reveals Mitigation of TLR4 Responses and Activation of TLR5 by Flagella

Successful bacterial pathogens have evolved to avoid activating an innate immune system in the host that responds to the pathogen through distinct Toll-like receptors (TLRs). The general class of biochemical components that activate TLRs has been studied extensively, but less is known about how TLRs interact with the class of compounds that are still associated with the live pathogen. Accordingly, we examined the activation of surface assembled TLR 2, 4, and 5 with live Tier 1 Gram-negative pathogens that included Yersinia pestis (plague), Burkholderia mallei (glanders), Burkholderia pseudomallei (melioidosis), and Francisella tularensis (tularemia). We found that Y. pestis CO92 grown at 28°C activated TLR2 and TLR4, but at 37°C the pathogen activated primarily TLR2. Although B. mallei and B. pseudomallei are genetically related, the former microorganism activated predominately TLR4, while the latter activated predominately TLR2. The capsule of wild-type B. pseudomallei 1026b was found to mitigate the activation of TLR2 and TLR4 when compared to a capsule mutant. Live F. tularensis (Ft) Schu S4 did not activate TLR2 or 4, although the less virulent Ft LVS and F. novicida activated only TLR2. B. pseudomallei purified flagellin or flagella attached to the microorganism activated TLR5. Activation of TLR5 was abolished by an antibody to TLR5, or a mutation of fliC, or elimination of the pathogen by filtration. In conclusion, we have uncovered new properties of the Gram-negative pathogens, and their interaction with TLRs of the host. Further studies are needed to include other microorganism to extend our observations with their interaction with TLRs, and to the possibility of leading to new efforts in therapeutics against these pathogens.

Genomic pattern analysis of Burkholderia mallei field isolates by pulsed-field gel electrophoresis (PFGE) discriminatory typing

Background and Objectives: Glanders is a serious zoonotic disease caused by Burkholderia mallei. Prevention, control, and treatment strategies of glanders are prerequisites for microbial source tracking. The present study was aimed to analyze the genomic pattern of B. mallei Iranian field isolates by pulsed-field gel electrophoresis (PFGE) typing. Materials and Methods: B. mallei isolates were aerobically cultured in nutrient broth/agar supplemented with glycerol 4% for 48 h at 37°C. API 20NE identification system was used for the biochemical characterization. Genomic DNA of bacterial isolates was extracted using OIE-recommended protocol. Molecular identification of bacterial isolates was done based on amplification of BimA and IS407-flip genes. PFGE was applied to prepare the genomic pattern of B. mallei isolates. The guinea pig was used as a suitable model for studying the histopathological characterization of B. mallei. Results: In both enzymatic digestion patterns by using Af1II and VspI, we found three different clonal types; І) PFGE type of B. mallei Razi 325 strain, ІІ) PFGE type of Tiger, Kordan, and Oshnavieh strains, and ІІІ) PFGE type of Semirom strain. B. mallei Razi 325 was categorized as unrelated strain which was belonged to the different cluster differing more than four bands. Conclusion: PFGE showed more discriminatory power and considerable reproducibility for molecular typing of B. mallei strains in our study. It is standardized the approaches for outbreak detection, pathogen phylogeny, molecular epidemiology, and population studies.

Preparation and transfer of Burkholderia mallei production strain 5584 in accordance with the biosafety requirements

The Veterinary Service of the Russian Federation takes measures to ensure regular control of livestock health status, to prevent infectious diseases and their introduction into the country; and if such diseases are diagnosed, it takes measures to prevent their spread and contain outbreaks as soon as possible. Success of the taken measures depends on the use of various diagnostic, preventive and therapeutic drugs. In order to produce such medicinal products, biofactories use production and reference strains with stable biological properties, which are stored in national collections of microorganisms. The only keeper of glanders strains is the Laboratory for Collection of Strains of Microorganisms in the FSBSI «FCTRBS-ARRVI», subordinated to the Ministry of Agriculture of the Russian Federation. The following steps were taken due to the official request from FKP Kursk Biofactory – BIOK Company for the transfer of Burkholderia mallei production strain 5584 from the collection of the institution: the strain was passaged in golden hamsters, its viability was determined and biological properties of the culture were studied. The strain was transferred in accordance with the established procedure and in compliance with the biosafety requirements. As the work progressed, Burkholderia mallei strain 5584 culture was isolated and freeze-dried. Before the transfer, biological properties of the freeze-dried Burkholderia mallei strain 5584 were studied for their compliance with the passport data. The obtained results showed that the Laboratory for Collection of Strains of Microorganisms in the FSBSI «FCTRBS-ARRVI» provides optimal conditions to preserve the strain viability and initial biological properties after 5 years of storage. Analysis of the data obtained during the transfer of Burkholderia mallei strain 5584 allowed us to assess the actions taken at all stages of the procedure. It was established that the transfer procedure for the requested glanders production strain complied with the biosafety requirements and regulatory framework regulating the process.