Proteomics-Based Safety Tool for Swine Brucellosis Diagnosis: A One-Health Perspective

Swine brucellosis, caused by Brucella suis, particularly biovars 1, 2, and 3, poses a significant threat to the swine industry and public health. In Europe, B. suis biovar 2 is prevalent, affecting both domestic pigs and wild boars. Infected swine experience reproductive disorders such as abortions in sows and orchitis in boars, leading to substantial economic losses. Although human infections from B. suis biovar 2 are relatively rare, the potential for transmission, especially in laboratory settings through airborne particles, underscores the importance of effective diagnosis.

Fig.1 2D electrophoresis of Brucellergene OCB (Rhône-Mérieux, France), an antigen produced from Brucella melitensis rough strain B115 employed in swine for in vitro serological tests. (Sagona S., et al., 2025)

Limitations of Conventional Diagnostic Methods

Traditional serological tests, including the Rose Bengal Test (RBT), Complement Fixation Test (CFT), and Enzyme-Linked Immunosorbent Assay (ELISA), have been the mainstay of brucellosis diagnosis. These tests detect antibodies against the smooth lipopolysaccharides (sLPS) of Brucella bacteria. However, they suffer from two major drawbacks.

Firstly, false positives are common due to cross-reactivity. Other Gram-negative bacteria like Yersinia enterocolitica O:9 have similar sLPS structures to Brucella. For example, in swine populations where Yersinia enterocolitica is present, the antibodies from this bacterium can react with Brucella tests, leading to misdiagnosis. Secondly, the production of antigens for these tests requires culturing live Brucella bacteria. This exposes laboratory personnel to the risk of airborne infections, as Brucella is highly infectious. While Brucellergene, a commercial antigen from a rough strain of B. melitensis B115, reduces cross-reactivity and is safer, it still necessitates Brucella culturing.

Proteomics: A New Frontier in Diagnosis

Proteomic techniques offer a novel approach to identifying specific Brucella antigens for diagnosis. Researchers analyzed Brucellergene, a mixture of over 20 cytoplasmic proteins from B. melitensis.

Protein Separation and Identification

Using Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and 2D electrophoresis, the proteins in Brucellergene were separated. SDS-PAGE revealed 16 distinct protein bands, while 2D electrophoresis showed over 20 spots, with molecular weights ranging from 11 to 115 kDa and isoelectric points between pH 4.8 and 7.8. Western blotting was then employed to identify proteins that bind to antibodies in infected swine serum. Four bands (B3, B13, B16, and I1) showed strong interactions. Mass spectrometry was used to determine the identities of the proteins in these bands.

The Promising Proteins

Four proteins emerged as potential diagnostic antigens:

Probable sugar-binding periplasmic protein

This protein likely plays a role in the transportation of sugars across the bacterial membrane. What makes it a standout candidate for diagnosis is its complete lack of similarity to proteins found in Yersinia. This absence of similarity significantly reduces the risk of false positives, a major advantage over traditional diagnostic methods.

Peptide ABC transporter substrate-binding protein

As part of a peptide transport system within the bacterium, this protein is remarkable for its production even under stressful conditions. ABC transporters are essential for the survival of bacteria in various harsh environments, such as nutrient-starved conditions or when under attack by the host’s immune system. This means that the peptide ABC transporter substrate-binding protein is likely to be present throughout the course of an infection, making it a reliable target for antibody detection, even in chronic cases where traditional tests often fall short.

GntR family transcriptional regulator

This protein is involved in regulating gene expression within the Brucella bacterium, including genes related to virulence. It has a relatively low similarity (less than 49%) to Yersinia proteins, further contributing to its potential as a specific diagnostic antigen. By influencing gene expression, it can provide insights into the bacterium’s activity during an infection, which can be crucial for accurate diagnosis.

Conserved hypothetical protein

Despite its unknown function, its conservation across different Brucella strains is a significant advantage. Moreover, it shares no similarity with Yersinia proteins, enhancing the specificity of potential diagnostic tests. The fact that it is conserved suggests that it may play an important, yet-to-be-discovered, role in the bacterium’s survival or pathogenesis, making it a valuable target for further research.

Advantages of the Newly Identified Proteins

These proteins overcome the limitations of traditional methods. Their lack of similarity to Yersinia and other cross-reactive bacteria significantly reduces false positives. Moreover, they can be synthesized in vitro using molecular biology techniques, eliminating the need for live Brucella culturing and ensuring laboratory safety. Proteins like the ABC transporter are expressed during different stages of infection, improving the sensitivity of detection in both acute and chronic cases.

Future Outlook

Although these findings are promising, further research is required. The next steps include in vitro synthesis of the proteins to ensure consistency and safety, field testing with samples from infected and non-infected swine, and development of user-friendly diagnostic kits. By advancing these steps, we can create a more accurate, safe, and accessible diagnostic tool for swine brucellosis, aligning with the One-Health framework and safeguarding both animal and human health.

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Reference

Sagona, Simona, et al. "Preliminary Investigation Towards a Safety Tool for Swine Brucellosis Diagnosis by a Proteomic Approach Within the One-Health Framework." International Journal of Molecular Sciences 26.4 (2025): 1517.

This article is for research use only. Do not use in any diagnostic or therapeutic application.