Research Article
Volume 6 Issue 5 - 2021
The Omp31r Recombinant Protein of Brucella melitensis and Immunological Evaluation for its Possible Use for the Diagnosis in Bovine Brucellosis
Annie Castillo Ochoa1, Rodolfo Fernández-Gomez2, Juan Carlos Freites3, Ana Teresa Serrano4 and Jose-David Rosales3*
1Simón Bolívar University, Caracas, Venezuela
2Biophysics Solutions CA, Caracas, Venezuela
3Centre of Agriculture and Alimentarium Security, Institute of Advanced Studies Foundation (IDEA), Caracas, Venezuela
4Faculty of Veterinary Sciences, Central University of Venezuela, Maracay, Venezuela
*Corresponding Author: Jose-David Rosales, Centre of Agriculture and Alimentarium Security, Institute of Advanced Studies Foundation (IDEA), Caracas, Venezuela.
Received: February 08, 2021; Published: April 30, 2021


In humans, Brucellosis is a potentially mortal disease, caused by intracellular Gram-negative bacteria, which belongs to the Brucella group and is worldwide distributed. In humans, the infection is mediated through the consumption of milk or fresh cheese no pasteurized and raw meat. Therefore, sanitary control in farm animals is greatly important, thus the serologic diagnosis is key to detect antibodies against Brucella´s lipopolysaccharide, by ELISA tests or agglutination tests. The aim of the work is testing a recombinant protein, to use it as a potential protein for serological diagnosis of brucellosis in bovine. Bioinformatics analysis results in the OMP31 gene identification from Brucella melitensis M15, forward cloned from genomic DNA. The OMP31 protein was expressed in a BL21 (DE3) system, using pET28a vector, and isolated by polyhistidine-NI affinity chromatography. Polyclonal antibodies against OMP31 were produced in rabbit and a western blot against Brucella abortus RB51, Brucella melitensis M15 and Brucella abortus 1119 were performed. The antibodies produced did recognize Brucella abortus RB51 and Brucella melitensis M15, but didn´t recognize the vaccine strain Brucella abortus 1119. A standardized ELISAi was performed, using healthy bovine´s serums and Brucella abortus positive bovine´s serums. The specificity and sensibility were estimated contrasting an ELISAi using OMP31r obtaining 90.6% and 77.17% and Brucella abortus 1119, 100%. The recognition of OMP31r by antibodies from serums of B. abortus infected bovines is quite promising, opening the possibility to produce both, rapids test kit and massive diagnosis to enable the control of bovine brucellosis.


Keywords: Brucella sp; Omp31r; Brucellosis; Recombinant Protein; Indirect ELISA


  1. Vizcaino N., et al. “Coding for an immunogenic major outer membrane protein”. Infection and Immunity 64 (1996): 3744-3751.
  2. Mantur BG., et al. “Review of clinical and laboratory features of human brucellosis”. Indian Journal of Medical Microbiology 25 (2007): 188-202.
  3. Cloeckaert A., et al. “Nucleotide sequence and expression of the gene encoding the major 25-kilodalton outer membrane protein of Brucella ovis: evidence for antigenic shift, compared with other Brucella species, due to a deletion in the gene”. Infection and Immunity 64 (1996): 2047-2055.
  4. Rossetti CA., et al. “Caprine brucellosis: A historically neglected disease with significant impact on public health”. PLOS Neglected Tropical Diseases 11 (2017): e0005692.
  5. Kalem F., et al. “Comparison of a New and Rapid Method: Brucella Coombs Gel Test With Other Diagnostic Tests”. Journal of Clinical Laboratory Analysis 30 (2016): 756-759.
  6. Schmoock G., et al. “Development of a diagnostic multiplex polymerase chain reaction microarray assay to detect and differentiate Brucella spp”. Diagnostic Microbiology and Infectious Disease 71 (2011): 341-353.
  7. Bricker BJ. “Diagnostic strategies used for the identification of Brucella”. Veterinary Microbiology 90 (2002): 433-434.
  8. Onilud OM., et al. “Development and application of dot-enzyme-linked immunosorbent (dot-ELISA) assay for detection of Brucella melitensis and evaluation of the shedding pattern in infected goats”. Journal of Immunoassay and Immunochemistry 38 (2017): 82-99.
  9. Thepsuriyanont P., et al. “ELISA for brucellosis detection based on three Brucella recombinant proteins”. The Southeast Asian Journal of Tropical Medicine and Public Health 45 (2014): 130-141.
  10. Perrett LL., et al. “Evaluation of competitive ELISA for detection of antibodies to Brucella infection in domestic animals”. Croatian Medical Journal 51 (2010): 314-319.
  11. Praud A., et al. “Assessment of the diagnostic sensitivity and specificity of an indirect ELISA kit for the diagnosis of Brucella ovis infection in rams”. BMC Veterinary Research 8 (2012): 68.
  12. Francisco J and Vargas O. “Brucellosis in Venezuela”. Veterinary Microbiology 90 (2002): 39-44.
  13. Lord V and Lord R. “Brucella suis infections in collared peccaries in Venezuela”. Journal of Wildlife Diseases 27 (1991): 477-481.
  14. Lord VR and Flores R. “Brucella from the capybara (Hydrochaeris hydrochaeris) in Venezuela: serologic studies and metabolic characterization of isolates”. Journal of Wildlife Diseases 19 (1983): 308-314.
  15. Muñoz PM., et al. “Efficacy of several serological tests and antigens for diagnosis of bovine brucellosis in the presence of false-positive serological results due to Yersinia enterocolitica O :9”. Clinical and Diagnostic Laboratory Immunology 1 (2005): 141-151.
  16. Al Dahouk S., et al. “Immunoproteomic characterization of Brucella abortus 1119-3 preparations used for the serodiagnosis of Brucella infections”. The Journal of Immunological Methods 309 (2006): 34-47.
  17. Nielsen K., et al. “Serological discrimination by indirect enzyme immunoassay between the antibody response to Brucella and Yersinia enterocolitica O:9 in cattle and pigs”. Veterinary Immunology and Immunopathology 109.1.2 (2006): 69-78.
  18. Pajuaba ACAM., et al. “Immunoproteomics of Brucella abortus reveals differential antibody profiles between S19-vaccinatedand naturally infected cattle”. Proteomics 12 (2012): 820-831.
  19. Tabatabai LB., et al. “Immunogenicity of Brucella abortus salt-extractable proteins”. Veterinary Microbiology 20 (1989): 49-58.
  20. Mahajan NK., et al. “Immunogenicity of major cell surface protein(s) of Brucella melitensis Rev 1”. Veterinary Research Communications 29 (2005): 189-199.
  21. Manat Y., et al. “Expression, purification and immunochemical characterization of recombinant OMP28 protein of Brucella species”. Open Veterinary Journal 6 (2016): 71-77.
  22. Mirkalantari S., et al. “Brucella melitensis VirB12 recombinant protein is a potential marker for serodiagnosis of human brucellosis”. Annals of Clinical Microbiology and Antimicrobials 16 (2017): 8.
  23. Lim JJ., et al. “Protective effects of recombinant Brucella abortus Omp28 against infection with a virulent strain of Brucella abortus 544 in mice”. Journal of Veterinary Science 13 (2012): 287-292.
  24. Tiwari AK., et al. “Evaluation of the recombinant 10-kilodalton immunodominant region of the BP26 protein of Brucella abortus for specific diagnosis of bovine brucellosis”. Clinical and Vaccine Immunology 18 (2011): 1760-1764.
  25. Kumar A., et al. “Optimization and efficient purification of recombinant Omp28 protein of Brucella melitensis using Triton X-100 and beta-mercaptoethanol”. Protein Expression and Purification 83 (2012): 226-232.
  26. Connolly JP., et al. “Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development”. Proteomics 6 (2006): 3767-3780.
  27. Adone R and Ciuchini F. “Brucella abortus RB51 and hot saline extract from Brucella ovis as antigens in a complement fixation test used to detect sheep vaccinated with Brucella abortus RB51”. Clinical and Vaccine Immunology 1 (2001): 119-122.
  28. Kim JY., et al. “Immunoproteomics of Brucella abortus RB51 as candidate antigens in serological diagnosis of brucellosis”. Veterinary Immunology and Immunopathology 160 (2014): 218-224.
  29. Schurig GG., et al. “Biological properties of RB51; a stable rough strain of Brucella abortus”. Veterinary Microbiology 2 (1991): 171-188.
  30. Ko KY., et al. “Immunogenic proteins of Brucella abortus to minimize cross reactions in brucellosis diagnosis”. Veterinary Microbiology Biology 156 (2012): 374-380.
  31. Sutula CL and JM Cuillet. “Interpreting ELISA data and establishing the positive negative theresfold”. Plant Disease 8 (1986): 722-726.
  32. Koyuncu I., et al. “Clinical immunology Diagnostic potential of Brucella melitensis Rev1 native Omp28 precursor in human brucellosis”. Central European Journal of Immunology 1 (2018): 81-89.
  33. Rosales Jose-David., et al. “Cloning, expression and immunological evaluation of the omp31 protein of Brucella melitensis and evaluation of its possible use for the diagnosis in bovine brucellosis”. Revista de Investigaciones Veterinarias del Perú 3 (2018): 996-1008.
  34. Navarro MC. “Expresión de la proteína Omp31 recombinante de Brucella ovis en el sistema de escherichia coli para su uso como antígeno en el diagnóstico de epididimitis contagiosa del carnero”. Maestría thesis, Universidad Autónoma de Nuevo León (2012).
  35. Ochoa AC., et al. “Prepare Escherichia coli powder and antibodies pre-absorption”. Bioprotocol “in press” (2020).
  36. Debbarh HS., et al. “Competitive enzyme-linked immunosorbent assay using monoclonal antibodies to the Brucella melitensis BP26 protein to evaluate antibody responses in infected and melitensis Rev.1 vaccinated sheep”. Veterinary Microbiology 53 (1996): 325-337.
  37. Cloeckaert A., et al. “Use of recombinant BP26 protein in serological diagnosis of Brucella melitensis infection in sheep”. Clinical and Vaccine Immunology 8 (2001): 772-775.
  38. Liu WX., et al. “Expression, purification, and improved antigenic specificity of a truncated recombinant bp26 protein of Brucella melitensis M5-90: a potential antigen for differential serodiagnosis of brucellosis in sheep and goats”. Biotechnology and Applied Biochemistry 58 (2011): 32-38.
  39. Thavaselvam D., et al. “Cloning and expression of the immunoreactive Brucella melitensis 28 kDa outer-membrane protein (Omp28) encoding gene and evaluation of the potential of Omp28 for clinical diagnosis of brucellosis”. Journal of Medical Microbiology 59 (2010): 421-428.
  40. Kumar S., et al. “Generation and characterization of murine monoclonal antibodies to recombinant 26-kDa periplasmic protein of Brucella abortus”. Hybridoma 26 (2007): 322-327.
  41. Cherwonogrodzky JW and Nielsen KH. “Brucella abortus 1119-30-chain polysaccharide to differentiate sera from B. abortus S-19-vaccinated and field-strain-infected cattle by agar gel immunodiffusion”. Journal of Clinical Microbiology 26 (1988): 1120-1123.
  42. Vizcaíno N., et al. “DNA polymorphism at the omp-31 locus of Brucella: evidence for a large deletion in Brucella abortus, and other species-specific markers”. Microbiology 143 (1997): 2913-2921.
  43. Gupta VK., et al. “Serological diagnostic potential of recombinant outer membrane protein (Omp31) from Brucella melitensis in goat and sheep brucellosis”. Small Ruminant Research2-3 (2007): 260-266.
Citation: Jose-David Rosales., et al. “The Omp31r Recombinant Protein of Brucella melitensis and Immunological Evaluation for its Possible Use for the Diagnosis in Bovine Brucellosis”. EC Veterinary Science 6.5 (2021): 06-16.

PubMed Indexed Article

EC Pharmacology and Toxicology
LC-UV-MS and MS/MS Characterize Glutathione Reactivity with Different Isomers (2,2' and 2,4' vs. 4,4') of Methylene Diphenyl-Diisocyanate.

PMID: 31143884 [PubMed]

PMCID: PMC6536005

EC Pharmacology and Toxicology
Alzheimer's Pathogenesis, Metal-Mediated Redox Stress, and Potential Nanotheranostics.

PMID: 31565701 [PubMed]

PMCID: PMC6764777

EC Neurology
Differences in Rate of Cognitive Decline and Caregiver Burden between Alzheimer's Disease and Vascular Dementia: a Retrospective Study.

PMID: 27747317 [PubMed]

PMCID: PMC5065347

EC Pharmacology and Toxicology
Will Blockchain Technology Transform Healthcare and Biomedical Sciences?

PMID: 31460519 [PubMed]

PMCID: PMC6711478

EC Pharmacology and Toxicology
Is it a Prime Time for AI-powered Virtual Drug Screening?

PMID: 30215059 [PubMed]

PMCID: PMC6133253

EC Psychology and Psychiatry
Analysis of Evidence for the Combination of Pro-dopamine Regulator (KB220PAM) and Naltrexone to Prevent Opioid Use Disorder Relapse.

PMID: 30417173 [PubMed]

PMCID: PMC6226033

EC Anaesthesia
Arrest Under Anesthesia - What was the Culprit? A Case Report.

PMID: 30264037 [PubMed]

PMCID: PMC6155992

EC Orthopaedics
Distraction Implantation. A New Technique in Total Joint Arthroplasty and Direct Skeletal Attachment.

PMID: 30198026 [PubMed]

PMCID: PMC6124505

EC Pulmonology and Respiratory Medicine
Prevalence and factors associated with self-reported chronic obstructive pulmonary disease among adults aged 40-79: the National Health and Nutrition Examination Survey (NHANES) 2007-2012.

PMID: 30294723 [PubMed]

PMCID: PMC6169793

EC Dental Science
Important Dental Fiber-Reinforced Composite Molding Compound Breakthroughs

PMID: 29285526 [PubMed]

PMCID: PMC5743211

EC Microbiology
Prevalence of Intestinal Parasites Among HIV Infected and HIV Uninfected Patients Treated at the 1o De Maio Health Centre in Maputo, Mozambique

PMID: 29911204 [PubMed]

PMCID: PMC5999047

EC Microbiology
Macrophages and the Viral Dissemination Super Highway

PMID: 26949751 [PubMed]

PMCID: PMC4774560

EC Microbiology
The Microbiome, Antibiotics, and Health of the Pediatric Population.

PMID: 27390782 [PubMed]

PMCID: PMC4933318

EC Microbiology
Reactive Oxygen Species in HIV Infection

PMID: 28580453 [PubMed]

PMCID: PMC5450819

EC Microbiology
A Review of the CD4 T Cell Contribution to Lung Infection, Inflammation and Repair with a Focus on Wheeze and Asthma in the Pediatric Population

PMID: 26280024 [PubMed]

PMCID: PMC4533840

EC Neurology
Identifying Key Symptoms Differentiating Myalgic Encephalomyelitis and Chronic Fatigue Syndrome from Multiple Sclerosis

PMID: 28066845 [PubMed]

PMCID: PMC5214344

EC Pharmacology and Toxicology
Paradigm Shift is the Normal State of Pharmacology

PMID: 28936490 [PubMed]

PMCID: PMC5604476

EC Neurology
Examining those Meeting IOM Criteria Versus IOM Plus Fibromyalgia

PMID: 28713879 [PubMed]

PMCID: PMC5510658

EC Neurology
Unilateral Frontosphenoid Craniosynostosis: Case Report and a Review of the Literature

PMID: 28133641 [PubMed]

PMCID: PMC5267489

EC Ophthalmology
OCT-Angiography for Non-Invasive Monitoring of Neuronal and Vascular Structure in Mouse Retina: Implication for Characterization of Retinal Neurovascular Coupling

PMID: 29333536 [PubMed]

PMCID: PMC5766278

EC Neurology
Longer Duration of Downslope Treadmill Walking Induces Depression of H-Reflexes Measured during Standing and Walking.

PMID: 31032493 [PubMed]

PMCID: PMC6483108

EC Microbiology
Onchocerciasis in Mozambique: An Unknown Condition for Health Professionals.

PMID: 30957099 [PubMed]

PMCID: PMC6448571

EC Nutrition
Food Insecurity among Households with and without Podoconiosis in East and West Gojjam, Ethiopia.

PMID: 30101228 [PubMed]

PMCID: PMC6086333

EC Ophthalmology
REVIEW. +2 to +3 D. Reading Glasses to Prevent Myopia.

PMID: 31080964 [PubMed]

PMCID: PMC6508883

EC Gynaecology
Biomechanical Mapping of the Female Pelvic Floor: Uterine Prolapse Versus Normal Conditions.

PMID: 31093608 [PubMed]

PMCID: PMC6513001

EC Dental Science
Fiber-Reinforced Composites: A Breakthrough in Practical Clinical Applications with Advanced Wear Resistance for Dental Materials.

PMID: 31552397 [PubMed]

PMCID: PMC6758937

EC Microbiology
Neurocysticercosis in Child Bearing Women: An Overlooked Condition in Mozambique and a Potentially Missed Diagnosis in Women Presenting with Eclampsia.

PMID: 31681909 [PubMed]

PMCID: PMC6824723

EC Microbiology
Molecular Detection of Leptospira spp. in Rodents Trapped in the Mozambique Island City, Nampula Province, Mozambique.

PMID: 31681910 [PubMed]

PMCID: PMC6824726

EC Neurology
Endoplasmic Reticulum-Mitochondrial Cross-Talk in Neurodegenerative and Eye Diseases.

PMID: 31528859 [PubMed]

PMCID: PMC6746603

EC Psychology and Psychiatry
Can Chronic Consumption of Caffeine by Increasing D2/D3 Receptors Offer Benefit to Carriers of the DRD2 A1 Allele in Cocaine Abuse?

PMID: 31276119 [PubMed]

PMCID: PMC6604646

EC Anaesthesia
Real Time Locating Systems and sustainability of Perioperative Efficiency of Anesthesiologists.

PMID: 31406965 [PubMed]

PMCID: PMC6690616

EC Pharmacology and Toxicology
A Pilot STEM Curriculum Designed to Teach High School Students Concepts in Biochemical Engineering and Pharmacology.

PMID: 31517314 [PubMed]

PMCID: PMC6741290

EC Pharmacology and Toxicology
Toxic Mechanisms Underlying Motor Activity Changes Induced by a Mixture of Lead, Arsenic and Manganese.

PMID: 31633124 [PubMed]

PMCID: PMC6800226

EC Neurology
Research Volunteers' Attitudes Toward Chronic Fatigue Syndrome and Myalgic Encephalomyelitis.

PMID: 29662969 [PubMed]

PMCID: PMC5898812

EC Pharmacology and Toxicology
Hyperbaric Oxygen Therapy for Alzheimer's Disease.

PMID: 30215058 [PubMed]

PMCID: PMC6133268

News and Events

May Issue Release

We always feel pleasure to share our updates with you all. Here, notifying you that we have successfully released the May issue of respective journals and the latest articles can be viewed on the current issue pages.

Submission Deadline for upcoming Issue

Ecronicon delightfully welcomes all the authors around the globe for effective collaboration with an article submission for the upcoming issue of respective journals. Submissions are accepted on/before May 24, 2021.

Certificate of Publication

Ecronicon honors with a "Publication Certificate" to the corresponding author by including the names of co-authors as a token of appreciation for publishing the work with our respective journals.

Best Article of the Issue

Editors of respective journals will always be very much interested in electing one Best Article after each issue release. The authors of the selected article will be honored with a "Best Article of the Issue" certificate.

Certifying for Review

Ecronicon certifies the Editors for their first review done towards the assigned article of the respective journals.

Latest Articles

The latest articles will be updated immediately on the articles in press page of the respective journals.

Immediate Assistance

The prime motto of this team is to clarify all the queries without any delay or hesitation to avoid the inconvenience. For immediate assistance on your queries please don't hesitate to drop an email to