Serological Diagnosis Of Brucellosis Research Articles

Smooth and Rough Brucella Infections in Dogs

Background: B. canis infections have been reported in many countries of the world. B.canis can cause infections both in dogs and human beings. It can be transmitted to human via laboratory accidents during handling and contact with sick dogs. is an infectious agent for humans and it is transmitted to humans by various ways including laboratory accidents and contact with sick dogs. Methods: In this study serum samples were collected by using stratified random sampling method from the Diyarbakýr Metropolitan Municipality Animal Care and Rehabilitation Center, where approximately 600 dogs were housed. Result: Out of 83 samples, 9.6% and 26.5% of serum samples were found as positive by B.canis RSAT and B.canis ELISA, respectively. While 6% and 9.6% of the samples were positive by B. abortus S99 RSAT and B. abortus ELISA, respectively. Sensitivity and specificity of B. abortus S99 ELISA was 98% and 95.5%, respectively. On the other hand, sensitivity and specificity of B.canis M(-) ELISA was 96.6% and 94%. As a result, it was concluded that B. canis infection is common in dogs and this should be considered both for dogs and public health. Unfortunately, there is no standardized commercial serological test for diagnosis of B.canis and other rough Brucella species currently. This is the main reason that rough brucella infections might not represent the real B.canis infection situation. Therefore, most of the cases have the possibility of being undetected. For this reason it is utmost important that B.canis RSAT and ELISA can be used as routine tests in the hospitals and veterinary clinics in the serological diagnosis of brucellosis.

Alfa-proteobacteria Grubuna Ait Bazı Bakteri Cinslerinden İzole edilen antijenlerin Brusellozisin Serolojik Tanısında Potansiyel Uygulanabilirliğinin Araştırılması

Brusellozis insanlarda ve hayvanlarda önemli enfeksiyonlara neden olan, halk sağlığını tehdit eden ve hayvancılık endüstrisine ciddi ekonomik kayıplara neden olan zoonoz bir enfeksiyondur. Bu çalışmada, Brucella suşları ile filogenetik yakınlığı bulunan alfa–Proteobacteria sınıfına ait Ochrobactrum anthropi, O. intermedium ve Rhizobium tropici türlerinden hazırlanan antijenlerin brusellosizin serolojik tanısında Brucella antijeni yerine kullanılıp kullanılamayacağının ortaya konulması amaçlandı. Çalışmada, test serumlarına pozitif ve negatif kontroller ile birlikte indirekt ELISA uygulandı. Sonuç olarak, sığır ve insan brusellozisi açısından test edilen antijenlerin hiçbiri güvenilir düzeyde sensitivite ve spesifisite göstermedi. Çalışmada, sadece R-LPS taşıyan O. intermedium antijeni ile ve sadece koyun ve köpeklerde kısmen kabul edilebilir sınırlarda bir tanısal performans alındı. Sonuç olarak, proteobakterilerin α-2 alt grubunda yer alan mikroorganizmaların brusellozisin indirekt teşhisinde bazı çapraz reaksiyonlara neden olabileceği ve bu durumun yanlış pozitiflik yaratabileceği saptandı. Öte yandan bu grup bakterilerden hazırlanan daha saf antijenlerin, B. canis ve B. ovis infeksiyonlarında kullanılma potansiyeli bulunabileceği ve bu yönde yapılacak çalışmalara ihtiyaç olduğu kanısına varıldı.

Brucella Coombs gel test: Can it replace other serological methods as a rapid test in serological diagnosis of brucellosis?

Brucella Coombs gel test: Can it replace other serological methods as a rapid test in serological diagnosis of brucellosis?

A novel recombinant multiepitope protein candidate for the diagnosis of brucellosis: A pilot study

Brucellosis is a zoonotic disease transmitted to humans from infected animals. As a systemic disease, it can harm any organ or system of the host body. Human brucellosis presents with various clinical symptoms, which makes diagnosis challenging. Serological diagnosis of brucellosis is based on ELISA or agglutination tests, which use colorimetry to detect antibodies generated against lipopolysaccharide (LPS) or extracts from whole-cell bacteria. To construct a protein that can specifically recognize Brucella, we analyzed hydrophilicity, accessibility, flexibility, antigenicity, and β-turns using a protein network server. Then, we chose the most abundant immunodominant epitopes of the outer membrane proteins omp31, BP26, omp2b and omp16. Based on the sequences of these major epitopes, fifteen major immunodominant epitopes were selected to construct a synthetic Brucella recombinant multiepitope outer membrane protein (rOmp) gene. This recombinant gene was expressed in E. coli, and the produced protein was purified by Ni-NTA affinity purification. The purified protein was tested in an indirect ELISA assay, demonstrating a high level of sensitivity and specificity. This technique is creating a unique antigen that, coupled with overexpression and low-cost purification, offers a promising diagnosis of both human and animal brucellosis, with the potential to avoid the disadvantages of whole brucellosis-antigen-based assays.

Comparison of Coombs Gel Test with ELISA and Standard Tube Agglutination Tests Used in Serological Diagnosis of Brucellosis

Objective: Serological tests are the most commonly used tests in the diagnosis of brucellosis; however, each serological test has some drawbacks. In this study, we aimed to determine the value of the Brucella Coombs gel test (BCGT) in the serological diagnosis of brucellosis in comparison with Standard tube agglutination (STA) and ELISA tests. Materials and Methods: The study included 42 patients who were considered to have brucellosis as a preliminary diagnosis. BCGT, Brucella-IgM/IgG ELISA, and STA tests were performed from serum samples of the patients. The correlation of the diagnostic tests was analyzed using Cohen’s Kappa Analysis. Results: Twenty-seven (64.2%) of 42 patients were diagnosed with brucellosis according to their medical history and clinical and serological tests. The sensitivity and specificity of BCGT to diagnose brucellosis was 96.2%, and 100%, respectively. The sensitivity and specificity for the diagnosis of brucellosis 62.9% and 100% for STA, respectively; 33.3% and 66.6% for Brucella-IgM; and 66.6% and 100% for Brucella-IgG. BCGT was significantly correlated with STA (κ= 0.590) and Brucella-IgG (κ=0.539) Conclusion: BCGT can be utilized as a simple and reliable test in the diagnosis of brucellosis with high sensitivity and specificity. Nevertheless, the sensitivity and specificity of BCGT should be demonstrated by comprehensive studies, including culture-confirmed cases and control groups.

Patho-epidemiological and molecular diagnosis of swine brucellosis in Punjab

Brucellosis is one of the major reproductive tract diseases of swine and causes a huge economic loss to pig farmers. In this study, serological, gross pathological, histopathological, immunohistochemical and Polymerase Chain Reaction technique(s) have been applied for diagnosis of brucellosis in pig. Seroprevalence studies showed prevalence of brucellosis to be 16.7% by Rose Bengal Plate Test (RBPT) and 12.5% by competitive ELISA(c-ELISA). In this study, comparison was done between RBPT and c-ELISA for serological diagnosis of brucellosis which revealed substantial degree of agreement (Kappa= 0.676, 95% CI= 0.372-0.826) between two tests. Histopathological examination revealed focal to diffuse mixed infiltration of neutrophils or lymphocytesin the fetal organs like lung especially in bronchi and interstitial septa. Lesions in the placenta were characterized by diffuse lympho-mononuclear cell infiltration. Histopathological changes in female reproductive tracts were characterized by cellular infiltration of the endometrial stroma, endometrial glands and hyperplasia of periglandular connective tissue. Immunohistochemical (IHC) staining revealed intracytoplasmic immunostaining of Brucella in bronchial epithelial cells of lung, endothelial cells in spleen, hepatocytes of liver, cells within the lymph node, kidney of fetus and placenta. In addition, positive immunostaining to Brucella was also seen in lamina propria cells of uterus. By PCR, eight out of 34 samples (2 from stomach contents, 2 from placental cotyledons and 4 from pooled tissue(s) produced 193 bpsized ampliconspecific for Brucella genus. In the present study, PCR and IHC provide a reliable test for the diagnosis of brucellosis in abortion cases on aborted fetal tissue and placental cotyledons. However, serology is important for detection of Brucella positive animal in a farm .

Serological diagnosis of brucellosis at the ruminants in Mostaganem (Algeria)

Serological diagnosis of brucellosis at the ruminants in Mostaganem (Algeria)

Serological Monitoring of Brucellosis in Female Calves Born from Infected Herds from Birth to their First Calving

The aim of this study was to determine the frequency of brucellosis contagion of female calves born from seropositive and seronegative cows in brucellosis-infected herds. Brucellosis was monitored by serological analysis of 192 female calves from ten stables. Eight of these stables corresponded to family dairy herds, which had been vaccinated with S19; one was a semi-intensive herd vaccinated with RB51 and the last one was an intensive herd vaccinated with both vaccines. Monthly blood samples were taken from the female calves, from birth up to nine months of age; later, blood samples were taken at 12, 15, 18, 21, 24 months, and during delivery or abortion. Blood samples were analysed with Rose Bengal, rivanol and radial immunodiffusion tests to detect the appearance of seropositivity. A total of 192 female calves were evaluated and only 23% (45/192) were seropositive to brucellosis during the entire study. Of the 45 serologically positive female calves, 47% (21/45) were daughters of seropositive cows, while 53% (24/45) were daughters of seronegative cows. In conclusion, only 23% of the female calves born from infected herds showed seropositivity to brucellosis from birth up to 24 months of age, of which less than half were daughters of seropositive mothers and more than half were daughters of seronegative mothers. Therefore, serological diagnosis of brucellosis should be considered at an early age, which is currently not regular practice. In addition, biosecurity measures should be established, mainly in family dairy herds.

Application of the immunochromatographic test-system for serological diagnosis of brucellosis based on recombinant antigen

Application of the immunochromatographic test-system for serological diagnosis of brucellosis based on recombinant antigen

The influence of canine brucellosis on morphofunctional features of epididymal spermatozoa: case report

ABSTRACT The present work reports a clinical case of a mongrel dog, with serological diagnosis of brucellosis, from which epididymal sperm analysis was performed. Sperm samples were collected from different segments of the epididymis (tail, corpus, and caput). Sperm samples were evaluated for computer-assisted motility analysis (CASA), spermatic morphology, mitochondrial activity and sperm plasmatic membrane and acrosomal integrity. Changes in sperm movement patterns were found (progressive motility, percentage of rapid sperm, percentage of rapid velocity, average pathway, curvilinear velocity, velocity straight line, amplitude of lateral head displacement, straightness and linearity), increase of total morphological defects (51%) and absence of sperm mitochondrial activity (20%) were verified, especially for cauda epididymides. We highlight that such changes can contribute to clinical diagnosis of Brucellosis in dogs and to the use of epididymal sperm in reproductive biotechnologies.

English

Brucellosis is an important and overlooked zoonotic disease especially in developing countries having great potential to badly affect the people occupationally exposed to disease. In humans, the disease is usually marked with undulant fever, headache, fatigues, back pain, general malaise and arthritis. There have been a number of published studies available focusing on the serological surveillance in Pakistan but very little has been done using serum as clinical specimen for Polymerase Chain Reaction (PCR) and advanced serological techniques like Fluorescence Polarization Assay (FPA). In this study we have used serum as clinical sample for molecular and serological diagnosis of brucellosis in occupationally exposed human population to address the limitations associated with safe handling of field samples and current diagnostic procedures. Sera from 110 volunteers were collected and analyzed by employing serology (Rose Bengal Test, competitive ELISA, FPA) and serum PCR assay. Seropositivity was observed in 4.5% (5/110), 9% (10/110) and 31% (34/110) of samples with RBT, cELISA and FPA, respectively. The PCR assay could detect about 38% (42/110) samples (p<0.05). Serum PCR and FPA were able to detect more positive cases in each category of human population sampled. We found serum could be used as more dependable and safer clinical specimen for both serological and molecular investigations. Together with serum PCR, this study strongly recommends the introduction of FPA into routine clinical diagnosis for brucellosis. Our findings described here will help to understand and let decision makers to adopt and implement better choices for future brucellosis control measures. © 2016 Friends Science Publishers

Immunoproteomics of Brucella abortus RB51 as candidate antigens in serological diagnosis of brucellosis

The current brucellosis serodiagnostic assays are chiefly based on detecting anti-LPS (lipopolysaccharide) antibodies. However, cross-reaction with some gram-negative bacteria can occasionally induce due to similar O-polysaccharide (OPS) structure. Therefore, the aim of the present study was to identify new candidate antigens from Brucella abortus RB51, a mutant strain lacking the LPS portion, which might be valuable in brucellosis diagnosis. To detect potential antigens, immobilized pH gradients (IPG) strips with three ranges (pH 3–5.6, 4–7 and 6–11) were applied. After separating the insoluble proteins of B. abortus RB51 using two-dimensional electrophoresis (2-DE), their immunogenicity was evaluated by western blotting using four types of antisera – B. abortus, Yersinia enterocolitica O:9 and Escherichia coli O157:H7-positive, and B. abortus-negative bovine sera. Among the several immunogenic spots, the spots showing specific reactivity with only the B. abortus-positive antisera, were considered as candidate antigens. Overall, eleven immuno-reactive proteins were identified, as follows: Cu/Zn superoxide dismutase, histidinol dehydrogenase, chaperonin DnaK, chaperonin GroES, beta-ketoadipyl CoA thiolase, two-component response regulator, the cell-division protein FtsZ, aldehyde dehydrogenase, 50s ribosomal protein L10 and invasion protein B. These selected highly immunogenic protein spots might be useful as alternative antigens for brucellosis and helpful in reducing the cross-reactivity.

Isolation of a field strain of Brucella abortus from RB51-vaccinated- and brucellosis-seronegative bovine yearlings that calved normally

A study was carried out in Pichucalco, Chiapas (Mexico) to determine whether recently calved cows or those that aborted shed Brucella. Serological diagnosis of brucellosis was made in all animals (209). Six of the cows that calved normally and two that aborted underwent a bacteriological study of milk and vaginal exudate. Brucella abortus was isolated from vaginal exudate samples in two 3- to 4-year-old seronegative first-birth cows that had calved normally. This was confirmed through bacteriological identification and PCR as a field strain and smooth phenotypes. We conclude that seronegative cows vaccinated with RB51 which calved normally and shed B. abortus in the vaginal exudate after calving could be a serious problem because these cows are overlooked in routine diagnoses and are a source of Brucella infection.

The false sero-negativity of brucella standard agglutination test: Prozone phenomenon

Objectives: We aimed to assess prozone phenomenon that is quite rare and causes false negativity in serological diagnosis of brucellosis with standard dilution titers. Materials and methods: In this study the tests of four cases that have false negative serological results were evaluated. Blood cultures were obtained from all cases while cerebrospinal fluid cultures were studied in the two cases. Standard agglutination test (SAT) and Coombs test were performed to all patients. Results: SAT and Coombs test was negative in titers up to 1/640 in all cases. The SAT and Coombs tests in cerebrospinal fluid (CSF) of the two cases with neurobrucellosis diagnosis were negative, as well. Since the clinical and laboratory findings suggested the brucellosis, the serums were restudied by diluting up to 1/10240 titer and we saw that the first 3 cases became positive at a titer of 1/1280. The fourth case remained negative and therefore, we applied high dilution Coombs test. This time the test gave a positive result at 1/10240 titer beginning from 1/2560 titer. B.melitensis was isolated from two cases. Conclusion: SAT and Coombs’ test must be diluted to titers 1/2560 or more in order to exclude false sero-negativity in cases with clinical and laboratory findings suggesting brucellosis. J Microbiol Infect Dis 2011; 1(3):110-113

The agreement rate of rose Bengal, modified rose Bengal and rapid Wright tests for detection of positive serum sample of Brucellosis

Background: Rose Bengal is a common test for serologic diagnosis of brucellosis. The aim of this study was to evaluate the agreement rate between modified rose Bengal, rose Bengal and rapid Wright tests for detection of antibody positive serum samples. Methods: C ross section study was designed. 941 serum sample of patients referred to the central medical laboratory of Kermanshah, were evaluated by rose Bengal, modified rose Bengal and rapid Wright, using convenient sampling method. Positive samples were confirmed and their titers were defined by Wright agglutination test. Results: The number of positive serum samples with modified rose Bengal was two fold higher than conventional rose Bengal test (P<0.001), which included all positive samples with rose Bengal. On the other hand, although the number of positive diagnosed serum samples with modified rose Bengal were nearly two times more than Wright test (P<0.001), but it did not include all positive samples detected by Wright test. All three tests showed acceptable agreement rate (k≥0.583). Conclusion: Results of this study confirmed that modified rose Bengal able to detect more positive serum sample than conventional rose Bengal and it would be substituted rose Bengal. However, the rapid Wright test should be used aside with modified rose Bengal, because it could not cover all positive samples detected by rapid Wright test.

Isolation of Brucella melitensis from a RB51-vaccinated seronegative goat

The purpose of this study is to determine the etiology of abortions presented in a goat herd declared as free of brucellosis and vaccinated with RB51 located in Mexico. The serological diagnosis of brucellosis in 33 animals was performed. The study included three goats that aborted in the last third of gestation and 15 goats that gave birth normally; samples of milk and vaginal exudate were subjected to bacteriological study. All animals were negative for serological diagnosis, and isolation of Brucella melitensis was achieved in a single goat from vaginal exudate. However, the particularity is that this goat was negative to the card, indirect ELISA, and radial immunodiffusion tests. Isolation of a field strain was confirmed by biochemical test resistance to rifampicin and PCR. It is concluded that a goat which aborted in the last third of gestation was found spreading B. melitensis through vaginal discharge despite being vaccinated with RB51 and seronegative for brucellosis.

A comparison of immuncapture agglutination and ELISA methods in serological diagnosis of brucellosis.

Different serological tests are used in serologic diagnosis of brucellosis. The most widely used of these are Standard Tube Agglutination and Coombs anti-brucella tests. Whereas ELISA Ig M and Ig G tests have been in use for a long time, immuncapture agglutination test has been recently introduced and used in serological diagnosis. The aim of this study was to compare diagnostic values of ELISA Ig M and Ig G and immuncapture agglutination tests with Coombs anti-brucella test. Sera from 200 patients with presumptive diagnosis of brucellosis were included into the study. Coombs anti-brucella test, ELISA Ig M and Ig G tests and Immuncapture test were investigated in these sera. Then, sensitivity, specificity, negative predictive and positive predictive values were calculated. Sensitivity, specificity, negative predictive and positive predictive values were found to be 90.6%, 76.3%, 94.2%, and 65.9% respectively for the Immuncapture test, whereas they were found to be 73.7%, 58.9%, 84.2%, and 42.8% for Ig G and 72.2%, 67.8%, 85.2%, and 48.7% for Ig M. The Immuncapture test was found to be compatible with ELISA Ig M and Ig G tests but it was statistically incompatible with Coombs anti-brucella test. Immuncapture agglutination test yields similar results to those of Coombs anti-brucella test. This test is a useful test by virtue of the fact that it determines blocking antibodies in the diagnosis and follow-up of brucellosis.

Utilidad de la determinación de anticuerpos IgG e IgM por ELISA e inmunocaptura en una serie clínica de brucelosis humana

The diagnostic difficulties of brucellosis makes the evaluation of new diagnostic tests necessary.Evaluation of different commercial tests in the serological diagnosis of brucellosis by ELISA and immunocapture antibodies in a clinical series of patients with brucellosis of the Health Network of the Catholic University of Chile.All the serums received in the Laboratory of Microbiology for suspicion of brucellosis during five years were studied. Two groups were obtained, one that fulfilled diagnostic criteria for brucellosis [clinical evidence, and/or positive blood culture and/or seroagglutination test (SAT) in titers > 1/160] and the control group. Each serum sample was analyzed using irnmunocapture-agglutination test (Brucellacapt), ELISA IgM and IgG.Of 10 patients with brucellosis, the serologic results were: 8/10 positives for ELISA IgG, 7/10 for Brucellacapt and SAT, and 5/10 for ELISA IgM.ELISA IgG alone was superior than SAT. The combination ELISA IgG/ Brucellacapt reaches the best detection performance (9/10) and can be an alternative to SAT.

Brucellosis: Epizootiologic and diagnostic challenge

Although it has been known as a separate etiological entity for almost 150 years, brucellosis is, on a global scale, one of the most frequent diseases that is transferred from animals to humans. It is present as an endemic disease in almost all countries of the Mediterranean Sea, which indicates that a large number of aspects in connection with the control and epizootiology of this disease still remain unexplained. Three of the six types of brucella have been officially confirmed in our country as well. They are Brucella melitensis biotip 3, Brucella suis biotip 2 and Brucella canis. Brucellosis is endemically present in Kosovo and Metohija province and in southern Serbia proper; over the past few years, the spread of brucellosis in sheep and goats as primary hosts for B. melitensis to new territories, mostly in Vojvodina province, has shown that risk analysis is one of the main factors in selecting and implementing control programmes. A correctly selected set of diagnostic tests yields reliable data in most cases, but interpretations of results are prone to result in subjective assessments as well. A special problem in the serological diagnosis of brucellosis is the cross reactivity of brucellas and some other bacteria, often a weak immunological response of the animal, or that the type of brucella that causes the infection determines the sensitivity and specificity of the applied tests, most often screenings tests. Due to the big economic losses resulting from disease control and eradication, and the serious risk to human health, brucellosis still poses an epizootiological, and, in particular, a diagnostic challenge.

FLUORESCENCE POLARIZATION ASSAY FOR THE DIAGNOSIS OF BRUCELLOSIS: A REVIEW

Fluorescence polarization assay (FPA) is based on the rotational differences between a small soluble antigen molecule in solution (labelled with a fluorochrome) and the antigen molecule complexed with its antibody. A small molecule will rotate randomly at a rapid rate, resulting in rapid depolarization of light, while a larger complex molecule will rotate slower and depolarize light at a reduced rate. The rate change in depolarization can be measured. The FPA is a homogeneous assay which does not require removal of unreacted reagents and can, therefore, be performed very quickly and, given portable equipment, in the laboratory and in the field. The latter obviates the need for shipping samples and eliminates waiting for results, as well as reducing test costs. The FPA technology has been developed and validated for the serological diagnosis of brucellosis in cattle, swine, sheep, goats, bison, and cervids. Sufficient cross reactivity of the common epitopes of Brucella abortus, B. melitensis and B. suis O-polysaccharide (OPS) allowed for the use of a single antigen for all species of smooth Brucella and animals. The OPS prepared from B. abortus S1119.3 was conjugated with fluorscein isothiocyanate (FITC). The FPA was initially developed for testing serum; however, the technology has been extended to testing whole blood and milk from individual animals or bulk tank samples pooled from 2000 or fewer animals. The accuracy of the FPA equalled or exceeded those obtained using other serological tests such as the buffered antigen plate agglutination test (BPAT), the milk ring test (MRT), the complement fixation test (CFT), the indirect enzyme immunoassay (IELISA), and the competitive enzyme immunoassay (CELISA).