Fluorescence Polarization Assay Tests Research Articles

Comparison of diagnostic tests for detecting bovine brucellosis in animals vaccinated with S19 and RB51 strain vaccines.

The diagnosis of bovine brucellosis in animals vaccinated with strain-19 (S19) and Rose Bengal (RB)-51 strain vaccines can be misinterpreted due to false positives. This study aimed to compare diagnostic tests for detecting bovine brucellosis in animals vaccinated with S19 and RB51 vaccine strains. Two groups of 12 crossbred Holstein calves between 6 and 8 months of age were used. On day 0, blood samples were collected from the animals, and the competitive enzyme-linked immunosorbent assay was used for serological diagnosis of bovine Brucellosis. All animals tested negative. After the first blood collection, the animals were subcutaneously vaccinated: one group received the S19 vaccine and the other received the RB51 vaccine. From the 3rd month after vaccination, all animals were sampled. Sampling was repeated every 2 months until the 7th month. Serological diagnosis of bovine brucellosis was performed using RB, tube serum agglutination test (SAT), SAT with 2-mercaptoethanol (SAT-2Me), and fluorescence polarization assay (FPA). Animals vaccinated with S19 showed positive results with the RB, SAT, and SAT-2Me tests in all months of post-vaccination diagnosis. In animals vaccinated with S19, FPA showed positive results at months 3 and 5 and negative results at month 7, indicating that this test discriminates vaccinated animals from infected animals 7 months after vaccination. Rose Bengal, SAT, SAT-2Me, and FPA tests showed negative results in animals vaccinated with RB51 in all months of diagnosis. Animals vaccinated with S19 may test positive for brucellosis using RB, SAT, or SAT-2Me tests 7 months later. Fluorescence polarization assay is an optimal alternative for diagnosing animals in the field, thereby preventing false positives, and consequently, unnecessary confiscations of animals. Animals vaccinated with RB51 tested negative with RB, SAT, SAT-2Me, and FPA tests in all months of diagnosis, confirming that the tests are ineffective for diagnosing brucellosis caused by rough strains.

Comparison of Fluorescence Polarization Assay with Rose Bengal Plate Agglutination test and Competitive Enzyme-Linked Immunosorbent assay for bovine brucellosis in Tanzania

Brucella are Gram-negative, facultative, intracellular bacterial species with B. abortus, B. melitensis, and B. suis carrying the smooth-lipopolysaccharide antigen. Accurate diagnostic results of brucellosis are needed for its control and eradication, however, they are primarily based on the serological testing of brucellosis in animals. The Rose Bengal Plate Agglutination Test (RBPT) and competitive Enzyme-Linked Immunosorbent Assay (c-ELISA) are the most commonly used tests for making such diagnosis. The use of Fluorescence Polarization Assay (FPA) in Tanzania is still in nascent stage. The purpose of this study was to compare RBPT, c- ELISA, and FPA in the diagnosis of bovine brucellosis. A total of 75 serum samples from cattle that were infected with Brucella in Kagera region were obtained. The FPA showed 90% (68 samples) prevalence, RBPT revealed 93% (70 samples) prevalence, and c-ELISA revealed 81% (61 samples) prevalence of brucellosis in the farm. The RBPT test has shown an inability to distinguish antibodies from cross-reacting organisms compared to the FPA test, while the c- ELISA was unable to pick a positive sample compared to the FPA test. FPA is very quick (5 min per sample), does not require specialized staff, and may be performed under field conditions. Therefore, FPA has a potential to overcome limitations in the detection of bovine brucellosis and can be used as a confirmatory test.

Evaluation of the Fluorescence Polarization Assay for the Diagnosis of Brucellosis in Goat Milk.

The milk ring test is a detection assay for antibodies against Brucella in bovine milk. It has good sensitivity but tends to give false positive results. In this study, we standardized the application of the fluorescence polarization assay (FPA) for the detection of antibodies against B. melitensis in goat milk. We obtained negative serum and milk samples from healthy goat flocks in the northern zone of Nuevo León. Positive milk and negative, weak, and strong controls were obtained by mixing volumes of positive control serum with negative control milk. Milk samples were treated with citric acid, after which an FPA was performed. Results were then compared with the Rose Bengal test and the FPA in serum. Milk treatment allowed the quantification of antibodies in samples. Significant differences were found between the 2%, 4%, and 6% groups, compared with the control group (F3, 67 = 17.45, p < 0.0001) but not between the 2% and 4% groups (p = 0.0718). The cut-off value was 74.1 mP, with a sensitivity (Se) of 95% and a specificity (Sp) of 100%. Se and Sp values in field milk samples were 84% and 74.55%, respectively. Despite the FPA test on milk samples showed lower Se and Sp than the FPA test on serum samples, its cutoff may be adjusted. It may be recommended as a screening test in goat milk and become useful for the control and eradication of the disease.

Presumptive Identification of Smooth Brucella Strain Antibodies in Canines.

Brucellosis is a zoonotic disease caused by a Gram-negative coccobacillus. There are four Brucella strains of zoonotic importance in our domestic species, subdivided by their culture phenotypes: Brucella abortus (B. abortus), B. melitensis, B. suis (smooth strains) and B. canis (rough strain). Dogs can serve as hosts for all four of the zoonotic strains; however, routine serologic testing in dogs has been limited to the identification of B. canis antibodies. The aim of our study was to identify smooth Brucella strain antibodies in canines. We hypothesize that the Brucella abortus Fluorescence Polarization Assay would be successful in identifying smooth Brucella strain antibodies in canines. Ninety-five dogs, including forty-five hog hunting dogs were screened for circulating antibodies to any of the four zoonotic strains of the bacteria utilizing a combination of Canine Brucella Slide Agglutination Test (CBSA), Brucella canis Agar Gel Immunodiffusion II test (AGIDII), Brucella abortus Card Agglutination Test (BCA), and the Brucella abortus Fluorescence Polarization Assay (FPA). Test interpretation results yielded a 0% (0/95) smooth Brucella strain seropositivity rate, with 2% (2/95) of dogs yielding inconclusive rough Brucella strain serology results (0–2% rough strain seropositivity rate). Additionally, a retrospective portion of the study was performed to identify sera containing circulating antibodies to any of the smooth strains of Brucella by testing previously banked canine serum samples stored at Cornell's Veterinary Diagnostic Laboratory from 2018 to 2019 via Brucella abortus FPA. Of the 769 serum samples tested, 13/769 (1.7%) yielded an inconclusive result, 725/769 (94.2%) were negative, 30/769 (4%) yielded a positive FPA test result, and 1/769 (0.1%) had to be excluded due to insufficient sample remaining to perform the diagnostic test. Of the 30 FPA positive canine serum samples, 97% (29/30) also tested positive on the CBSA test. Additionally, there was a statistically significant (p < 0.0001) likelihood of altered (spayed/neutered) and mixed breed dogs to be FPA positive when compared to intact, purebred dogs, respectively.

Association of brucellosis to abortions in humans and domestic ruminants in Kagera ecosystem, Tanzania.

Brucellosis is a worldwide zoonotic disease of socio-economic importance. Understanding the association of this disease with pregnancy outcome has the potential of contributing to the reduction of its reproductive burden in humans and animals among pastoral communities in Tanzania. A prospective cohort study was conducted in Kagera Region on pregnant women (n=76) and gravid ruminants (121 cattle, 125 goats and 111 sheep). Exposed and non-exposed groups to brucellosis were followed for 6months (from 15 November 2017 to 15 April 2018). Sera were collected and analysed using Rose Bengal Test (RBT) and Fluorescence polarization assay (FPA) test. Measures of effect, univariable and multivariable logistic regression analyses were computed. Positivity to both RBT and FPA tests was 21% (95% CI: 12.5-32) in pregnant women and 5% (95% CI: 3.1-8) in gravid ruminants. Among aborted cases, four women (out of nine), two cows (out of seven), two goats (out of 26) and zero sheep (out of 11) were positive to brucellosis. The abortion rate in humans and ruminants was 11.8% and 12.3%, respectively. Seropositivity to brucellosis was similar in aborted and non-aborted cases in humans (p=.08) and in ruminants (p=.2). At the population level, brucellosis was associated with abortions (population attributable risk: PAR) at 3.5% in pregnant women and at 0.5% in gravid ruminants in the study area. Infections to brucellosis were increased in exposed pregnant women (OR=19; 95% CI: 1.8-203, p=.01) and in cattle (OR=11; 95% CI: 1.3-88, p=.02). There is an indication that brucellosis could be contributing to abortions in pregnant women and domestic ruminants Kagera Region. Molecular tools could support more the results from serological tests to avoid cross-reaction with other pathogen agents. Control of brucellosis in animals is likely to reduce the threat of abortions in humans.

Comparative evaluation of fluorescence polarization assay and competitive ELISA for the diagnosis of bovine brucellosis vis-a-vis sero-monitoring

Brucellosis is an important zoonosis that constitutes a serious public health hazard which is caused by a bacterium belonging to the genus Brucella. In the present study, two highly specific serological tests for brucellosis diagnosis, fluorescence polarization assay (FPA) and competitive ELISA (cELISA) were standardized in the laboratory, evaluated and compared with rose bengal plate test (RBPT), indirect ELISA (iELISA) and commercial cELISA kit. For test evaluation, 1386 serum samples [apparently healthy animals (n = 260), samples from Brucella infected farms (n = 701) and B. abortus S19 vaccinated animals (n = 425)] were analyzed to assess suitable diagnostic test in B. abortus S19 post vaccinated bovine population. In apparently healthy brucellosis free farms, RBPT, iELISA, in-house FPA and cELISA were found to be highly specific than commercial cELISA. Commercial cELISA kit was comparatively more sensitive than other serological tests in samples collected from infected farms. The FPA showed sensitivity nearly equal to RBPT and in-house cELISA showed greater sensitivity than RBPT in infected farms. In animals with persistent vaccinal antibodies, only in-house FPA and cELISA recorded higher specificity of 87.64 and 90.27%, respectively. The other tests, RBPT and iELISA displayed similar reactivity with vaccine antibodies to that of infection antibodies whereas commercial cELISA kit showed an intermediate specificity of 47.69%. With these findings, RBPT, iELISA and cELISA are suggested for screening infected herds, and in-house developed FPA and cELISA tests with a proven specificity can be used for confirmatory diagnosis of brucellosis in B. abortus S19 post vaccinated animal populations.

English

Mycobacterium avium subspecies paratuberculosis (Map), is the etiological agent of paratuberculosis, a disease that affects cattle and causes economic losses to the animal husbandry industry. Its opportune diagnosis, in herds, is part of the control measures of the disease; therefore, the objective of this work was to compare paratuberculosis detection of infected bovines with the Fluorescence Polarization Assay (FPA) and Enzyme-linked Immunosorbent Assay (ELISA). Six-hundred and three sera and feces samples, from bovines older than 2 years old were studied. The sera were assessed with the FPA technique using as antigen a protein fraction of 35 kDa obtained from the raw extract of the Map strain 3065, and for ELISA the protoplasmic antigen of the same strain was used. DNA was obtained from the feces and assessed by nested PCR. The correlation of results was established by Kappa Test.&nbsp; The FPA test had sensitivity (Se) of 88.50% and specificity (Sp) of 91.42% (p &le;0.000); for ELISA Se 83.86% and Sp 89.87% (p &le;0.000) were obtained. Concordance (K) between tests was 0.6742%, and when compared with nested polymerase chain reaction (PCR), the FPA test had K = 0.7314%, while for ELISA it was 0.5771%. The FPA technique using as antigen the protein fraction of 35 kDa showed a higher sensitivity and specificity, moreover it was a simple technique for the determination of the antigen-antibody interaction, and therefore it becomes an alternative diagnostic tool to detect paratuberculosis infected bovines. Key words: Paratuberculosis, diagnosis, fluorescence polarization assay (FPA), enzime-linked immunoabsorbent assay (ELISA). &nbsp

Fluorescence polarization assay: Diagnostic evaluation for porcine brucellosis

Brucellosis in pigs, caused by the bacterium Brucella suis, is an important zoonotic infection. In the present study, fluorescence polarization assay (FPA) was standardized and compared with indirect enzyme linked immunosorbent assay (iELISA) and competitive ELISA (cELISA) for diagnosis of porcine brucellosis. Test performances were evaluated using representative panel (n = 100), samples from swine brucellosis outbreak (n = 300), samples from brucellosis suspected animals (n = 291) and sera samples from apparently healthy animals (n = 1121). With panel samples, the FPA cut-off ≥11ΔmP was arrived with sensitivity (Se) and specificity (Sp) of 95.00 and 98.75%, respectively. Testing of samples from swine brucellosis outbreak, the diagnostic Se and Sp of 100 and 95.14% by iELISA, 73.91 and 100% by cELISA and 86.96 and 100% by FPA, respectively were recorded. Similarly, in case of swine brucellosis suspected samples, relative performance of FPA with cELISA had revealed higher kappa value of 0.864 with an accuracy of 93.47. Indirect ELISA was found to be highly sensitive but showed cross reactivity mainly for Yersinia enterocolitica O9 antibodies than cELISA and FPA. The high specificity of FPA test recorded in various types of samples in the study indicated that, FPA could serve as confirmatory test for individual animal diagnosis, outbreak confirmation, surveillance and quarantine of swine brucellosis cases.

Prevalência sorológica de Brucella spp. em porcos ferais e bovinos em simpatria no Pantanal do Mato Grosso do Sul, Brasil

The objective of this study was to determine the prevalence of anti-Brucella antibodies in feral pigs and bovines simpatrics in the Pantanal subregions of Paiaguás and Nhecolândia. The study was conducted in the municipality of Corumbá, Mato Grosso do Sul State, Brazil. A total of 105 feral pigs and 256 cattle were sampled in 12 farms, in all animals blood samples were collected for the serological diagnosis with Rose Bengal Test (RBT) for screening, 2-Mercaptoethanol (2-ME) confirmatory test and comparative test with Fluorescence Polarization Assay (FPA). The prevalence of positive feral pigs were 1% (1/105) in the RBT and FPA and no positive AAT results were confirmed in the 2-ME test. The prevalence of positive cattle sampled was 11.32%, 4.3% and 7.42% in the RBT, 2-ME and FPA tests respectively. The degree of agreement obtained between the serological tests used in cattle was Kappa = 0.506 (p &lt;0.001), 95% CI (0.282 - 0.729). The results of the serological tests demonstrated that brucellosis is widespread in bovine herds of the region studied, but the same type of exposure to the agent did not occur in feral pigs according to the diagnostic tests used.

Effectiveness of Rose Bengal test and fluorescence polarization assay in the diagnosis of Brucella spp. infections in free range cattle reared in endemic areas in Zambia

The effectiveness of Rose Bengal test (RBT) and fluorescence polarization assay (FPA) in diagnosing cattle brucellosis in endemic areas was assessed and RBT and FPA test agreement was compared (n = 319). The sensitivity of RBT and FPA in detecting low Brucella titres were evaluated in paired sera (n = 34). A logistic regression model was constructed to predict cattle test result in FPA using RBT as the main predictor and incorporating bio-data and animal history. There was 79.3% agreement between the RBT and FPA (Kappa = 0.59; Std error = 0.05; p = 0.000) and a high correspondence between high RBT scores and positive FPA results suggesting that sera with high RBT score may not require confirmation with tests such as competitive-ELISA or CFT. High FPA cut-off points were more likely to miss animals with low antibody titres. The RBT had a reduced ability in detecting low antibody titres compared to the FPA. FPA test interpretation was improved if a priori information, such as sex and age was used. Under the challenging disease surveillance conditions prevailing in rural Africa, field-testing methods that are sensitive and specific; allow single animal contact, low technical skills in data interpretation are suitable.

Validation of a fluorescence polarization assay (FPA) performed in microplates and comparison with other tests used for diagnosing B. melitensis infection in sheep and goats

Fluorescence polarization assay (FPA) is a relatively new test for the serological diagnosis of Brucella spp. infection in animals. FPA, carried out in 96-well microplate format, was validated here for diagnosing B. melitensis infection in sheep and goats. This study included sera from 1933 sheep and goats, from animals reared in naturally infected flocks (verified by culture) and showing a positive reaction to two different tests conducted in parallel. In addition, 2154 sera originating from healthy sheep and goats, reared in areas where B. melitensis had never been isolated, were assayed. The optimum cut-off value offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp) was determined at 15 mP over the mean value of the buffer control used in each microplate as determined by receiver operating characteristic analysis. The DSn and DSp of the FPA for small ruminants carried out in microplates at this cut-off value were calculated to be 95.9% and 97.9% with 95% confidence intervals (95% CI) of 94.9–97.7% and 97.2–98.4%, respectively. The accuracy of the FPA, as expressed by determination of the area under the curve, was 0.991. Indirect ELISA and FPA tests offered the highest DSn when compared with the Rose Bengal test, the complement fixation test, the modified Rose Bengal test and competitive ELISA. The parallel or serial combination of FPA with indirect ELISA offers the highest DSn and DSp. As temperature can affect the results of the FPA, all reagents must be at the same temperature and the standard for comparison must always be read under the same conditions as the sera under test. FPA performed in microplates is a promising assay; the DSn and accuracy are better than those of the tests currently approved for diagnosing B. melitensis in small ruminants. Because of its simplicity, speed, and accuracy, this test can improve capacity for laboratory testing and the efficacy of an eradication program based on a test-and-slaughter policy.

A COMPARISON OF FOUR SEROLOGIC ASSAYS IN SCREENING FOR BRUCELLA EXPOSURE IN HAWAIIAN MONK SEALS

A survey for Brucella spp. antibodies was undertaken on 164 serum samples from 144 Hawaiian monk seals (Monachus schauinslandi) from the northwestern Hawaiian Islands collected between 1995 and 2002. The buffered antigen plate agglutination test (BPAT), the indirect enzyme immunoassay (I-ELISA), the competitive enzyme immunoassay (C-ELISA), and the fluorescence polarization assay (FPA) were compared with regard to their ability in detecting antibodies to Brucella spp. in the serum samples. Overall, antibodies were detected in 28 (17.1%) animals, using the BPAT test, 25 (15.2%) by the C-ELISA, and 19 (11.6%) in the I-ELISA and the FPA test, using thresholds established for cattle. No evidence of gross pathology consistent with clinical brucellosis was noted in any of the seropositive animals tested. Although further work would be necessary to validate these tests for use with monk seals it appears that both the C-ELISA and the FPA tests would be appropriate as diagnostic screening tests for detection of antibodies to Brucella spp. in this species.