Judy A. Stack

Validation of FPA and cELISA for the detection of antibodies to Brucella abortus in cattle sera and comparison to SAT, CFT, and iELISA

The fluorescence polarisation assay (FPA) is a recently described test for the serological diagnosis of Brucella infection. It has many methodological advantages over older, more established tests and can be performed in a fraction of the time. To validate the FPA, serum samples from 146 confirmed (by culture) Brucella-infected cattle were tested in conjunction with serum samples from 1947 noninfected cattle. The competitive ELISA (cELISA) was validated using these positive reference samples and 1440 negative samples, while data for the indirect ELISA (iELISA) was generated from 6957 negative samples plus the positive sera. Published diagnostic specificity (DSp) data for the complement fixation test (CFT) and serum agglutination test (SAT) was used in conjunction with the test results on the positive sera to obtain diagnostic specificity plus diagnostic sensitivity (DSn). After selection of a cutoff for the FPA and cELISA, the diagnostic specificity and sensitivity total for each test were compared. The results, with 95% confidence intervals, were: FPA (195.7+/-2.79), iELISA (195.0+/-2.70), cELISA (194.9+/-3.48), CFT (191.7+/-4.45), and SAT (180.4+/-6.33). The data presented supports the use of the FPA in diagnosis of brucellosis and questions the use of the SAT and CFT for either screening or confirmatory testing.

Phenotypic and molecular characterisation of Brucella isolates from marine mammals

BackgroundBacteria of the genus Brucella are the causative organisms of brucellosis in animals and man. Previous characterisation of Brucella strains originating from marine mammals showed them to be distinct from the terrestrial species and likely to comprise one or more new taxa. Recently two new species comprising Brucella isolates from marine mammals, B. pinnipedialis and B. ceti, were validly published. Here we report on an extensive study of the molecular and phenotypic characteristics of marine mammal Brucella isolates and on how these characteristics relate to the newly described species.ResultsIn this study, 102 isolates of Brucella originating from eleven species of marine mammals were characterised. Results obtained by analysis using the Infrequent Restriction Site (IRS)-Derivative PCR, PCR-RFLP of outer membrane protein genes (omp) and IS711 fingerprint profiles showed good consistency with isolates originating from cetaceans, corresponding to B. ceti, falling into two clusters. These correspond to isolates with either dolphins or porpoises as their preferred host. Isolates originating predominantly from seals, and corresponding to B. pinnipedialis, cluster separately on the basis of IS711 fingerprinting and other molecular approaches and can be further subdivided, with isolates from hooded seals comprising a distinct group. There was little correlation between phenotypic characteristics used in classical Brucella biotyping and these groups.ConclusionMolecular approaches are clearly valuable in the division of marine mammal Brucella into subtypes that correlate with apparent ecological divisions, whereas conventional bioyping is of less value. The data presented here confirm that there are significant subtypes within the newly described marine mammal Brucella species and add to a body of evidence that could lead to the recognition of additional species or sub-species within this group.

Competitive ELISA for bovine brucellosis suitable for testing poor quality samples.

Unutbefor tesin * A COMPETITIVE enzyme-linked immunosorbent assay (CELISA) was evaluated as an alternative confirmatory test to the complement fixation test (CFT) for the diagnosis of brucellosis in cattle. The aim was to show that the cELISA was capable of testing poor quality serum samples which were unsuitable for diagnosis by the CFT. An M dominant epitope lipopolysaccharide (LPS) antigen was extracted from Brucella melitensis strain 16M, by the hot phenol method (Cherwonogrodzky and others 1991). An anti-M epitope monoclonal antibody (mAb) (Greiser-Wilke and others 1985) was conjugated with horseradish peroxidase (HRP) by a method adapted from Nakane and Kawaoi (1974). HRP (20 mg) was dissolved in 5 ml distilled water and 1-2 ml freshly prepared 0*1M sodium periodate was added. After stirring for 20 minutes at room temperature, the activated HRP was dialysed against lmM sodium acetate (pH 4.0) for 15 to 20 hours. The dialysed HRP and 20 mg mAb were adjusted to pH 9-0 before being combined, and then stirred for two hours at room temperature, after which 0 5 ml ascorbic acid (4 mg/ml) was added. After four hours incubation at 4°C, the mixture was dialysed for 15 to 20 hours against several changes of 0-1M phosphate buffered saline. The conjugate was passed through a 100 kD filter, added to equal volumes of glycerol and stored at -20°C. The optimal concentration of antigen and conjugate was determined by titration against standard sera from a known Brucella-free sheep and serum from a goat experimentally infected with B melitensis. The optimum concentrations selected were those that gave the highest ratio between the optical density (OD) of the negative and positive standards in conjunction with an OD of less than 0.100 for the positive standard and greater than 0*700 for the negative standard. The M dominant B melitensis antigen and anti-M mAb were selected as they have been shown to be less affected by false positive serological reactions caused by cross-reacting bacteria, such as Yersinia enterocolitica 0:9, which affects all Brucella serodiagnostic assays (A. P. MacMillan, unpublished observations). Brucella abortus infection is detected by this combination of antigen and conjugate, as antibodies specific for the M epitope are produced. The CELISA was adapted from the method described by MacMillan and others (1990). LPS antigen (100 VI), suspended in carbonate buffer (pH 9.6) aqa predetermined dilution, was added to each well of a 96-well polystyrene polysorp plate (Nunc Life Technologies) and incubated overnight at 40C. Unbound antigen was removed by washing with phosphate buffered saline (PBS) (pH 7.2) containing 0.01 per cent Tween 20 (wash solution). Undiluted test and control sera (20 ill) were dispensed into respective duplicate wells and mixed with 100 VI of conjugate at a predetermined dilution. The reaction took place at room temperature for 30 minutes on a rotary shaker at 160 revs/minute, after which the plates were washed three times with the wash solution. The reaction was developed with 100 il1 of substrate/chromogen solution comprising 30 mg o-phenylenediamine dihydrochloride (OPD) and 300 VIl 3 per cent hydrogen peroxide suspended in 75 ml distilled water. The reaction was stopped after 15 minutes incubation *Numberf sampls whichwere unsuitbl for tsigdeto ecessNive haemolysi andu/oraticomWlplemelntarativt

A new homogeneous assay for high throughput serological diagnosis of brucellosis in ruminants.

The control and eradication of brucellosis is highly desirable but heavily resource intensive as high throughput serological testing is required. The aim of this study was to meet the needs of high throughput screening laboratories involved in this process through the development of a new assay. An existing cELISA used for the serodiagnosis of brucellosis in ruminants was converted to an AlphaLISA homogenous proximity based assay. This assay requires no separation steps and can be performed in low volume microtitre format. The Brucella AlphaLISA was validated on a panel of bovine, ovine and caprine sera from infected and uninfected animals. The diagnostic sensitivities (>96%) and specificities (>98%) obtained compared well to those from cELISA, iELISA and FPA performed on the same samples. The AlphaLISA met the testing criteria set for ELISAs as defined by the OIEELISA standards and had an analytical sensitivity similar to that of the parent cELISA. The method was also used on a small panel of serum samples from cattle that were experimentally infected with Yersinia enterocolitica O:9. Some false positive reactions were obtained as was also the case with results from FPA, iELISA, cELISA, CFT and SAT. Despite this, the methodological advantages of the AlphaLISA mean that this assay is well suited to high throughput serodiagnosis. This report is the first description of the use of AlphaLISA to detect pathogen specific antibodies. Furthermore, the relative ease with which the cELISA was converted to this platform indicates that this technology is ready to meet the high throughput testing requirements for the diagnosis of many other diseases.

Knowledge and practices related to bovine brucellosis transmission amongst livestock workers in Yewa, south-western Nigeria.

Brucellosis is an endemic disease in the animal population in Nigeria and of major public health importance, particularly amongst livestock workers who are ignorant of the risk of Brucella infection. Therefore, to gain insight into the knowledge and practices related to brucellosis transmission amongst livestock holders (LH) and livestock marketers (LM) in Yewa, an international livestock trading centre in south-western Nigeria, we conducted an interviewbased study using a cluster sampling technique. In all, a total of 157 respondents comprising 54 LH and 103 LM were interviewed. Two-thirds (69.5%) of the two groups had poor knowledge of brucellosis with no significant difference between them (p = 0.262). Furthermore, consumption of unpasteurised milk, uncooked meat and its products, co-habitation with animals, and poor hygiene were significant risk practices identified as possible means of transfer of Brucella infection from animals to humans amongst these livestock workers (p < 0.05). In conclusion, our findings revealed that poor knowledge and practices related to the consumption of unpasteurised or unboiled dairy products, contaminated beef, and unhygienic practices are factors that will facilitate Brucella infections amongst livestock workers in Nigeria. Therefore, there is a need for more public health enlightenment programmes, as well as implementation of brucellosis control measures in the cattle populations.

Competitive Electrochemiluminescence Wash and No-Wash Immunoassays for Detection of Serum Antibodies to Smooth Brucella Strains

ABSTRACT Brucellosis is a bacterial zoonotic disease of major global importance. Natural hosts for Brucella species include animals of economic significance, such as cattle and small ruminants. Controlling brucellosis in natural hosts by high-throughput serological testing followed by the slaughter of seropositive animals helps to prevent disease transmission. This study aimed to convert an existing competitive enzyme-linked immunosorbent assay (cELISA), used for the serodiagnosis of brucellosis in ruminants, to two electrochemiluminescence (ECL) immunoassays on the Meso Scale Discovery (MSD) platform. The first assay employed a conventional plate washing step as part of the protocol. The second was a no-wash assay, made possible by the proximity-based nature of ECL signal generation by the MSD platform. Both ECL wash and no-wash assays closely matched the parent cELISA for diagnostic sensitivity and specificity. The results also demonstrated that both ECL assays met World Organization for Animal Health (OIE) standards, as defined by results for the OIE standard serum (OIEELISASPSS). This report is the first to describe an ECL assay incorporating lipopolysaccharide, an ECL assay for serodiagnosis of a bacterial infectious disease, a separation-free (no-wash) ECL assay for the detection of serum antibodies, and the use of the MSD platform for serodiagnosis. The simple conversion of the cELISA to the MSD platform suggests that many other serodiagnostic tests could readily be converted. Furthermore, the alignment of these results with the multiplex capability of the MSD platform offers the potential of no-wash multiplex assays to screen for several diseases.

Time-Resolved Fluorescent Resonance Energy Transfer Assay for Simple and Rapid Detection of Anti-Brucella Antibodies in Ruminant Serum Samples

ABSTRACT Brucellosis is a globally significant zoonosis, the control of which is difficult and resource intensive. Serological tests form a vital part of a multifactorial approach to control and are often performed in large numbers. The aim of the present study was to develop a new assay to improve the efficiency, ease, and effectiveness of serological testing. An existing competitive enzyme-linked immunosorbent assay (cELISA) was adapted to a completely homogeneous time-resolved fluorescent resonance energy transfer (TR-FRET) assay. This was achieved by labeling an anti-Brucella monoclonal antibody with a long-lifetime donor fluorophore and Brucella smooth lipopolysaccharide with a compatible acceptor and optimizing the reading conditions. The assay was performed in a 96-well plate with a single 30-min incubation period and no separation (wash) steps and was concluded by a single plate-reading step. The performance of the assay was evaluated with a panel of serum samples from infected (n = 73) and uninfected (n = 480) sources and compared to the performance of the parent cELISA, an indirect ELISA (iELISA), and fluorescence polarization assay (FPA). The performance of the TR-FRET assay matched the performance of the iELISA, which had 100% diagnostic sensitivity and specificity, and surpassed the performance of the cELISA and the FPA. The results also demonstrated that the TR-FRET technique is effective with poor-quality serum samples from the field. To the knowledge of the authors, this is the first homogeneous TR-FRET assay to detect antibodies raised against an infectious disease. The technique appears to be sufficiently adaptable to meet the needs of many other similar testing requirements to identify infectious diseases.

An evaluation of the capability of existing and novel serodiagnostic methods for porcine brucellosis to reduce false positive serological reactions.

Porcine brucellosis is a zoonotic disease of truly global significance because even in countries without the disease the occurrence of false positive serological reactions (FPSRs) creates significant problems. Statutory diagnostic testing is required in many disease free countries or regions and is often a prerequisite for the movement of live animals. Currently this testing is dependent almost entirely on serological assays and these may result in a significant number of FPSRs. The aim of this study was to examine existing and novel serodiagnostic assays to evaluate their diagnostic sensitivity and resilience to FPSRs. The existing assays evaluated were the RBT, smooth lipopolysaccharide (sLPS) indirect (i) ELISA, sLPS competitive (c) ELISA, and the FPA. The novel assays evaluated were the sLPS TR-FRET assay, a rough (r) LPS iELISA, a recombinant protein BP26 iELISA and a cytoplasmic protein extract (Brucellergene™) iELISA. Four populations of sera were evaluated: those from Brucella suis infected swine (n=34), randomly selected samples from non-infected swine (n=161), sera from non-infected swine within herds exhibiting FPSRs (n=132) and sera from swine experimentally infected with Yersinia enterocolitica O:9 (n=4). The results show that all the assays dependent on the sLPS O-polysaccharide (OPS) for their sensitivity (the RBT, sLPS ELISAs, FPA and the sLPS TR-FRET) had significantly reduced diagnostic specificity when applied to the FPSR population, the RBT being most affected. Of the two rapid homogeneous assays, the TR-FRET was diagnostically superior to the FPA in this study. Neither of the protein based iELISAs demonstrated sufficient diagnostic sensitivity to resolve the FPSRs. The rLPS iELISA showed no cross reaction with the FPSRs and had diagnostic sensitivity similar to that of the OPS based assays.

Survey of the seroprevalence of brucellosis in ruminants in Kosovo

A cross-sectional survey of the seroprevalence of brucellosis in sheep, goats and cattle in Kosovo was made in January 2001. A total of 12,000 serum samples, from 7941 cattle, 3548 sheep and 511 goats, were screened using the Rose Bengal test. Doubtful and positive results were further tested with competitive and indirect ELISAs. The overall serological prevalences derived from the samples positive to all three tests, were 6.26 per cent (95 per cent confidence intervals [CI] 5.5 to 7.1 per cent) for sheep, 7.24 per cent (5.3 to 9.8 per cent) for goats and 0–58 per cent (0.43 to 0.77 per cent) for cattle. The survey covered 26 of the 29 municipalities and showed that brucellosis was widely but unevenly distributed throughout the province. Seropositive animals were found in 25 per cent (19 to 32 per cent) of 162 villages surveyed. The risk of cattle being infected on holdings where both cattle and sheep were kept was greater, with a risk ratio of 4.6 (2.2 to 9.6), than on holdings where only cattle were kept. Brucella melitensis probably predominates as the cause of brucellosis in ruminants in the province of Kosovo.

Investigating the use of protein saver cards for storage and subsequent detection of bovine anti-Brucella abortus smooth lipopolysaccharide antibodies and gamma interferon.

ABSTRACT Brucella abortus, a smooth strain of the genus Brucella, is the causative agent of bovine brucellosis. To support the ongoing development of diagnostic tests for bovine brucellosis, the use of Protein Saver cards (Whatman) for bovine blood serum and plasma sample collection has been evaluated. These cards offer significant logistical and safety alternatives to transporting and storing liquid samples and may aid in diagnostic programs and validation studies. To evaluate the utility of these cards, 204 bovine blood serum samples from Brucella-infected and noninfected animals were stored on and eluted from the Protein Saver cards. Anti-Brucella smooth lipopolysaccharide (sLPS) antibody titers for the serum eluates were compared to those of the unprocessed original serum samples by indirect enzyme-linked immunosorbent assay (ELISA). The results showed a highly significant correlation between titers from the serum eluates and the unprocessed sera. Therefore, under these circumstances, serum eluates and unprocessed serum samples may be used interchangeably. Blood plasma from 113 mitogen-stimulated whole-blood samples was added to and eluted from the Protein Saver cards. The gamma interferon (IFN-γ) titers in the plasma eluates were compared to those of the unprocessed plasma samples obtained by IFN-γ ELISA. The results showed a significant correlation between the plasma eluates and the unprocessed plasma samples. To derive a signal in the plasma eluate, it was necessary to develop a novel and highly sensitive ELISA for the detection of IFN-γ. The serum samples stored on cards at room temperature over a 10-day period showed little variation in antibody titers. However, the plasma eluates showed a progressive loss of IFN-γ recovery over 10 days when stored at room temperature.