Methods

The present document describes the requirements for validation (fitness for purpose) of ELISA kits for brucellosis diagnostic. The fulfilment of the OIE and EU requirements regarding the validation of indirect and competitive enzyme-linked immunosorbent assays (I-ELISA and C-ELISA) kits for the detection of antibodies specific to smooth Brucella species (especially B. abortus, B. melitensis and B. suis) in sera/milk samples from various animal species. The standardisation and validation are critical steps when a method is intended for routine diagnostic use in multiple laboratories. Therefore, customers can have confidence in the results produced by the test. All diagnostic assays should be validated for the species which they will be used for, according to the new OIE and EU principles and methods. For validation, the manufacturer must submit a file to the National Reference Laboratory for Brucellosis (NRL) including a descriptive administrative part of the reagent(s) and a technical part. The evaluation criteria are described in the document (3. Criteria for ELISA kits validation).

The present document describes a standard technique aiming at controlling the fulfilment of OIE and EU requirements regarding the standardisation of indirect enzyme-linked immunosorbent assays (iELISA) kits for the detection of antibodies specific to smooth Brucella species (especially B. abortus, B. melitensis and B. suis) in bovine individual sera or pools of sera.

The present document describes a standard technique aiming at controlling the fulfilment of OIE and EU requirements regarding the standardisation of diagnostic antigens for the detection of antibodies specific of smooth Brucella species (especially B. abortus, B. melitensis and B. suis):
- in animal individual sera by the Rose Bengal Test (RBT) and the Complement Fixation Test (CFT);
- in bovine pooled milk samples by the Milk Ring-Test (MRT).

The present document describes a standard technique aiming at detecting antibodies specific of Brucella ovis by the complement fixation test in ovine sera.

The present document describes a standard technique that allows establishing species and biovars of an identified Brucella culture. Species identification is based on two main sets of properties: lysis by phages and oxidative metabolic profiles on selected amino acid and carbohydrate substrates. The described identification techniques enable to establish species and biovar of specific Brucella culture.

The present document describes a standard technique that allows research and bacteriological identification of the Brucella genus, in animal samples (ruminants, equidae, suidae, camelidae and carnivores, marine mammals, both wild and domestic). This is an isolation technique on solid culture agar medium. The described identification techniques enable a presumptive identification of bacteria of the Brucella genus.

Bruce Ladder and Bruce Ladder suis

The Bruce Ladder (BL) and the Bruce Ladder Suis (BLS) are two molecular typing tools designed to identify Brucella species for BL (Garci-Yoldi et al., 2006 ; Lopez-Goñi et al., 2008 ; Lopez-Goñi et al., 2011) and to differentiate Brucella suis biovars, Brucella canis and Brucella microti for the BLS (Lopez-Goňi et al. 2011 ; OIE Terrestrial Manual 2018, Chapter 3.1.4). These methods are applied on DNA extracted from calibrated suspensions of isolated strains.

Principle

Both methods consist in the separation of different DNA fragment by electrophoresis previously amplified by a multiplex Polymerase Chain Reaction.

A PCR multiplex is a method allowing the amplification of several distinct DNA fragments in one reaction, with all primers related to the amplification of different loci introduced into the same reaction tube.

Primers used for the PCRs are:

Once amplified, the DNA fragments are deposited on an agarose gel and are separated according to their size by electrophoretic migration

Interpretation

After migration, a picture is taken and the DNA fragments of samples tested are compared with the fragments of reference strains (used as controls).

The Real Time Polymerase Chain Reaction (PCR) or Quantitative PCR is a molecular diagnostic tool used to detect nucleic acids, and in this case Brucella DNA (Bounaadja et al., 2009). This method is applied on DNA extracted from specimens (lymph nodes, genital swab, fluids, organs...) or from calibrated suspension of an isolated strain.

 

Principle

This method consists of an exponential amplification of DNA, mediated by a Polymerase Chain Reaction. As a quantitative method, it allows to estimate the initial amount of DNA in a sample.

Samples are previously inactivated and DNA is extracted using appropriate methods: manual or automated, silica column or magnetic beads, …

This PCR is based on Taqman technology, with the hydrolysis of oligonucleotide probe marked by fluorochromes. Fluorescence is released during each cycle of Taq Polymerase proportionally to targeted DNA amplification  and is measured at each cycle. This increase of fluorescence is translated into an amplification curve which makes possible to determine a Cycle Threshold (Ct) value.

To detect Brucella with this PCR, different genes are targeted: IS711 (multicopy), bcsp31 and per (monocopy) using specific primers (forward and reverse) and specific probes.

 

To validate the PCR run, several controls should be included in each test:

- An Internal Positive Control (IPC) = an exogenous DNA introduced in each sample before the extraction step, which can indicate potential inhibition during the extraction steps; its presence validates the extraction

- A negative extraction control = negative control (as Phosphate Buffer Saline Solution) extracted at the same time as samples, which allows to check the presence of possible contamination during the extraction steps;

- A positive extraction control = well-characterised sample that contains target DNA, which confirms the correct extraction of DNA. (This control must be calibrated around 35 (Ct value) to limit the risk of contamination);

- A PCR positive control = known target DNA previously extracted and included to ensure the good running of PCR;

- A PCR negative control = ultrapure water instead of sample to validate the no-contamination of the PCR.

 

Interpretation:

- An IPC amplification curve should be found in each well, except for the PCR positive control. Homogeneity of IPC curves should be checked to check potential inhibitions. For PCR on DNA from specimens, samples are tested pure and at a dilution of 1/10. The expected difference in IPC curves in terms of Ct is approximately 3 (1 log). If the difference is greater than 3 Ct, an inhibition is probable.

- Extraction negative control should be negative for the target(s) but positive for IPC

- Extraction positive control should be positive for the target(s) and positive for IPC

- PCR positive control should be positive for the target(s) and negative for IPC

- PCR negative control should be negative (no amplification curve)

- Sample should be positive for IPC and are considered as:

- Positive if there is an amplification curve for IS711 only or for IS711 and at least one other target (bcsp31 or per) with a Ct value lower than 38

- Doubtful if there is an amplification curve for IS711 and at least one other target (bcsp31 or per) with a Ct value between 38 and 40. Samples should be retested.

- Negative if the Ct value is higher than 40 or if there is no amplification curve for all targets.

- Inconclusive if there is an amplification curve for bcsp31 or per only. Samples should be retested.

C-ELISA

An ELISA is a plate-based laboratory technique making use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample. ELISAs are typically performed in 96-well (or 384-well) polystyrene plates, which will passively bind antibodies and proteins.

 

Antibodies also known as immunoglobulins (Ig) are gamma globulin proteins that are found in blood. These specialized immune proteins are produced following the introduction of an antigen into the body and possess the remarquable ability to combine with the antigen that triggered its production (specific affinity). The antibody recognises and bind to the antigenic determinant region of the antigen.

Enzyme-linked immunosorbent assays rely on specific antibodies to bind the target antigen, and a detection system to indicate the presence and quantity of antigen binding.

 

Brucella antigens (for review, Ducrotoy et al., 2016, DOI: 10.1016/j.vetimm.2016.02.002).

Like other gram negative bacteria, Brucella consist of cytoplasm surrounded by a cell envelope made of an inner membrane, periplasm and an outer membraine. The outer membrane contains two main classes of antigens: lipopolysaccharide (LPS) and detergent-soluble proteins (referred to as outer membrane proteins [Omp]). Structurally, the LPS can be either smooth (S) or rough (R) depending on the Brucella species. These two LPS differ in the presence in the former and absence in the latter of a long polysaccharide section, the O-polysaccharide

 

The antigens relevant to ruminant brucellosis can be classified into 2 categories: S-LPS and the related haptenic polysaccharides (O-polysaccharide linked with most core oligosaccharide sugars or core-O-polysaccharides ; the native haptens (NH) and the polysaccharide B) ; and protein antigens. The O-polysaccharide carries three basic antibody reactivities: A, M and C (Douglas and Palmer, 1988). In diagnosis, the criticial concept is the existence in the serum of an infected animal of a range of antibody reactivities (A>M, A= M and A<M) that in practice correspond to overlapping epitopes highly repeated in the O-polysaccharide (hence their colloective name as Brucella C epitope ; Weynants et al., 1997). Brucella O-polysaccharides cross-react with gram-negative bacteria that contain variable proportions of N-substituted perosamine. Among these bacteria, Yersinia enterocolitica serotype O:9 generates the strongest cross-reactivity.

 

Principle of C-ELISA (source: Laboratoryinfo.com)

This type of ELISA depends on the competitive reaction between the sample antigen and antigen bound to the wells of microtiter plate with the primary antibody.

1-First, the primary antibody is incubated with the sample. This results in the formation of Ag-Ab complex which are then added to the wells that have been coated with the same antigens.

2-After an incubation, unbound antibodies are washed off. The more antigen in the sample, more primary antibody will bind to the sample antigen. Therefore there will be smaller amount of primary antibody available to bind to the antigen coated on well.

3-Secondary antibody conjugated to an enzyme is added, followed by a substrate to elicit a chromogenic signal.

 

Concentration of color is inversely proportional to the amount of antigen present in the sample.

 

Before starting the work, read kit instruction carefully!

 

Results

The ELISA assay yields three different types of data output:

  1. Quantitative: ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a known, purified antigen) in order to precisely calculate the concentrations of antigen in various samples.
  2. Qualitative: ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen.
  3. Semi-Quantitative: ELISAs can be used to compare the relative levels of antigen in assay samples, since the intensity of signal will vary directly with antigen concentration.

 

 

Serological tests for Brucellosis [relayed from OIE Terrestrial Manual]:

The buffered Brucella antigen tests (rose bengal test and buffered plate agglutination test), the complement fixation test, the enzyme-linked immunosorbent assays (ELISA) or the fluorescence polarisation assay, are suitable tests for screening of herds/flocks and individual small ruminants, camelids and bovines (cattle and buffaloes). However, no single serological test is appropriate in each animal species and all epidemiological situations, and some of these tests are not adequate for diagnosing brucellosis in pigs. Therefore, the reactivity of samples that are positive in screening tests should be assessed using an established confirmatory or complementary strategy. The indirect ELISA or milk ring test performed on bulk milk samples is effective for screening and monitoring dairy cattle.

 

Several variations of the C-ELISA, using S-LPS or O-PS as antigens, have been described for cattle, small ruminants and pigs employing different antiglobulin-enzume conjugates, substrate or chromogens and antigens prepared from different smooth Brucella strains. Nevertheless, the technique used and the interpretation of results must have been validated in accordance with OIE requirements. It has been shown that the C-ELISA eliminates some but not all False Positive Serological Reactions (FPSR) caused by cross-reacting bacteria in cattle (Muñoz et al., 2005) and swine (Praud et al., 2012). In some cases, in ruminants or pigs, FSPR may be observed in C-ELISA while not in other S-LPS-based tests, including I-ELISA. Moreover, the C-ELISA reduces but not fully eliminates the reactions caused by antibodies produces in response to vaccination.

Some protocols are less sensitive or less specific than others; therefore results obtained from different assays are not always comparable. C-ELISA for diagnosing anti-Brucella antibodies in small ruminants and swine is essentially te same as that described for cattle, but the cut-off should have been properly establhised for these species using the appropriate validation techniqes.

 

Whatever the C-ELISA format used:

i) a positive and a negative control are included in each plate. Optical Density (OD) ranges to be obtained with these two controls must be established to define the criteria for validating each plate results. The OD of the positive control is the one with which the OD of each test serum is compared to establish the final result -negative of positive).

ii) an additional positive serum (internal control) must be included in each plate to validate the repeatability of the test from plate to plate and from day to day (control chart).

 

CFT

The  complement fixation test (CFT) is used to detect the presence of specific antibodies in the patient’s serum. This test is based on the use of complement, a Biologically labile serum factor that causes the immune cytolysis i.e. lysis of antibody coated cells.

 

Principle (source Laboratoryinfo.com)

It is the nature of the complement to be activated when there is formation of antigen-antibody complex.
The first step is to heat the serum at 56°C to destroy patient’s complement.

A measured amount of complement and antigen are then added to the serum.

If there is presence of antibody in the serum, the complement is fixed due to the formation of Ag-Ab complex. If no antibody is present then the complement remains free.

To determine whether the complement has been fixed, sheep RBCs and antibodies against sheep RBCs are added.

Interpretation

Positive test : The available complement is fixed by Ag-Ab complex and no hemolysis of sheep RBCs occurs. So the test is positive for presence of antibodies.

Negative test : No Ag-Ab reaction occurs and the complement is free. This free complement binds to the complex of sheep RBC and it’s antibody to cause hemolysis, causing the development of pink color.

 

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