LA TROBE UNIVERSITY 27 Practical Exercises: In the first two practical classes, you will study the release of antigens into faeces during natural fluke infections in cattle as well as the serum...

Lab report for an Animal Health subject


LA TROBE UNIVERSITY 27 Practical Exercises: In the first two practical classes, you will study the release of antigens into faeces during natural fluke infections in cattle as well as the serum antibody responses of cattle to liver fluke antigens resulting from the infection. Antigen and antibody responses will be evaluated using the following two techniques: 1. Coproantigen Sandwich ELISA (Practical 1) 2. Serology ELISA (Practical 2) Both these tests rely on the specificity of an antibody to react with its target antigen. These techniques can be used as experimental tools to quantitate the release of antigen in faeces as well as antibody responses to antigens following infection. Practical 1 Coproantigen Sandwich ELISA The BIO-X Bovine Fasciola hepatica coproantigen ELISA kit allows the detection of coproantigens present in faecal material of cattle infected with F. hepatica. These coproantigens can be found in the faeces even outside the fluke’s egg-laying period. The previously mentioned serology ELISA assays allow detection of specific antiparasite circulating antibodies but tests of this type are of limited diagnostic value in endemic areas because antibody titers remain at high levels even when animals have been successfully treated (eg see Figure 4 in Brockwell et al. 2013). Thus, the advantage of using a coproantigen sandwich ELISA assay compared to a serology indirect ELISA assay is that the samples will test positive only if adult flukes are present in the bile ducts of sheep and cattle and antigens are passed out in the faeces of infected animals. The coproantigen ELISA technique is carried out as follows: detailed methods are provided below: The coproantigen sandwich ELISA is an indirect assay using faeces as the source of the parasite antigen. Alternate rows of the 96-well microplate have been sensitised with a specific polyclonal antibody against F. hepatica antigens that are shed in the infected animal faeces. This antibody thus captures the antigens in the faecal material, known as ‘coproantigens’. The remaining rows in the microplate have been sensitised with a polyclonal antibody that is not specific for the parasite antigens (see Fig 3). These control rows allow differentiation between a specific immunological reaction and non-specific binding to eliminate false positives. Faecal material is diluted in dilution buffer, added in duplicate to the wells of the microplate and incubated. A specific monoclonal antibody (coupled to biotin) produced against an antigenic A B C D E F G H Fig 3. In the strip, rows A, C, E and G have been coated with specific polyclonal antibody against F. hepatica and rows B, D, F and H have been coated with a control antibody (polyclonal antibody not specific for parasite). 28 determinant of F. hepatica is added to the wells. A second conjugate, a peroxidase-coupled avidine specific to biotin is added and the plate is incubated. At the end of the incubation period the chromogen solution, tetramethylbenzidine (TMB), is added. If F. hepatica coproantigens are present and the two conjugates have bound in the F. hepatica-specific antibody-coated wells, the enzyme catalyses the transformation of the colourless chromogen into a pigmented compound (blue). The intensity of the resulting blue colour is proportionate to the amount of coproantigen bound in the well. The enzymatic reaction is stopped by acidification, which turns the compound yellow and the resulting optical density at 450 nm can be read using a spectrophotometer. The signals recorded for the negative control wells are subtracted from the corresponding positive wells. A control positive antigen is provided with the kit so we can validate that the experimental materials are functioning correctly i.e. validates the results. This control antigen is composed of lyophilised ground flukes and will elicit a strong positive result when used in the ELISA. 1. Add the diluted faecal samples to the plate coated with capturing antibodies specific for the F. hepatica faecal antigen (shown in red) and incubate for 1 hour to allow antibody binding. Capture antibody 2. Wash any unbound antigen from the plate. 3. Incubate the plate with the biotin-linked detection antibody (it will bind to the antigen that is already bound to the capturing antibody). Bound antigen Biotin-linked detection antibody Biotin 29 4. Wash the unbound biotin-linked detection antibody from the plate. 5. Incubate with avidine-peroxidase conjugate. 6. Wash the unbound avidine-peroxidase conjugate from the plate. 7. Incubate with the substrate (TMB) of the enzyme (peroxidase) linked to avidine. 8. Add the stop solution and read the result on a microplate reader. Biotin-linked detection antibody Biotin Avidine Peroxidase 30 Practical 1 Exercise History: A farmer has a mob of cattle and is concerned that they may be suffering from a parasitic infection. The mob consists of 300 dairy cattle aged 12-15 months with an average bodyweight of 320kg. Animals have been grazing on irrigated pasture for the previous six months consisting mainly of perennial ryegrass and clover. A few animals appear to be losing condition and have a “bottle jaw” plus at least half of the mob have coats that are rough and dull in appearance. In the last month they received a pour-on roundworm drench (Cydectin) and had a booster vaccine of 5 in 1 administered. The owner does not think the animals have improved since the pour-on treatment and is worried that the herd may be infected with liver fluke as he has heard that this parasite can be a problem in his area of Maffra, Victoria. The farmer has sent 24 faecal samples from his herd for fluke analysis. Each team will receive 2 cattle faecal samples for evaluation and the data from the practical class you are in will be used to assess the fluke status in the herd (e.g. if you are in Group A, use only Group A results). Aims: To determine the relative prevalence and intensity of infection in the herd and to advise the farmer whether it is economical to drench his herd for liver fluke. Samples: Each team will receive 2 faecal samples sent by the farmer for liver fluke testing; the class will analyse 24 samples altogether and share the data Test: Samples will be tested using the Bio-X liver fluke Coproantigen ELISA kit Bio-X liver fluke Coproantigen ELISA Materials and Method Materials: Please wear a pair of gloves before handling any of the below mentioned materials! ✓ 200 μl Micro Pipette ✓ 200 μl Micro Pipette tips (Yellow) ✓ 1x 8 well ELISA strip (BIO-X Bovine Fasciola hepatica coproantigen ELISA kit) ✓ 2 x tubes of Cattle faecal sample extracts ✓ 1 x tube of negative control ✓ 1 x tube of Positive antigen reference solution (this is available to only 1 team per class) ✓ 1 x bottle of washing solution ✓ 1 x tube of diluted Biotin-linked anti-Fasciola hepatica conjugate ✓ 1x tube of diluted avidine peroxidase conjugate Chromogen (TMB) solution ✓ 1x tube of Stop solution (1M H3PO4 acid – handle with care) ✓ 1x piece of cling film ✓ 1 x piece of Aluminium foil ✓ Paper towel 31 Method: Steps 1 – 4 have already been completed for you. Your exercise begins from step 5 onwards; 1. Dilute the 5 x concentrated dilution buffer 5 fold by taking 25 ml of 5 x concentrated dilution buffer and adding 100 ml of distilled water (dH2O). 2. Dilute the given cattle faecal samples in the dilution buffer (2 g of faeces + 2 ml of dilution buffer). 3. Centrifuge at 1,000 g for 10 minutes. 4. Collect the supernatants into labelled centrifuge tubes. Complete steps 5 – 20 during your practical class 5. Pipette 100 μl aliquots of the diluted samples to the wells of your strip (e.g. sample 1 in wells A1 and B1; sample 2 in wells C1 and D1). Proceed in the same manner for the positive reference (G1 and H1). Samples 1 and 2 are unknown samples sent by the farmer to be tested for liver fluke infection (please see table below). 6. 7. Cover strip with cling film and incubate at room temperature for 1 hour. 8. Empty the wells of their contents by flipping the strip over a sink and then tap the strip upside down against a piece of clean absorbent paper to remove all the liquid. 9. Wash strip by adding washing solution. Fill all the used wells to the top with the washing solution using a squirt bottle. 10. Empty the wells completely once more by turning the strip over sharply over a sink and then tap as described in step 7. 11. Repeat steps 8 and 9, twice more (in total the strip must be washed 3 times), taking care to avoid the formation of bubbles in the microwells. Team Number A Sample 1 B Sample 1 C Sample 2 D Sample 2 E Negative control F Negative control G Positive Reference H Positive Reference Samples 1