The most common horse tapeworm, Anoplocephala perfoliata, predominantly attaches to the ileocaecal junction in the gut which is a narrow section between the small intestine and the caecum. This localised attachment causes damage to the intestines and the presence of large numbers of tapeworms cause intestinal obstruction and clinical disease, resulting in colic (Back et al., 2013).
Diagnosis of tapeworm infections
Diagnosing tapeworm infections in horses has been difficult until recently, as tapeworms are not reliably detected by standard worm faecal egg counts (FEC) due to the way in which they release eggs. However, saliva or serum antibody tests have been shown to be reliable and accurate.
The serum tapeworm test detects tapeworm-specific antibodies which have been shown to have strong positive correlation to tapeworm infection intensity (Spearman’s correlation, 0.78). The ELISA format includes a calibration curve to accurately generate a ‘serum score’ for each sample as well as provide a quality control for each ELISA carried out. Serum scores are categorised into ‘low’, ‘borderline’ and ‘moderate/high’ diagnoses based on validation (Lightbody et al., 2016, see the Peer Reviewed Papers tab) of the test and anti-tapeworm treatment is recommended for borderline and moderate/high results.
Uses of the Serum Tapeworm Test
- Identification of horses with tapeworm infections for targeted treatment.
- Diagnosis of horses with clinical symptoms.
Limitations
- The test does not provide exact numbers of tapeworms present, but provides a low, borderline or moderate/high diagnosis.
- Antibody levels in blood reduce over time in response to treatment so the test cannot be used to monitor anthelminitic efficacy. It has been demonstrated that blood antibody levels are not reduced to low diagnosis until at least 4 months after treatment. However, this is complicated by horses becoming reinfected or juvenile tapeworm maturing after treatment.
Testing recommendations
- Do not carry out the first test until 4 months after the last treatment.
References
Back H, Nyman A, and Osterman Lind E. (2013) The association between Anoplocephala perfoliata and colic in Swedish horses–a case control study. Vet. Parasitol. 197: 580–585.
Lightbody, K.L., Davis, P.J. and Austin, C.J. (2016) Validation of a novel saliva-based ELISA test for diagnosing tapeworm burden in horses. Vet. Clin. Path. 45: 335-346.
Serum Tapeworm ELISA validation
- Validation is published in Veterinary Clinical Pathology. (Lightbody, K. L. et al. (2016) Vet. Clin. Path., 45: 335–346).
- 85% sensitivity and 78% specificity is obtained for a 1+ tapeworm cut-off.
- 15% of horses were misclassified as negative, but no false negatives were obtained for horses with a pathogenic burden (20+ tapeworms).
ELISA storage and shelf life
- ELISA plate storage: -20°C.
- Other kit components: 4°C.
- 6 months shelf life at recommended storage temperatures.
- 4 weeks shelf life once ELISA plate seal is broken.
- Storage inter-variability: 5.5% CV for 25 calibrator: 5.9 - 10.9% CV range for samples (n=6) (17 test kits over 24 week stability trial).
ELISA precision
- Intra-variability: 2.5% CV for 25 calibrator: 1.3 - 5.1% CV range for samples (n=8).
- Inter-variability*: 4.6% CV for 25 calibrator: 2.0 - 13.0% CV range for sample (n=8) (8 ELISA plates over 5 days).
Reagents and materials provided
- ELISA plate pre-sensitised with tapeworm antigens.
- Vacuum packed ELISA plate in stripwell format allowing three separate uses of the kit if required.
- 3 uses = up to 33 samples
- 2 uses = up to 38 samples
- 1 use = up to 43 samples
- Calibration reagent supplied – anti-tapeworm IgG
- Calibration curves must be performed for each ELISA test for quality control and to calculate serum scores.
- Anti-horse IgG(T):Horseradish Peroxidase (HRP) conjugate.
- 3,3’,5,5’-Tetramethylbenzidine (TMB) Liquid Substrate System.
Key Materials and Equipment required but not provided include:
- Microbalance
- Incubator
- Plate reader capable of absorbance readings at 450 nm
- Pipettes including multichannel pipette
- Plate washer (preferable)
- PBS tablets
- Tween 20
- Bovine Serum Albumin (Sigma – A6947)
- Plate sealing films if running >1 test
Protocol Outline
- Prepare all reagents, samples and calibrators as detailed in the instructions.
- Add blocking buffer to each well.
- Incubate for 1 hour at 25°C.
- Add samples and calibrators to each well in duplicate.
- Incubate for 1 hour at 25°C.
- Add TMB to each well.
- Incubate for 10 minutes at 25°C.
- Add stop solution to each well.
- Read plate at 450nm.
The link below is password protected. You will be provided with the password with your first kit order.
Validation of the serum tapeworm ELISA has been published in peer reviewed journal, Veterinary Clinical Pathology.
Validation paper serum ELISA highlights
Serum samples (139) were collected from horses at a UK abattoir. The ileocecal junction and cecum were visually examined for tapeworms and any present were counted. Samples were analysed using the serum ELISA. The test results were compared to tapeworm numbers and the data sets were statistically analysed.
Using Mann–Whitney tests, comparison of the Low-burden group to the Moderate/High-burden group (1+ tapeworm cut-off) for the test resulted in P-values of <.0001, demonstrating a high statistically significant difference (Figure 1).
Area under the curve (AUC) data, from ROC curve analysis, was significantly different from 0.5 (P < .001) demonstrating that both the serologic ELISA was able to differentiate between samples using either a 1+ or 20+ tapeworm cutoff.
When a 1+ tapeworm cutoff was assigned for serum ELISA data, the optimum sensitivity, specificity, PPV, and NPV were obtained with a serum ELISA cutoff score of 2.7.
The link below is password protected. You will be provided with the password with your first kit order.
Cost: £300.00 ex VAT
Number of samples tested by one kit:
- three separate uses: 33
- two separate uses: 38
- one use: 43
Download the technical PDF for more details here.
Contact info@austindavis.co.uk to order or for more information.
Note: This ELISA kit is not available in the UK.