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The EquiSal Tapeworm Test

EquiSal Tapeworm is a simple-to-use saliva test for detecting tapeworm infections in horses. Developed and marketed by Austin Davis Biologics, this ELISA-based test measures tapeworm molecule-specific antibodies which have been shown to correlate with tapeworm burdens [1].

 

The test works like a blood test but uses saliva that horse owners collect using a specifically designed saliva collection swab. The swab is posted back to the laboratory in preservative which keeps the sample stable for at least three weeks. At the laboratory, samples are analysed on a part robotic system which ensures high reproducibility and accuracy in the testing process. Samples are analysed within three days of arrival at the laboratory and, following detailed QC checks, test results are returned via email on the same day.

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Analysis of the commercial dataset for the EquiSal Tapeworm test service indicates that only one in three tests is positive, substantially reducing anthelmintic use [2], which is critical considering recent reports of anthelmintic resistance in tapeworm [3].

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How the test works

The EquiSal Tapeworm test measures Anoplocephala-specific antibodies (IgG(T)) in horse saliva to indicate the level of tapeworm infection. The test includes three ELISAs: 'Specific', 'Non-Specific Binding' (NSB), and 'Total' IgGT ELISA. These ELISAs contribute data to an algorithm that calculates a 'saliva score'. The Specific ELISA targets IgG(T) antibodies against A. perfoliata 12/13kDa antigens. The NSB and Total ELISAs assess non-specific binding and overall IgG(T) to adjust for saliva variability.

 

Saliva production rates can significantly affect sample concentration, potentially causing false negatives. By measuring total IgG(T) and incorporating it into the saliva score calculation the EquiSal Saliva test addresses this issue. Saliva can make ELISA wells 'sticky' which the NSB ELISA, serving as a control, accounts for, so that the saliva score reported is an accurate reflection of an individual's antibody response to A. perfoliata. 

It is advisable to test horses biannually, preferably during spring and autumn.

 

Spring testing is crucial for identifying horses with infections which can lead to pasture contamination through the shedding of eggs during the peak activity period of the oribatid mite intermediate host. Testing in autumn helps detect horses carrying substantial tapeworm burdens, informing treatment decisions to reduce the risk of tapeworm-related disease.

 

Where a low tapeworm risk is determined, either by consistent negative test results or after comprehensive risk assessment, it may be adequate to test annually, in spring or autumn.

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An initial EquiSal Tapeworm test is recommended to be conducted four months following the last anthelmintic treatment for tapeworms. Horses that report with borderline or moderate/high results should undergo a subsequent test three months after treatment to verify a decline in salivary antibody levels.

 

Horses that consistently test positive for tapeworms are likely to be grazing highly contaminated paddocks. Rapid reinfection is often seen on poorly managed paddocks. This indicates a need to assess and improve management practices, such as increasing the frequency of dung removal or decreasing the number of horses per area, to reduce tapeworm transmission through oribatid mites. In these cases, targeted treatments can reduce egg shedding and, consequently, the paddock's infected mite population, thereby lowering opportunities for infection.

 

Continued contamination may arise from other, non-tested, herd members. It is recommended to test all horses in a group at the same time to identify those with higher burdens act as the main sources of environmental contamination. If saliva testing all horses is impractical, treating the non-tested horses simultaneously could be considered.

 

Introducing new horses that carry moderate to high parasite loads into a group can lead to contamination, as can visiting other sites (club camps, showgrounds) that are infested with mites containing cysticercoids. Contamination can also occur from infected oribatid mites originating from adjacent fields or manure heaps.

 

Under-dosing with anthelmintics can result in persistent infections; therefore, it is crucial to adhere to best practice procedures when administering wormers. This includes accurately determining the weight of the animal before dosing.

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When horses consistently have positive test results, particularly where pasture management is considered excellent or has been improved, anthelmintic resistance may be an issue. In these circumstances, it is crucial that owners work with veterinary surgeons to assess all options for tapeworm control, including evaluating the effectiveness of different types of anthelmintic.

When to EquiSal test and addressing horses that are consistently test-positive

Before its commercial launch, the EquiSal Tapeworm test was thoroughly validated using gold standard samples from horses at post-mortem where tapeworms were counted [1]. The resultant data was used to establish cut-off values for diagnosing low, borderline, or moderate/high tapeworm burdens.

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In these studies, the test successfully identified all horses with potentially pathogenic tapeworm burdens (i.e., those with >20 A. perfoliata) and only misdiagnosed horses as having no burden when less than 20 tapeworms were present. This level of A. perfoliata burden is considered to be non-pathogenic. However, it is important to keep in mind that false negatives and positives are possible in all tests that are used to diagnose infection.

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Serological ELISA tests were also performed for each horse in the validation study, allowing a comparison between the serum and saliva based ELISA formats and also with tapeworm numbers. Comparison of the data from the EquiSal Tapeworm test and serological tapeworm test showed that there was a strong positive correlation between the two types of tests. The results from both tests did not agree only in a few horses with non-pathogenic burdens (<20 tapeworms). Overall, the EquiSal Tapeworm test and serological tapeworm test were found to have the same level of accuracy for diagnosing tapeworm burdens.

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Subsequent studies in collaboration with other veterinary parasitologists have confirmed these results from the initial validation studies and have demonstrated the value of applying saliva or blood tests in populations of horses in support of evidence based tapeworm control. 

The EquiSal Tapeworm test is supported by independently peer-reviewed published research

Our study, published in the Equine Veterinary Journal in 2018 [4] reported findings from 237 horses based at a welfare sanctuary in which EquiSal Tapeworm testing was used to inform anthelmintic treatment decisions over a year. The approach reduced anti-tapeworm treatments by 86% compared to a 6-monthly interval treatment strategy. Most horses diagnosed below the treatment threshold in their first test and remained below this threshold in the following two tests; 168 horses (71%) required no anti-tapeworm treatments over the course of a year. No increases in tapeworm infection prevalence was observed during the study period, with only seven horses indicated for treatment following all three tests, suggesting that some horses are more susceptible to tapeworm infections than others.

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The results indicate a substantial decrease in the use of anti-tapeworm treatments and emphasise the importance of regular monitoring by testing. The majority of horses required no anti-tapeworm treatments over the course of a year and prompt treatment was able to limit paddock contamination and exposure of the rest of the herd.

Peer-reviewed published field studies demonstrate the value of using EquiSal testing to reduce anthelmintic use

FAQs

Tapeworm eggs are distinguishable by their unique shape in FEC tests; however, the shedding of proglottids containing eggs by tapeworms is intermittent, and only a fraction of adult tapeworms release eggs, while juvenile worms may constitute the majority of the burden [2].

 

Consequently, FECs do not correlate well with tapeworm burdens, and these tests have low sensitivity for detecting infection, leading to a high incidence of false negatives, particularly in horses with lower burdens (i.e., <20) of tapeworms.

Tapeworm eggs can be detected in faecal egg count tests, so why use EquiSal Saliva testing?

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The EquiSal Tapeworm test uses three ELISA formats for each sample. These measure tapeworm antigen-specific antibodies and account for individual saliva flow rates and the inherent variability of saliva matrices in ELISA plates. A calibration curve using total IgG antibody standards is included on each plate so that results for each sample report as a relative concentration rather than just an optical density measure. This ensures that results are reproducible and tightly controlled between tests. The final saliva score is generated using an algorithm that combines the results from each ELISA format so the final score does not have units.

Why are EquiSal test results reported as scores and not as antibody concentrations or optical densities?

Validation of the EquiSal Tapeworm test against the gold standard of counting tapeworms at post-mortem demonstrated that the test identifies 1+ tapeworm burdens (Saliva Score >-0.09) with a sensitivity of 83% and specificity of 85%. Saliva scores show a strong positive correlation to tapeworm numbers (Spearman’s rank correlation, 0.74) and identify 20+ tapeworm burdens (Saliva Score >0.6) with a sensitivity of 86% and specificity of 79%. Statistical analysis identified a narrow range between Saliva Score -0.09 and 0.6 which is considered a borderline result. When the 1+ tapeworm threshold is applied, all horses with a burden of 20 or more tapeworms were correctly identified. The table below summarised how results are provided by the EquiSal Tapeworm test service; treatment is recommended as detailed.

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How does the EquiSal Tapeworm saliva score relate to tapeworm burden, diagnosis and treatment recommendation?

The Saliva Score is not an optical density value and negative scores (such as -0.09 stated for the 1+ tapeworm cut-off) are a result of test data being subject an algorithm which combines results from three ELISA formats that take into account tapeworm-specific antibody, background binding of saliva to the ELISA plate, and variations in saliva flow.

How can the Saliva Score have a negative value?

The narrow borderline region between -0.09 and 0.6 was assigned as a result of the saliva score thresholds for 1+ and 20+ tapeworms. A saliva score of -0.09 was identified as the cut off for 1+ tapeworm threshold and 0.6 as the cut off for 20+ tapeworms threshold, the narrow region between these thresholds contained an equal number of low, 1-20 tapeworm and 20+ tapeworm burdened horses so this is designated a borderline range.

Why is there a borderline zone in the reporting system?

An extremely high Saliva Score can reach into the hundreds, but an average moderate/high score is around 9. There is strong positive correlation (Spearman’s rank, 0.74) between tapeworm numbers and the test Saliva Score, but the test does not report a result for an exact value of tapeworm numbers as each horse has an individual immune response to a burden.

What is the average Saliva Score for a moderate/high burden diagnosis?

Based on an in-depth analysis of the validation data, it was found that using the 1+ tapeworm burden cut-off did not misdiagnose any high burden horses (i.e. those with >20 tapeworms). While studies have shown that the severity of intestinal lesions is associated with the number of tapeworms present [5-8], it is generally accepted that the presence of 20 worms or less is not pathogenic. Reports also indicate that not only does infection intensity relate to clinical impact, but the site of attachment of the worms within the intestine, particularly the ileo-caecal junction, where even a small number of worms can result in lesions [9].  

 

EquiSal Tapeworm testing service results indicate that two-thirds of tested horses in the UK fall below the 1+ burden threshold, meaning that, using the 1+ tapeworm burden cut-off, fewer horses need to receive anthelmintics compared to blanket worming strategies.

Why is treatment recommended for 1+ tapeworms – why not recommend for high burdens only (>20 tapeworms)?

Horses can be infected with tapeworms year-round if they graze paddocks that are infected with intermediate host oribatid mites containing tapeworm larvae. The level of tapeworm-infected mites on the paddock will largely determine the prevalence (number of horses infected) and infection intensity (level of burden in individual horses) in a grazing population. Independent studies that enumerated tapeworm burdens and assessed parasite population dynamics in horses at post-mortem [14, 15] have shown that immature and sterile adult worms dominate tapeworm populations at most times during the year, and that egg-laying adult tapeworms are more likely to be present in the intestine in spring (see figures below adapted from these studies). It is therefore recommended to test horses in spring to identify those individuals with higher gravid adult tapeworm burdens that will likely contaminate paddocks during the optimal summer transmission period through oribatid mites. In doing this, the objective is to disrupt the tapeworm life cycle so that large numbers of mites do not become infected with cestode eggs during spring and summer.  These independent studies also demonstrated that higher levels of tapeworm burdens are more likely to be found in autumn and winter. Therefore, tapeworm testing is also recommended in autumn to identify those horses with worm burdens that might result in clinical disease such as colic, keeping in mind that 20 or more tapeworms in the intestine have been shown to be associated with severe damage that may cause disease [7].

What is the rationale behind testing twice a year and the timing of testing?

Twice a year testing rationale 1_edited.
Twice a year testing rationale 2_edited.

Based on a study conducted on horses diagnosed with tapeworm, EquiSal Tapeworm testing showed that 50 of horses' saliva scores reduced to low levels within 6 weeks, 90% within 10 weeks, and the remaining 10% within 12 weeks. This suggests that tapeworm-specific salivary antibodies reduce more rapidly than those in blood, and horses can be saliva tested from 3 months after tapeworm treatment. The reason for this is that these antibodies are probably part of a mucosal immune response, secreted through mucosal epithelial cells by transcytosis [10].

 

It is likely that tapeworm-specific antibodies measured in the EquiSal Tapeworm test are produced in salivary glands by plasma cells that originated as recirculating (via the lymphatic system) B-blasts triggered in the gut submucosa at the site of infection [11].  It remains possible that low levels of blood antibodies could leak into the saliva by transudation or passage through the gingival crevicular space; however, pilot trial data (Austin et al. unpublished) suggest that this is not a significant factor. In contrast to humoral antibody responses (detected in blood testing), mucosal antibody responses have a shorter persistence.

The situation is complicated if a horse becomes reinfected after treatment due to exposure to a infected mite-contaminated paddock. In these circumstances, for horses that repeatedly test positive 3 months after treatment, the pasture's management should be improved. This is because rapid reinfection can occur due to ingesting infected mites from contaminated paddocks, especially because wormers used to treat tapeworms do not have a persistent effect.

How long after worming are tapeworm-specific antibodies present in saliva?

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While the test is anticipated to be effective for all equids, such as donkeys, it has not been specifically validated in these animals. Validation studies to date have been conducted solely with horse saliva samples.

Can the EquiSal Tapeworm test be used to test donkeys?

EquiSal Tapeworm Saliva samples may be collected before dental procedures begin; however, they should not be collected after dental work. The presence of blood in the mouth, which leads to contamination of the saliva with blood antibodies, can impact the test outcomes.

Can EquiSal testing be carried out at the same time as dental work?

The EquiSal Tapeworm saliva test and the serological test show good agreement with each other with moderate to strong correlations reported [1,12,13], however different diagnoses between the two assays can occur. In the validation studies [1], when the EquiSal Tapeworm test was compared to the serological test, the diagnoses did not agree only when horses had <20 tapeworms (a level considered non-pathogenic).  When salivary antibodies were monitored 2-weekly after worming, data suggested that salivary antibodies reduced more rapidly than previously reported for blood antibodies. Together with scientific understanding of the horse's immune system, this suggests that blood tests are more likely to pick up previous infections than the EquiSal Tapeworm test. 

Under what circumstances does the serological test diagnose a high burden when EquiSal Tapeworm test does not?

  1. Lightbody KL, Davis PJ, Austin CJ. Validation of a novel saliva-based ELISA test for diagnosing tapeworm burden in horses. Vet. Clin. Pathol. 2016;45:335-346.

  2. Matthews JB, Peczak N, Lightbody KL. The use of innovative diagnostics to inform sustainable control of equine helminth infections. Pathogens 2023;12:1233.

  3. Nielsen MK. Apparent treatment failure of praziquantel and pyrantel pamoate against anoplocephalid tapeworms. Int. J. Parasitol. Drugs Drug Resist. 2023;22:96-101.

  4. Lightbody KL, Matthews JB, Kemp-Symonds JG, Lambert PA, Austin CJ. Use of a saliva-based diagnostic test to identify tapeworm infection in horses in the UK. Equine Vet J. 2018;50:213-219.

  5. Edwards GB. The role of tapeworms in Equine colic. Pferdeheilkunde 1999 4;15:309-312

  6. Rodríguez-Bertos A, Corchero J, Castaño M, Peña L, Luzón M, Gómez-Bautista M, Meana A. Pathological alterations caused by Anoplocephala perfoliata infection in the ileocaecal junction of equids. Zentralbl. Veterinarmed. A. 1999;46:261-269.

  7. Pavone S, Veronesi F, Piergili Fioretti D, Mandara MT. Pathological changes caused by Anoplocephala perfoliata in the equine ileocecal junction. Vet. Res. Commun. 2010;34 Suppl 1:S53-56.

  8. Hreinsdóttir I, Hreinsdóttir A, Eydal M, Tysnes KR, Robertson LJ. Anoplocephala perfoliata infection in horses in Iceland: Investigation of associations between intensity of infection and lesions. J. Parasitol. 2019;105:379-386.

  9. Pearson GR, Davies LW, White AL, O'Brien JK. Pathological lesions associated with Anoplocephala perfoliata at the ileo-caecal junction of horses. Vet Rec. 1993;132:179-182.

  10. Pittaway CE, Lawson AL, Coles GC, Wilson AD. Systemic and mucosal IgE antibody responses of horses to infection with Anoplocephala perfoliata. Vet. Parasitol. 2014;199:32-41.

  11. McGhee JR, Fujihashi K. Inside the mucosal immune system. PLoS Biol. 2012;10:e1001397.

  12. Jürgenschellert L, Krücken J, Austin CJ, Lightbody KL, Bousquet E, von Samson-Himmelstjerna G. Investigations on the occurrence of tapeworm infections in German horse populations with comparison of different antibody detection methods based on saliva and serum samples. Parasit. Vectors. 2020;13:462.

  13. Burcáková L, Königová A, Kuzmina TA, Austin CJ, Matthews JB, Lightbody KL, Peczak NA, Syrota Y, Várady M. Equine tapeworm (Anoplocephala spp.) infection: evaluation of saliva- and serum-based antibody detection methods and risk factor analysis in Slovak horse populations. Parasitol. Res. 2023;122:3037-3052.

  14. Meana, A., Pato, N.F., Martín, R., Mateos, A., Pérez-García, J., Luzón, M.,  2005. Epidemiological studies on equine cestodes in central Spain: infection pattern and population dynamics. Vet. Parasitol. 130, 233-240. 

  15. Rehbein S., Visser M., Winter R., 2013. Prevalence, intensity and seasonality of gastrointestinal parasites in abattoir horses in Germany. Parasitol Res. 112, 407-413.

References

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