Featured News New, Improved USDA-Licensed HerdChek* Mycobacterium paratuberculosis Antibody ELISA Test Kit Expert Opinion: ELISA Value for Paratuberculosis Control		MORE INFORMATION Newsletter Archive Subscribe now Print this page

As a part of an ongoing commitment to deliver the highest quality products, IDEXX is completing the first round of product improvements to the HerdChek* M. pt. ELISA kit.

Mycobacterium avium subspecies paratuberculosis, the causative agent of Johne’s disease, results in a chronic, contagious infection in the intestinal tract of ruminants. The disease affects many ruminant species worldwide, causing considerable economic losses due to weight loss and decreased milk production. Beyond the animal health impact, there is an ongoing debate among scientists regarding the potential association of M. paratuberculosis with human Crohn’s disease. The combined animal-health impact of Johne’s disease and its potential connections to human health support the need for increased testing and participation in disease-control programs worldwide.

The original IDEXX M. pt. ELISA kit has been in the market for several years and continues to serve as an important screening tool, providing excellent specificity on typical populations. However, some recent studies have shown specificity challenges with certain isolated herds.¹

IDEXX’s primary goal in developing an improved M. pt. test kit was to enhance assay specificity on these isolated problematic herds without impacting other areas of performance. The following summary highlights data from IDEXX studies conducted to evaluate the performance of the improved HerdChek M. pt. Antibody ELISA Test Kit.

MATERIALS AND METHODS

Kits from a commercially released lot of the original HerdChek M. pt. ELISA Test Kit (defined as “original kit” in this study) were used for comparison against the improved kit manufactured by the IDEXX research-and-development group.

Serum samples were obtained from multiple U.S. and European sources for evaluation. Many of the samples were obtained from either known-infected herds, or herds presumed to be negative based on herd histories. Negative herd-history information ranges from participation in Johne’s disease control programs to sampling from a country that claims to be free of disease. Johne’s-positive samples were identified by positive cultures (using either liquid or semisolid culture methods) of fecal samples that were obtained at the time of blood sampling.

Tests were run using the protocol defined in the M. pt. antibody test kit instruction manual. No changes were made to the protocol or to the methods used for data analysis and interpretation.

RESULTS

Performance data presumed-negative populations

Nine populations, resulting in 962 negative samples, were examined to assess assay performance of the improved kit. For these studies, a few populations that have previously shown elevated S/P ratios with the original kit were targeted.

A summary of the population means and standard deviations is shown in Table 1. In all cases, the improved kit resulted in better separation of the negative population from the cutoff (S/P = 0.25). The number of standard deviations from the cutoff was greater for all populations assessed using the improved kit.

Table 1. M. pt. antibody performance data for presumed-negative populations Nine populations (n = 962) tested with the original kit (highlight color X) or the improved kit (highlight color Y). nd represents statistics not determined due to small population size.

Performance on typical populations

With typical populations, the original kit shows a normally distributed population with adequate separation from the assay cutoff. Figure 1 demonstrates that the improved kit delivers consistent specificity, and results in greater population separation from the assay cutoff.

Figure 1. Distributions of typical populations The distributions for negative population G (n = 255) when tested with the original kit or improved kit are shown. The red line represents the location of the assay cutoff.

Performance on high-background negative herds

Figure 2 illustrates the population distributions obtained using serum samples from a presumed-negative herd that demonstrated elevated S/P ratios with released batches of the M. pt. ELISA kit. In cases where known-negative herds exhibited elevated S/P ratios, both the original kit and the improved kit reported broader populations, and resulted in a reduced number of standard deviations from the cutoff. Even with this observation, the improved kit allows for better separation of the negative population from the assay cutoff.

Figure 2. Performance on high-background negative herds The distributions for negative population D (n = 207) when tested with the original kit or improved kit are shown. The red line represents the location of the assay cutoff. M. pt. Ab—Performance Data Problem Populations

Known-infected herds and culture-positive individuals

To ensure that the improvements to the M. pt. ELISA test kit did not impact assay sensitivity, serum samples from culture-positive animals in population J were tested with the original and improved versions of the kit. Table 2 shows good agreement with respect to results interpretation between the two versions of the kit. Only 16 of the 36 culture-positive samples were ELISA-positive when tested with either kit version. The improved kit generally yielded higher S/P ratios (seen with 12 of the 16 ELISA-positives) than the original kit. Only two discrepant test results were noted (samples 9 and 18); which were samples whose S/P ratios fluctuated around the assay cutoff. Because the improved kit assay background is reduced on negative populations, the ability to detect samples with moderate humoral responses to M. pt. antigens is improved, thus modestly increasing assay sensitivity. Follow-up studies are needed to substantiate this effect.

Table 2. Performance on culture-positive samples shown are the S/P ratios reported for samples in population J tested with the current or improved kit. ELISA-positive results are red. M. pt. Ab—Performance Data Sensitivity Unaffected by Kit Changes

Comparison of discrepant results

Table 3 summarizes the discrepant test results obtained in this study. In addition to known-infected animals from population J, three other known-infected herds (designated K, L and M) were evaluated. In general, the S/P ratios obtained with the current kit and the improved kit on known-infected herds were in agreement. It is interesting that with samples coming from presumed-negative herds or fecal-culture-negative animals, all of the discrepancies reflect elevated S/P ratios with the original kit that are suppressed when the improved kit is used. With culture-positive samples, on the other hand, the test result discrepancies are evenly distributed between the two kit versions, and a number of the discrepancies are with samples showing S/P ratios close to the cutoff. These results suggest that the improved kit has its greatest impact on test specificity.

Table 3. Summary of discrepant test results shown are the discrepancies between the original kit and the improved kit observed with all populations tested. The S/P ratios resulting in a positive test interpretation are red. Disease status was defined as FCpos, fecal culture-positive sample; FCneg, fecal culture-negative sample; PNH, presumed-negative herd (herd of known-negative history and/or status 3 or 4 participant in a Johne’s control program). M. pt. Ab—Performance Data Discrepant Results

Summary of sensitivity and specificity

Using the data collected for negative herds A through I, and fecal-culture-positive animals in herds J through M, sensitivity and specificity calculations were made. Fecal-culture-negative animals identified in known-infected herds were not included in the evaluation because their true disease status is unclear.

Sensitivity calculations were made using the following two groupings of negative populations, one containing only U.S. herds and a second with all herds tested in the study. The sensitivity and specificity are summarized in Figure 3. Regardless of which populations are examined, the improved M. pt. ELISA kit shows better specificity (99.58%) than the original kit.

Figure 3. 2x2s—performance summary M. pt. Ab—Performance Data 2x2s Performance Summary

SUMMARY

The improved IDEXX HerdChek M. pt. Antibody ELISA Test Kit delivers better specificity compared to the original IDEXX kit, with comparable sensitivity and performance in other areas.

TIMING AND NEXT STEPS

The new M. pt. kit is now USDA-licensed and is expected to be available for sale in mid-July 2006. For more information, contact IDEXX Customer Support at 1-800-548-9997 or your IDEXX sales representative.

Validation work for similar improvements to the IDEXX HerdChek M. pt. Antibody ELISA produced at the IDEXX Europe facility are underway, with launch expected later in 2007.

Studies conducted by IDEXX Laboratories; data on file.

Control of paratuberculosis in dairy herds requires improvements in calf-raising practices to limit opportunities for transmission of M. paratuberculosis from cows to calves. In most herds, this must be supported by diagnostic testing to identify the most infectious cows to be culled from the herd or managed differently than the test-negative cows. For this purpose, ELISA is the diagnostic technology of choice. It is fast, low-cost, and S/P results are directly related to the likelihood of a cow being infected, the level of M. paratuberculosis shedding in feces, and the infection cost to the producer in lost milk production. This article provides an update on research showing the value of ELISAs in paratuberculosis control and future opportunities for product improvement and application.

Speed of diagnosis is important to both producers and laboratories. No diagnostic technology can match ELISA in speed. From start to finish, the ELISAs can be performed in less than two hours. Turnaround time for clients, from sampling a cow to receiving results, is dictated more by the speed of the sample courier than by the assay. For producers, the sooner that infected cows can be detected, the sooner transmission of the infection from those cows can be halted. Speed in the lab also translates to higher throughput and lower cost to the client.

Dairy clients are very price-sensitive when it comes to veterinary diagnostics. In my experience, above a price of roughly $15.00, herd owners become very reluctant to do much testing for paratuberculosis. There are no available diagnostic technologies that can deliver results for less per-assay cost than ELISA.

Throughput translates to capacity, which is critical to most diagnostic laboratories, particularly those in states with sizeable dairy industries. For example, in Wisconsin on January 1, 2005, the inventory of dairy cows was 1,480,000 (National Agricultural Statistics Service). If a laboratory had to test all of these cows once per year and worked five days a week with no holidays, it would require running 5,692 assays per day. Even if only 10% of the cows were tested, 569 assays per day surpasses what is feasible for assay systems other than ELISA.

ELISAs for paratuberculosis have been criticized for lack of sensitivity. However, cows that test ELISA-positive are those that are shedding significant numbers of M. paratuberculosis in their feces and are, therefore, the more infectious cows in the herd. Moreover, the ELISA S/P is directly related to the likelihood that the cow is infected and the rate of fecal shedding by the cow.^1,2 In addition, studies by the USDA have shown that cows with S/P values >1.00 have significant reductions in milk production.³ Thus, the “strong-positive” cows are the ones that are: 1) most infectious, 2) likely to soon break with clinical paratuberculosis, and 3) causing economic losses to dairy producers. So, while a sensitivity of 30% may sound low, the fact is that the sensitivity of detecting the most important cases of bovine paratuberculosis is very high.

ELISA specificity is also important. Lower specificity means more frequent false-positive results. False-positives can be costly to dairy producers because they trigger management actions based on test results that may include cow segregation, discarding the cow’s colostrum or even culling. A mistaken cull and replacement of a dairy cow in today’s market is a $1,200 to $1,400 mistake. The new improved IDEXX ELISA reduces the likelihood of false-positive detection that was observed with the current kit after extensive field evaluation.²

Quantitative interpretation of the ELISA is crucial for maximizing the value of the assay to dairy producers. IDEXX was the first to introduce multilevel ELISA interpretation, which has been well-received by both bovine practitioners and dairy producers. Progressive dairy producers now record the ELISA S/P value in their computer records and use this as a herd-management tool. The magnitude of the ELISA S/P can be incorporated with other quantitative data on the cow, such as milk production and somatic cells counts in milk, to prioritize cows for replacement. Multiple scientific studies have now confirmed the importance of reporting ELISA results quantitatively, like S/P values, instead of dichotomously (positive or negative).^2,4

*HerdChek is a trademark or a registered trademark of IDEXX Laboratories, Inc. in the United States and/or other countries. All other product and company names are trademarks or registered trademarks of their respective holders.

‡Not available through IDEXX Europe