Letters to the Editor

Dr. Pijoan's response follows...

Mycoplasma hyopneumoniae ELISA cut point

This letter expresses my concern regarding the results reported in the article, "Effect of Mycoplasma hyopneumoniae sow vaccination on piglet colonization at weaning" (Journal of Swine Health and Production, May-June, 2003, pp 131-135).¹ I am confused and concerned about the authors' use of a sample-to-positive (S:P) value of 1.0 as the positive cut point for the Tween 20 ELISA. This cut point is approximately two times higher than values published in the original paper² describing the Tween 20 ELISA, which is referenced by the authors, and those for the modified assay³ currently performed in many laboratories, including my laboratory at Iowa State University. Both papers describe their results in terms of optical density values (ODs); however, if S:P ratios are calculated, in each case the result would be a positive cut point of approximately 0.5. Use of S:P ratios helps to standardize assays, allowing for constant values and minimizing variability between assay results.

It was unclear where the Tween 20 ELISA assays described in this paper were performed. Currently, the diagnostic laboratory at the University of Minnesota obtains the reagents for the Tween 20 M hyopneu-moniae assay from my laboratory at Iowa State University. If the positive control serum used in the assays described in the paper originated from my laboratory, the OD of the positive sera should be approximately 0.400, resulting in a positive S:P cut point between 0.5 and 0.6. Ratios of 0.4 to 0.5 are considered suspect. Whatever the source of the control serum used in the assays described in this article, using a positive control serum that results in a positive S:P cut point of 1.0 would decrease the sensitivity of the assay and potentially increase the number of false negative results.

In any ELISA assay, an important goal is to have the positive control sample (which is used as a procedural control to guarantee assay accuracy) within the linear range of the assay. Therefore, the positive control sera used in most ELISA assays are selected to result in an S:P cut point of approximately 0.5. With an S:P cut point of 1.0, the sensitivity would decrease, as all values below that of the positive control would be interpreted as negative.

In a recent study,⁴ a graduate student in my laboratory, Keith Erlandson, compared three M hyopneumoniae antibody ELISA assays currently used in the United States, including the Tween 20, the HerdChek Mycoplasma hyopneumoniae Antibody Test Kit (Idexx Laboratories, Westbrook, Maine), and the Dako Mycoplasma hyopneumoniae ELISA (Dako Corp, Carpenteria, California). He found that when the published and validated S:P cut points were used, sensitivity values for all three assays were low (0.35, 0.37, and 0.49, respectively, for the Tween 20, the Idexx HerdChek, and the Dako ELISA), and the potential number of false negative results would be high. Increasing the positive S:P cut point to 1.0 would further lower the sensitivity of the assay and increase the number of false negative results.

There is a current interest in developing M hyopneumoniae-free swine herds. Increasing the positive S:P cut point of the M hyopneumoniae serological assay would increase the potential number of false negative results, thus increasing the risk of introducing infected animals into negative herds. This might result in significant economic impact and disease in the herds of unsuspecting producers who are trying to establish or maintain M hyopneumoniae-free herds.

In summary, the use of a positive S:P cut point of 1.0 for the Tween 20 is inaccurate and inconsistent with the published protocols established for this assay. Use of this cut point might significantly increase the number of false negative results reported to veterinarians, with possibly devastating consequences. It is recommended that the use of this cut point be re-examined by the authors and modified to the published values.

Eileen L. Thacker, DVM, PhD
Iowa State University
Ames, Iowa

References - refereed

1. Ruiz AR, Utrera V, Pijoan C. Effect of Mycoplasma hyopneumoniae sow vaccination on piglet colonization at weaning. J Swine Health Prod. 2003;11:131-135.

2. Nicolet J, Paroz P, Bruggman S. Tween 20 soluble proteins of Mycoplasma hyopneumoniae as antigen for an enzyme-linked immunosorbent assay. Res Vet Sci. 1980;29:305-309.

3. Bereiter M, Young TF, Joo HS, Ross RF. Evaluation of the ELISA and comparison to the complement fixation test and radial immunodiffusion enzyme assay for detection of antibodies against Mycoplasma hyopneumoniae in swine serum. Vet Microbiol. 1990;25:177-192.

References - non refereed

4. Erlandson K, Thacker B, Wegner M, Evans R, Thacker E. Evaluation of three serum antibody ELISA tests for Mycoplasma hyopneumoniae. Proc IPVS. Ames, Iowa. 2002:74.

Mycoplasma hyopneumoniae ELISA cut point - response

I have read with interest Dr Thacker's letter regarding the ELISA cutoff points reported in our paper "Effect of Mycoplasma hyopneumoniae sow vaccination on piglet colonization at weaning" (Journal of Swine Health and Production, May-June, 2003, pp 131-135).¹ I find it rather surprising that Dr Thacker is so concerned about these cutoff points, since they were not a relevant part of the study. As indicated in the paper, we used the test and its interpretation as performed in the University of Minnesota Veterinary Diagnostic Laboratory, since the accuracy of the Tween 20 test was not the purpose of our study.

Independently of the cutoff values, the study clearly demonstrated that sow vaccination resulted in a significant increase in S:P ratios and that this increase was also seen in litters from vaccinated sows, but not in litters from nonvaccinates. I think these results are obvious, expected, and not surprising, regardless of how the lower limits of the test are being interpreted.

More importantly, I think that Dr Thacker totally missed the point of the study, which was to demonstrate that sow vaccination significantly reduced piglet colonization. Piglet colonization was determined by nested polymerase chain reaction, not by serology. The serological assays are not central to the study, nor do they invalidate the conclusions. On the other hand, the real importance of this study is to open the possibility of sow vaccination in the control of M hyopneumoniae infections in farms that use strict off-site production. I believe that this is a major contribution to the control of M hyopneumoniae, since sow vaccination not only significantly reduces vaccine usage, but also addresses the important problem of M hyopneumoniae vaccination in the nursery. Nursery pigs are commonly vaccinated for M hyopneumoniae at a time that coincides with circulation of porcine reproductive and respiratory syndrome virus, aggravating the problems caused by this virus.

Carlos Pijoan, DVM, PhD
College of Veterinary Medicine
University of Minnesota

Reference - refereed

1. Ruiz AR, Utrera V, Pijoan C. Effect of Mycoplasma hyopneumoniae sow vaccination on piglet colonization at weaning. J Swine Health Prod. 2003;11:131-135.