Comparison of subjective and objective test evaluations for use of Mycobacterium phlei-adsorbed serum in a dot-enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in cattle

S. Bech-Nielsen From the National Veterinary Laboratory, PO Box 373, DK-1503, Bulowsvej 27, Copenhagen V, Denmark (Berg Jorgensen, Frandsen, Feld, Ahrens) and the Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210 (Bech-Nielsen, Shulaw).

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W. P. Shulaw From the National Veterinary Laboratory, PO Box 373, DK-1503, Bulowsvej 27, Copenhagen V, Denmark (Berg Jorgensen, Frandsen, Feld, Ahrens) and the Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210 (Bech-Nielsen, Shulaw).

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P. L. Frandsen From the National Veterinary Laboratory, PO Box 373, DK-1503, Bulowsvej 27, Copenhagen V, Denmark (Berg Jorgensen, Frandsen, Feld, Ahrens) and the Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210 (Bech-Nielsen, Shulaw).

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J. B. Jorgensen From the National Veterinary Laboratory, PO Box 373, DK-1503, Bulowsvej 27, Copenhagen V, Denmark (Berg Jorgensen, Frandsen, Feld, Ahrens) and the Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210 (Bech-Nielsen, Shulaw).

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N. C. Feld From the National Veterinary Laboratory, PO Box 373, DK-1503, Bulowsvej 27, Copenhagen V, Denmark (Berg Jorgensen, Frandsen, Feld, Ahrens) and the Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210 (Bech-Nielsen, Shulaw).

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P. Ahrens From the National Veterinary Laboratory, PO Box 373, DK-1503, Bulowsvej 27, Copenhagen V, Denmark (Berg Jorgensen, Frandsen, Feld, Ahrens) and the Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210 (Bech-Nielsen, Shulaw).

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Summary

Use of a dot-elisa with serum adsorbed with Mycobacterium phlei or with nonadsorbed serum was compared. In addition, results attained using visual observation were compared with those obtained using a densitometer. Infection status of cattle was determined by results of culture of feces from a number of cattle with various degrees of exposure (low prevalence and test-negative) and disease manifestation (clinical suspect vs subclinical infection). Two paratuberculosis-negative herds, fecal culture-confirmed clinically suspect cases of paratuberculosis, and cows from 2 paratuberculosis-infected herds with diagnosis confirmed on the farm (low infection rate) were tested.

Significant (P < 0.05) increase in the dot-elisa response was found in cattle with heavy M paratuberculosis shedding when nonadsorbed and adsorbed sera were used, compared with the response in cattle that were fecal culture-negative or were shedding M paratuberculosis at lower amounts. Paratuberculosis was diagnosed by visual determination in 29 of 44 (65.9%) of fecal culture-positive, clinically suspect cattle when nonadsorbed serum was used. Results of the visual test were negative in 85 of 93 (91.4%) of the fecal culture-negative cattle when nonadsorbed serum was used. However, when using M phlei-adsorbed serum, the sensitivity of the visual determination decreased to 34.1% (15/44), and the specificity increased to 97.8% (91/93). When taking into consideration the amount of bacterial shedding (ie, colonies per gram of feces isolated by culture of feces), 22 of 29 (75.9%) cattle were heavy shedders, with > 1,500 colonies/g of feces at the time of serologic testing when nonadsorbed serum was used, and 11 of 15 (73.3%) cattle were heavy shedders when adsorbed serum was used. Thus, about 75% of paratuberculosis-positive cattle, detected by use of the dot-elisa, were heavy shedders.

Effects on sensitivity and specificity of using various cut-off points for the various test groups were determined by use of video densitometric measurement, because sera were not discretely segregated into distinct groups of positive and negative results. Specificity of the elisa in the 2 fecal culture-negative herds was 96.2% at elisa cut-off optical density value ≥ 0.4 (OD) for adsorbed serum. For nonadsorbed serum, specificity was 78.8% at similar OD cut-off value.

Comparison of dot-elisa results determined by visual vs objective densitometric measurement indicated compatible results for test specificity; adsorption of serum with M phlei increased test specificity. Test sensitivity using visual evaluation was 66%, and was 87.5% using the objective densitometric evaluation for nonadsorbed serum at elisa cut-off OD value of 0.2. This difference was even more pronounced when adsorbed sera were used—34.1 and 82.5%, respectively.

Summary

Use of a dot-elisa with serum adsorbed with Mycobacterium phlei or with nonadsorbed serum was compared. In addition, results attained using visual observation were compared with those obtained using a densitometer. Infection status of cattle was determined by results of culture of feces from a number of cattle with various degrees of exposure (low prevalence and test-negative) and disease manifestation (clinical suspect vs subclinical infection). Two paratuberculosis-negative herds, fecal culture-confirmed clinically suspect cases of paratuberculosis, and cows from 2 paratuberculosis-infected herds with diagnosis confirmed on the farm (low infection rate) were tested.

Significant (P < 0.05) increase in the dot-elisa response was found in cattle with heavy M paratuberculosis shedding when nonadsorbed and adsorbed sera were used, compared with the response in cattle that were fecal culture-negative or were shedding M paratuberculosis at lower amounts. Paratuberculosis was diagnosed by visual determination in 29 of 44 (65.9%) of fecal culture-positive, clinically suspect cattle when nonadsorbed serum was used. Results of the visual test were negative in 85 of 93 (91.4%) of the fecal culture-negative cattle when nonadsorbed serum was used. However, when using M phlei-adsorbed serum, the sensitivity of the visual determination decreased to 34.1% (15/44), and the specificity increased to 97.8% (91/93). When taking into consideration the amount of bacterial shedding (ie, colonies per gram of feces isolated by culture of feces), 22 of 29 (75.9%) cattle were heavy shedders, with > 1,500 colonies/g of feces at the time of serologic testing when nonadsorbed serum was used, and 11 of 15 (73.3%) cattle were heavy shedders when adsorbed serum was used. Thus, about 75% of paratuberculosis-positive cattle, detected by use of the dot-elisa, were heavy shedders.

Effects on sensitivity and specificity of using various cut-off points for the various test groups were determined by use of video densitometric measurement, because sera were not discretely segregated into distinct groups of positive and negative results. Specificity of the elisa in the 2 fecal culture-negative herds was 96.2% at elisa cut-off optical density value ≥ 0.4 (OD) for adsorbed serum. For nonadsorbed serum, specificity was 78.8% at similar OD cut-off value.

Comparison of dot-elisa results determined by visual vs objective densitometric measurement indicated compatible results for test specificity; adsorption of serum with M phlei increased test specificity. Test sensitivity using visual evaluation was 66%, and was 87.5% using the objective densitometric evaluation for nonadsorbed serum at elisa cut-off OD value of 0.2. This difference was even more pronounced when adsorbed sera were used—34.1 and 82.5%, respectively.

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