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Field testing of an enhanced direct-fecal polymerase chain reaction procedure, bacterial culture of feces, and a serum enzyme-linked immunosorbent assay for detecting Mycobacterium avium subsp paratuberculosis infection in adult dairy cattle

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  • 1 Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843
  • | 2 Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843
  • | 3 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX 77843
  • | 4 Texas Veterinary Medical Diagnostic Laboratory, 1 Sippel Rd, College Station, TX 77843
  • | 5 Texas Veterinary Medical Diagnostic Laboratory, 1 Sippel Rd, College Station, TX 77843
  • | 6 Texas Veterinary Medical Diagnostic Laboratory, 1 Sippel Rd, College Station, TX 77843
  • | 7 Texas Veterinary Medical Diagnostic Laboratory, 1 Sippel Rd, College Station, TX 77843
  • | 8 Department of Animal Sciences, College of Agriculture and Life Sciences, Texas A&M University, Dallas, TX 75252

Abstract

Objective—To estimate the sensitivity (Se) and specificity (Sp) for an enhanced direct-fecal PCR procedure, bacterial culture of feces (BCF), and a serum ELISA for detecting Mycobacterium avium subsp paratuberculosis (MAP) infection in adult dairy cattle.

Sample Population—Fecal and serum samples were collected from 669 adult cattle randomly selected from a 4,000-cow dairy herd known to contain animals infected with MAP.

Procedures—Serum samples were evaluated for MAP-specific antibodies via ELISA. Fecal samples were evaluated by BCF and enhanced PCR methods (both gel-based [GB]-PCR and quantitative real-time [qRT]-PCR assays). Fecal samples also were pooled (5:1) and then subjected to GB-PCR assay. Bayesian statistical methods were used to estimate Se and Sp for each diagnostic test without knowledge concerning true MAP infection status.

Results—Adjusting for Se conditional dependence between serum ELISA and BCR, overall Se and Sp were estimated at 33.7% and 95.9%, 51.3% and 99.0%, and 32.2% and 100% for serum ELISA, qRT-PCR, and BCF, respectively.The GB-PCR assay yielded positive results for 38.3% of the pools known to contain feces from at least 1 cow that had positive GBPCR results.

Conclusions and Clinical Relevance—Estimated Se values for the serum ELISA and BCF were slightly lower than those reported elsewhere. The enhanced qRT-PCR method offered relative improvements in Se of 52% and 59% over serum ELISA and microbial culture, respectively. Pooling of fecal samples and testing with the GB-PCR assay are not recommended. Additional studies with qRT-PCR and fecal pools are required.

Abstract

Objective—To estimate the sensitivity (Se) and specificity (Sp) for an enhanced direct-fecal PCR procedure, bacterial culture of feces (BCF), and a serum ELISA for detecting Mycobacterium avium subsp paratuberculosis (MAP) infection in adult dairy cattle.

Sample Population—Fecal and serum samples were collected from 669 adult cattle randomly selected from a 4,000-cow dairy herd known to contain animals infected with MAP.

Procedures—Serum samples were evaluated for MAP-specific antibodies via ELISA. Fecal samples were evaluated by BCF and enhanced PCR methods (both gel-based [GB]-PCR and quantitative real-time [qRT]-PCR assays). Fecal samples also were pooled (5:1) and then subjected to GB-PCR assay. Bayesian statistical methods were used to estimate Se and Sp for each diagnostic test without knowledge concerning true MAP infection status.

Results—Adjusting for Se conditional dependence between serum ELISA and BCR, overall Se and Sp were estimated at 33.7% and 95.9%, 51.3% and 99.0%, and 32.2% and 100% for serum ELISA, qRT-PCR, and BCF, respectively.The GB-PCR assay yielded positive results for 38.3% of the pools known to contain feces from at least 1 cow that had positive GBPCR results.

Conclusions and Clinical Relevance—Estimated Se values for the serum ELISA and BCF were slightly lower than those reported elsewhere. The enhanced qRT-PCR method offered relative improvements in Se of 52% and 59% over serum ELISA and microbial culture, respectively. Pooling of fecal samples and testing with the GB-PCR assay are not recommended. Additional studies with qRT-PCR and fecal pools are required.

Contributor Notes

Supported in part by the USDA Animal and Plant Health Inspection Service (grant No. 03-9100-0803-GR).

Presented in part at the 86th Annual Meeting of the Conference of Research Workers in Animal Diseases, St Louis, December 2005.

The authors thank Drs. Sangeeta Khare and L. Garry Adams for technical assistance.

Address correspondence to Dr. Scott.