Diagnosis and Control of Johne's Disease (2003) / Chapter Skim
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3. Diagnostics
3. Diagnostics
Pages 45-65
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From page 45... ...
Despite considerable research effort, all methods are fraught with difficulties that have impeded the control and eradication of JD. Diagnostic test interpretation and evaluation are important subjects that are discussed in greater detail in Appendix A
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From page 46... ...
At the herd level, tests will tend to perform better as the proportion of individuals in more advanced stages of disease increases. For control programs, it is important to make this distinction between test performance at the individual animal level and test performance at the herd level.
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From page 47... ...
Table 3-3. Utility of Diagnostic Tests in Clinical Stages of Johne's Disease Test Stage I Stage II Stages III, IV Pathologic lesions Generally useful Generally useful Useful Signs of disease n/a n/a Very useful Fecal culture n/a Very useful Extremely useful PCR Limited utility Useful, depending on Useful clinical progression Acid-fast bacilli n/a n/a Useful, depending fecal smear on clinical progression IFN-y Limited utility Generally useful, Useful depending on clinical progression Serology Generally n/a Generally useful, Useful depending on clinical progression Notes: N/A: not applicable PCR: polymerase chain reaction IFN-~: gamma interferon
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From page 48... ...
and because of laboratory errors, such as sample misidentification or cross-contamination. Because of the relatively low sensitivity of conventional fecal culture in subclinically infected animals, and because Map grows very slowly on artificial media, much has been done to improve performance of the procedure and, more recently, to develop hybrid test procedures (e.g., Collins et al., 1990b; Secott et al., 1999~.
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A variety of methods and parameters have been evaluated over the years to improve the sensitivity of Map detection by culture. The following discussion briefly highlights the most common methods and techniques for isolating Map from clinical specimens.
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A molecular based confirmatory test, such as PCR to detect the Map marker sequence IS900, can be used to confirm positive specimens. Automated Culture Radiometric Systems The radiometric system in greatest current use is the BACTEC system (Becton Dickinson Laboratories, Sparks, Maryland, USA)
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From page 51... ...
Using individual fecal culture as the gold standard, the authors reported a sensitivity of 86 percent and specificity of 96 percent higher than expected. The authors concluded that pooling of the samples and using fecal culture influenced diagnostic sensitivity and that strategic pooling of samples would considerably reduce testing costs when a herd is not suspected of being infected.
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From page 52... ...
evaluated five conventional and radiometric culture methods. The authors reported that the Whitlock decontamination to BACTEC medium was most sensitive for detecting shedder cattle, followed in order by Whitlock decontamination to HEYM, modified Whitlock decontamination to BACTEC medium, conventional decontamination with sedimentation to HEYM, and conventional decontamination and filtration to BACTEC medium (Eamens et al., 20004.
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From page 53... ...
(Manning and Collins, 2001~. Lymphocyte Blastogenesis and Transformation Lymphocyte blastogenesis transformation is a relatively complex in vitro bioassay that uses the antigen Johnin PPD to stimulate lymphocytes present in fresh bovine whole blood co-incubated with 12 I-5-iodo-2'deoxyuridine (Buergelt et al., 1977, 1978a)
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Serologic Tests to Detect the Humoral Response Serologic tests for Map are most useful in establishing the herd prevalence of infection, for presumptive identification of infected animals, and for confirming the diagnosis of ID in animals that demonstrate compatible clinical signs (Nielsen et al., 2001~. A variety of tests can detect humoral antibodies to Map in bovine serum, but because only the agar gel immunodiffusion (AGID)
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From page 55... ...
was developed after CFT as a quick test for animals that were showing clinical signs of JD (Sherman et al., 1984, 1989~. Positive test results correlate well with clinical signs of JD, but failure to detect subclinically infected animals is a major drawback.
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From page 56... ...
Ranking of the values obtained can be used to determine the likelihood ratio that cattle are infected with Map or are fecal shedders (Collins, 1996; Nielsen et al., 2001; Spangler et al., 1992~. Test Result Measures Fixed-Decision Thresholds or Cutoffs For serologic tests with a continuous result, such as conventional ELISA, a positive test is one that yields a signal of sufficient intensity (optical density)
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From page 57... ...
One approach to reducing the effect of procedure variability is to run replicates of each specimen or, to minimize total testing costs, to repeat those tests that yield results within a given range of the cutoff value. Information Cost and Degree of Result Categorization When a result from a laboratory procedure that is inherently continuous, such as a colony count or an analyte concentration, is classified into one of a few categories (positive, suspect, negative)
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From page 58... ...
Little work has been published that relates serologic test results to infection states and to the associated production and health; however, because of their rapid turnaround, the use of such tests is increasing. Likelihood Ratios and ROC Curves Likelihood ratios that correspond to specific test results are a useful approach for reporting test procedures that yield a continuous quantitative result, such as optical density from conventional-format ELISA (Radack et al., 1986~.
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Although not directly applicable to establishing ROC curves for laboratory tests that yield continuous results, Obuchowski (2000) provides tables containing some smaller sample sizes as preliminary estimates.
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Studies of mycobacterial strain collections showed the presence of multiple copies of IS900 in all isolates of Map and in no other mycobacterial species analyzed. However, recent studies have shown that insertion sequences similar to IS900 exist and can yield falsepositive test results if stringent procedures are not followed (Cousins et al., 1999)
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avium isolates derived from human clinical sources and reported positive amplification reactions in 15 of 28 isolates. The results were confirmed by hybridizing the PCR reaction products with a labeled plasmid probe derived from Map.
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EPIDEMIOLOGIC TOOLS Test Protocol Standardization Because of the relatively poor epidemiologic sensitivity and specificity of current laboratory tests for subclinical infections, considerable research is focused on improving test performance. Some have proposed methods for optimizing conventional Map culture of bovine-origin specimens (Whipple et al., 1991; Whitlock and Rosenberger, 1990)
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From page 63... ...
Laboratory Proficiency Evaluation The variability in ELISA test performance makes laboratory proficiency testing and quality control all the more important for JD control programs, but commercial test design can run counter to this goal. In a study involving eight laboratories using an earlier version of this ELISA kit, the within-plate coefficient of variation averaged 7 percent, but it ranged from 5 percent to 29 percent across laboratories.
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From page 64... ...
The result will be an increase in confidence in JD test results. At the request of the Laboratory Certification Subcommittee of the Johne's Disease Committee of the United States Animal Health Association, in 1996 NVSL began an annual check testing program for veterinary diagnostic laboratories to become approved for ID fecal culture or serology of bovineorigin specimens (Whitlock et al., 2000a)
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From page 65... ...
voluntary cattle herd status program recommends that all tests be performed in an approved laboratory (USAHA, 1998~. No such check testing program exists for veterinary diagnostic laboratories that test JD specimens originating from other ruminant livestock species.
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