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Johne's
disease, Article Four, part three
Johne's Disease Diagnostic Tests ' Fecal Culture Prepared and edited by Christine Rossiter and Don Hansen From the National Johne's Working Group Culture methods Mycobacterium avium subs. paratuberculosis is the name of the microbe that causes Johne's disease. In this article it will be referred to as MAP. Culturing fecal samples for MAP has been the anchor of Johne's disease diagnosis for decades. There are two major methods currently in use:
Despite our extensive reliance on this test, conventional culture methods are not standardized and laboratories vary in their capabilities to provide the most accurate and highest quality results. Two major improvements have increased the sensitivity of conventional culture: centrifugation and double incubation. NVSL recommends that laboratories use a referenced centrifugation method versus sedimentation. Conventional culture MAP is fastidious and difficult to grow. Several steps are involved in the conventional process. Centrifugation concentrates MAP and greatly improves detection, but increases contaminating organisms as well. Samples are decontaminated to selectively minimize growth of competing organisms. Incubation with antibiotics further reduces contaminating organisms, but inhibits MAP to some extent. Typically four tubes of Harold's egg yolk agar are inoculated and incubated for 12 to16 weeks. Samples are routinely checked for MAP growth throughout the period. MAP is identified based on colony morphology, acid fast staining, slow growth rate and dependence on mycobactin J growth factor. Some laboratories do additional DNA testing to confirm positive cultures with an IS900 sequence probe that is specific for most MAP isolates. Radiometric culture (BACTEC, by Becton Dickinson) This method is semi-automated and requires sophisticated instrumentation and radioisotopes. It has similar sensitivity to culture but has the advantage of a shorter growth time of only 4-7 weeks. Growth must be confirmed by IS900 probe. It is used mostly in research facilities. Culture Results Normally these mycobacteria tend to clump together during their growth phase forming a colony unit. MAP growth on solid media is measured by counting the number of colony forming units or CFUs that grow. In order to better quantify results, NVSL recommends that each sample contain a gram of feces so that CFU counts may be reported as CFUs per gram of feces. If a standard centrifugation method is used, CFU counts range from 1-100 per tube, with 100 equivalent to TNTC (too numerous to count). Total CFUs for a standard four-tube set could range from 1 to 300, or TNTC per gram of feces processed. The lower limit of detection for fecal culture is about 10 CFU per gram of feces. NVSL recommends that labs report the number of CFUs per gram of feces, not just as positive or negative growth. Accuracy of fecal culture Recall the four stages of progression for Johne's disease. Most animals in Stages I or II will not shed enough organisms to be detected and have a false negative test result. However, fecal culture has been used for decades and the sensitivity is thought to be 30-50%. When employed in a typical herd or for the National Johne's Herd Status Program, the National Johne's Working Group expert panel recommended a sensitivity estimate of 40% for fecal cultures. The specificity of conventional fecal culture can be 100%. With a specificity of 100%, a hallmark advantage to fecal culture is that a positive result indicates a true infection. A 100% specificity assumes that sample collection and procedures have been performed properly. Cross contamination of fecal samples can occur and strict precautions should be followed during handling and processing. This also assumes that "once infected, always infected" and that a simple mycobacterial pass through episode has not occurred. Although, Whitlock cultured MAP from feces of cattle 24 hours after feeding them manure from a high level (TNTC) shedder, the extent to which this pass through phenomenon occurs has not been determined. It may be possible in heavily contaminated environments but such high-risk environments also imply that many animals are already infected and are shedding high numbers of microbes. A more complete history and risk assessment would be required to determine the actual plausibility and occurrence of pass through events in a specific herd. Pass through could effect the interpretation of culture results for that herd. Fecal culture tends to detect infection earlier than serological tests, i.e., in Stage II. The majority of low fecal shedders will not be detected by the Johne's ELISA. Fecal culture will detect infection in immature animals who have unusually advanced infection and who may be shedding MAP at 12-24 months of age. However its routine use in immature animals is not recommended unless specific circumstances dictate need for maximum early detection. The influence of prevalence on interpretation of results With a specificity of 100% there is little doubt about interpreting a positive result. The chance that a positive culture is correct is nearly always 100% regardless of disease prevalence. The predictive value for a positive test is always 100% when properly performed. The influence of prevalence on the confidence practitioners can have in an individual negative fecal culture result is similar to the ELISA. In a low prevalence herd or situation (±1%), the chance that a negative culture indicates non-infection in a mature animal is 99.7%. In a high prevalence situation, i.e., 30%, the chance that a negative culture indicates non-infection in a mature animal drops to 79%. Use of fecal culture Of all currently available tests, culture provides the most accurate, definitive, and quantitative information about the status of an individual or herd. For this reason it has the highest value for some uses: 2. Confirmation of infection in ELISA positive cattle 3. Aggressive detection of infected cattle, especially if used with serology 4. Quantify shedding status of individual cattle or groups Occasionally a truly infected animal with a positive ELISA result will have a negative result on a single fecal culture test. With time repeat cultures will detect the infection and have positive results. Three disadvantages likely preclude high volume use of culture in Johne's control strategies. High volume culturing would require laboratories to make substantial investments in equipment, space, and technical time that are well beyond their existing capacities or resources. Culture requires 12-16 weeks to complete and bars its use when information is needed quickly. Conventional culture can be expensive to use for sampling large numbers of animals. Across the U.S. costs range from $7 to $25 per sample An advantage to culture is that fecal shedding can be detected early in Stage II, usually at low levels (10 CFU/gram). Colony counts will increase in animals that progress to Stage III and IV and eventually will be TNTC. Assessing disease Stage is another advantage of fecal culture. Animals with TNTC results have disseminated infection, are progressing toward clinical disease, and are shedding high levels of mycobacteria (103 to 108 organisms per gram of feces)! High CFU counts also correlate with high risk that MAP will be secreted in milk and colostrum or infect the fetus. TNTC results are often reported by 8 weeks of culture, yet these cattle have often already been removed for clinical disease. Cattle that progress more slowly can have TNTC results on repeated fecal samples. In contrast, low shedders (i.e. 1-10 CFU per gram) appear to have intermittent shedding patterns and may not be positive when cultured a second or third time. Long-term follow-up studies by Whitlock suggest that 30% of low shedders progress to Stage III or IV in 2-4 years. Thus, culture results with standardized colony counts can be useful to differentiate animals in advanced compared to early infection. This can help prioritize animal removal or management decisions when eradication is not the immediate goal. Low-level fecal shedders may be candidates for management or monitoring whereas high shedders are candidates for immediate removal or separation.
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