Dr. Bill Shulaw, Extension Beef and Sheep Veterinarian, The Ohio State University
Since the middle of last summer, a new diagnostic process for Johne's disease fecal cultures has been in place at the Animal Disease Diagnostic Laboratory at the Ohio Department of Agriculture in Reynoldsburg. At the center of this process is the Trek ESP liquid culture system.
In contrast to the older system of culture on solid media, this system utilizes a liquid media, or "broth" as it is sometimes called, in which to grow the causative bacteria - Mycobacterium avium subspecies paratuberculosis (MAP). Following standard fecal sample preparation procedures, a small quantity of the processed sample is placed in a sealed vial of the liquid media along with specific growth supplements and certain antibiotics to control non-specific bacterial and fungal growth. The vial is then placed into a specialized incubator and connected to sensors that monitor changes in pressure which signal growth inside the vial. Several of these incubators are connected to a computer that monitors the changes in each vial several times each hour. These data are stored in the computer and continually matched to a preprogrammed formula that mimics the typical growth of MAP. When the match is close enough, the computer signals the microbiologist that the sample is positive.
Liquid from the positive vials is stained to look for typical organisms and stain-positive specimens are then subjected to a procedure called PCR which stands for polymerase chain reaction. This reaction tests for the presence of DNA specific for MAP. In fact, two different PCR reactions are used to be sure positive samples really contain MAP. Although samples may be positive as soon as 10 to 14 days, the vials which are not identified as positive by the computer are incubated for a total of 42 days. At the end of this time, they are also stained to look for the typical bacteria. If they are seen, the sample is tested by PCR in the same way. This is important, because not all positive fecal specimens will be detected without this step. The final accounting and reporting are complete about eight weeks after the process begins. This is a significant improvement in the incubation time required for the older solid media cultures which were incubated for a total of 16 weeks before the final report was issued.
Perhaps, the most significant advantage of the liquid media-based system over that of the solid media method is its apparent overall increase in positive fecal samples. An exhaustive comparison of this system with the solid media method has not been done, but research has suggested that the improvement should be at least 40 to 50% and possibly considerably more depending on herd history and specific situations. This is similar to the experience of other laboratories using this system and research done in Ohio with other liquid culture systems. Although not all the factors are known, it is likely that the liquid supports the growth of MAP better than the solid media.
For producers, the increase in sensitivity may be a "good news, bad news" situation. The good news is that more cows shedding MAP in their manure will be detected, especially those shedding low numbers. For producers attempting to eliminate the infection, more rapid progress can be made. In addition, we can have increased confidence in the status of "test-negative" herds that have been cultured. The possibility of detecting animals shedding low numbers of MAP in pooled samples from several animals is improved, thus facilitating some kinds of testing strategies. The "bad news" is that for some producers, it may appear that their control efforts are going in the wrong direction. For example, a producer annually testing 100 cows who has typically found 10 test-positive animals may see 15 positive animals on the next test using the new culture method, assuming their situation stayed the same. A similar increase in sensitivity of culture occurred a few years ago when the laboratory adopted an improved sample preparation technique that resulted in more MAP being available to inoculate on the media.
An additional advantage of this liquid culture-based method is that it reduces labor by eliminating the need to pull thousands of solid media tubes out of walk-in incubators and visually inspect them for growth. This is partially offset by the need to routinely stain all liquid cultures and perform the PCR tests on positive samples. However, the net effect of this method, along with some changes in laboratory procedures, is to allow a modest increase in the number of samples that can be processed each week.
The biggest disadvantage of the liquid-based culture method is cost. The supplies and equipment needed to perform the cultures are somewhat more expensive than the solid media and the supplies to conduct the confirmatory PCR tests are also fairly expensive. This means that in times of tight state and federal budgets, it will become increasingly difficult to provide the high level of testing support currently enjoyed by Ohio's producers.
For years, producers and veterinarians have asked for better tests for Johne's disease. Science continues to provide incremental improvements, and tests will continue to get better as has fecal culture. Will we ever have a test that will positively identify an infected heifer at six months of age with a single test? Although this is highly desirable, the answer is, "Probably not." The biology of this disease and the nature of the animal's response to the infection suggest that this will be very difficult. Consider the situation with human tuberculosis caused by the related bacteria, Mycobacterium tuberculosis. This disease affects about one-third of the world's population, and someone is newly infected every second of every day. One person will develop active tuberculosis every three seconds. The diagnostic screening tests for human tuberculosis are still a skin test and a chest x-ray, and culture of the patient's respiratory secretions remains the confirmatory test. No satisfactory vaccine for human tuberculosis exists. Although our tests for Johne's disease get better nearly every year, and this newest method of culture is very good, testing and culling alone will never be enough to control or eradicate the disease.