Herds that are "culture negative" on all samples are eligible for certification at "Status Level 1" if they also meet the other criteria outlined in the PA Johne's Herd Certification User's Guide. Herds that have one or more culture positive samples can decide, in consultation with their veterinarian, if further culture of animals is warranted to determine which animals in the herd are infected.
A total of six composite samples should be collected from areas where a large proportion of the mature cow herd is commingled. These should consist of two samples from each of the following areas:
- mature cow housing areas (alleyways, crossovers, gutters)
- manure storage areas (lagoons, piles, pits, or manure spreader) and
- manure concentration and mixing areas (eg. hospital pens, travel lanes, holding pen)
Proper collection of the samples is crucial to ensure validity of the results. The objective of environmental sampling is to collect composite samples which represent the whole group of animals being tested, and to maximize the probability of finding MAP organisms if they exist in the herd. Therefore, whenever possible mixed manure should be collected rather than individual cow samples. Each composite environmental sample should contain approximately 50g of fecal matter collected by means of at least six (preferably more) different 'grab samples' within each sampling location. (Each grab sample should consist of mixed manure rather than manure from an individual.)
In freestall barns and travel lanes, crossovers (Figure 1), areas around waterers, alley corners, and ends of scrape lanes are excellent places to collect well-mingled manure grab samples. Figure 2 demonstrates how two composite samples could be assembled from a freestall housing facility, with each composite consisting of 8 grab samples from the floor. Grab samples 1 through 8 would be placed into a pail, rectal sleeve, or other container and thoroughly mixed.
Figure 1. Sampling sites at crossover.
Figure 2. Collection of 2 composite environmental samples.
Approximately 50 g of this composite sample would then be placed into a container for submission to the laboratory. A new or cleaned container would then be used to collect the next set of grab samples.
In tie stall barns, the grab samples used to assemble the composite environmental samples should be obtained from all gutters containing manure from cows 24 months of age and older. Other suitable locations for grab sampling are places where the gutter cleaner turns a corner, exits the barn, or goes up a ramp to a manure spreader or manure stack. (Of course, other mature cow housing, holding, or travel areas can also be sampled.) All samples must contain a high proportion of fecal matter - samples with excessive bedding content should be avoided.
Manure lagoons and storage areas should be sampled (using personal precautions for safety!) from at least four locations around the perimeter by submersing a sampling device up to 10 cm below the surface. Samples may also be collected using three 4 x 4 gauze squares tied to fishing line with fishing weight and soaked at least 10 cm below the manure's surface. Samples from stacked manure piles should be obtained from at least four different sites up to 10 cm beneath the surface. Avoid sampling manure that has obviously been heated or composted. Samples may be collected from a manure spreader if the manure is fresh (ie. not dried and exposed to sun/heat for any period of time).
Collecting samples from bedded pens and packs can present challenges depending on the relative amounts of manure and bedding in the pens. Samples should also represent as many animals as possible - therefore a composite sample should not be made up of manure from only one or two animals. If a pen is regularly and thoroughly cleaned, it may not be appropriate to attempt to gather a valid sample from that area - instead an additional sample should be collected from other housing areas. The samples must also contain a high proportion of fecal matter - samples with excessive bedding content should be avoided. The top layer of bedding may need to be pushed aside to uncover enough manure for sampling, and grab samples can often be obtained in the pen corners or from areas around waterers. Excessively heated or composted manure should be avoided.
All samples should be labeled with the sampling location and herd identifier, and transported to the laboratory on ice. A properly completed AAI-11 form should accompany the samples, with the samples being clearly identified as being "environmental" in origin. For example, "Environmental Sample #1 - Freestalls" and "Environmental Sample #2 - Freestalls" could be written in the 'Animal Name' column for the samples collected from the mature cow housing area shown in Figure 2. Samples that are not in proper containers or are otherwise inappropriately submitted will be discarded!
If you have any questions regarding the use, appropriate implementation, or interpretation of environmental cultures in the PA Johne's Disease Herd Certification Process please contact Dr. Ernest Hovingh (814-863-8526, firstname.lastname@example.org).