Part I: Definitions

Accredited Veterinarian:   A veterinarian approved by the Deputy Administrator of USDA/APHIS/VS to perform functions required by State-Federal-Industry cooperative programs. For the Nebraska Voluntary Johne's Disease Control Program, the accredited veterinarian must have completed the required Johne's training, and his/her duties include annual herd visits, animal testing, and producer education. All samples for program testing must be collected by an accredited veterinarian or State or Federal animal health official.

Approved Laboratory:   A laboratory that has passed an annual check test for Johne's disease administered by the National Veterinary Services Laboratories. All program testing must be conducted by a laboratory approved for the specific test being used.

Anniversary Date:   The date of the initial herd test for entry into the program. Once established, the anniversary date remains the same, regardless of subsequent herd tests.

Biosecurity:   Animal husbandry and hygiene practices designed to limit opportunities for exposure to M. paratuberculosis.

Commingling:   Physical contact or exposure to manure or raw milk of susceptible species. For example, all cattle and other susceptible species grazed together or on the same area of a property or farm at any time during any 12-month period, are considered to be commingled. Susceptible species include domestic and exotic ruminants such as sheep, goats, cervids, and camelids. Exposure to manure via contaminated water or feed sources is also considered commingling.

Designated Johne's Coordinator (DJC):   A person who has demonstrated the knowledge and ability to perform the functions required under these program standards and who is responsible for implementing the Nebraska Voluntary Johne's Disease Control Program.

ELISA:   Enzyme-linked immunosorbent assay.

Exposure:   Contact with known infected animals; contact with the manure or raw milk of infected or exposed animals of susceptible species; or contact with infected herds via contaminated water or feed sources.

Herd:   A group of cattle managed as a separate and discrete unit not commingled with other groups of susceptible species. All cattle on two or more premises geographically separated but on which cattle have been interchanged or where there has been contact between the premises is considered one herd. Contact of animals between separated premises under common management is assumed to have occurred unless complete separation and biosecurity measures between premises can be established by the herd owner or manager. The DJC will make the final determination of the herd test-negative level of a group of animals.

Herd Management Plan:   A written plan, produced by the Johne's certified veterinarian in conjunction with the producer, which includes animal husbandry and hygiene practices specific to that herd and that is designed to limit opportunities for exposure to Mycobacterium avium subsp. Paratuberculosis. This includes preventing the introduction of JD into the herd and reducing transmission of the disease among animals within the herd. A copy of the Herd Management Plan must be submitted to the DJC for review and final approval.

Herd member:   Any susceptible species of animal that is commingled with the herd.

Herd Risk Assessment:   Before developing an individual Herd Management Plan, a Johne's Certified Veterinarian must conduct a risk assessment to identify aspects of management of all production areas likely to introduce or spread JD infection throughout the herd. This also includes estimation of the risk of JD infection based on herd history, known infection status, and biosecurity concerns. A copy of the risk assessment must be submitted to the DJC with the Herd Management Plan. Risk Assessments are required on years 1, 2, 4, 6, and 10 of program participation.

Herd Test-Negative Levels:   Herds may achieve test-negative levels of 1, 2, 3, or 4. Each level of increase indicates higher confidence in the Johne's disease free status of the herd.

Infected Animal:   An animal that has been confirmed by an official Johne's disease test to be infected with Mycobacterium avium subsp. paratuberculosis.

Johne's Certified Veterinarian:   An accredited veterinarian who has completed training approved by the Nebraska DJC for Johne's disease epidemiology, testing, test interpretation, herd risk assessments, and development of herd management plans, as well as State and Federal Program requirements. Certified Veterinarians will be required to take a JD refresher course approved by the DJC at least once every three years.

Johne's Disease (JD):   Intestinal bacterial disease caused by Mycobacterium avium subsp. paratuberculosis. Clinical signs, which appear after a long incubation period, include long-lasting or chronic diarrhea and weight loss despite a good appetite.

Level Achievement Year:   The year a herd in the testing and classification element of the program obtained it current classification. Level achievement year is noted because continued monitoring increases confidence the herd is not infected.

Management Herd:   A herd that has completed a risk assessment and herd management plan that satisfies the requirements of the DJC but has not completed the requirements for the Test-Negative Levels or test-positive management.

National Program Coordinator:   A USDA staff veterinarian who will assist state Johne's epidemiologists, State Johne's Advisory Committees, and the USAHA Johne's Committee with the administration and review of the Voluntary Johne's Disease Herd Control Program.

Official Identification:   An official eartag or official identification device or method as identified in 9 CFR and approved by APHIS as being tamper resistant and providing unique identification for each animal. An official identification may conform to the alphanumeric National Uniform Eartagging System, or it may bear the valid premises identification used in conjunction with the producer's livestock production numbering system to provide a unique identification number.

Official Johne's Disease Test:   An organism-detection test approved by the Administrator and conducted in a laboratory approved by the Administrator. The Administrator approves laboratories to conduct an official Johne's disease test only after determining that the laboratory meets the check-test proficiency requirements prescribed by NVSL. Approval continues as long as such check-test proficiency requirements are met on an annual basis.

Premises Identification Number:   A unique number assigned by the State animal health official to a livestock production unit that is, in the judgment of the State animal health official and AVIC, epidemiologically distinct from other livestock production units.

Program:   Voluntary Bovine Johne's Disease Control Program (VBJDCP).

Random Sample:   Thirty (30) animals (or all if herd size is less than 30) consisting of cows over three (3) years old and bulls over two (2) years old selected to be representative of the herd in regard to age and sex.

Screening Test:   A Johne's disease test approved by the Administrator for use in the Program and conducted in a laboratory validated through an approval process by NVSL. Screening tests are tools that have been developed to aid in determining the presence or absence of M. avium supsp. paratuberculosis within a herd. Animals found positive to these tests should be considered suspect unless they show clinical signs of Johne's disease (in which case they are considered positive) or they are confirmed positive or negative by an official JD test.

State Johne's Disease Advisory Committee:   An appointed committee to assist in the oversight and coordination of the State's Voluntary Johne's Disease Control Program. The committee may be comprised of any or all of the following:

• Dairy producers - purebred, commercial, and commodity groups;
• Beef producers - purebred, commercial, and commodity groups;
• University extension - beef and dairy;
• Diagnostic Laboratory - Director and personnel;
• Veterinary practitioners - beef and dairy; and
• Regulatory veterinary medicine - state, federal, and/or field services.

Statistical Subset:   An ELISA test on or fecal culture of a statistically determined number of animals. The following table shows the number of animals to include in Program ELISA and fecal culture statistical subset testing:

No. Cows in Herd >3 Years Old and Bulls >2 Years Old	Number of Animals to Sample
	ELISA	Fecal Culture
< = 300	Test all	Test all
301 - 400	Test all	Test all up to 313
401 - 500	Test all	324
501 - 600	Test all up to 531	332
601 - 700	540	338
701 - 800	547	342
801 - 900	552	345
> 901	580	360

In smaller herds, all cattle three years or older must be tested. In herds with fewer than 30 animals 36 months old or older, animals 24 months and older must also be tested.

Sample numbers above are based on the following assumptions:

• The cattle to be tested are in second or higher lactation.
• ELISA tests are assumed to have 25 percent sensitivity and fecal cultures are assumed to have 40 percent sensitivity. (These were consensus estimates of the Herd Status Committee of the National Johne's Working Group of the United States Animal Health Association Committee on JD for cows with subclinical infections in first or higher lactation, and no changes were made for older populations sampled.)
• ELISA and fecal culture are assumed to have 100 percent test specificity (given follow-up of all ELISA positives with fecal culture).
• The confidence of detecting infection (at least one test-positive cow), if present at a true prevalence of two percent, is 95 percent.
• The calculations are based on sampling without replacement.

Test-Negative Level:   Level 1, 2, 3, or 4 with each increase indicating a lower probability of Johne's disease in the herd.

Test-Positive Herd:   A herd that is enrolled in the program and meets the test- positive component requirements described in this document.

USDA:   United States Department of Agriculture

Veterinary Services (VS):   The division of APHIS in charge of animal health activities within the United States. Part II: Requirements for Entry into the Program

A.   Herds enrolling in the herd testing and classification element must have completed a Herd Risk Assessment and developed a Herd Management Plan using the guidelines outlined in the NJWG Handbook for Veterinarians and Beef and Dairy producers, and the "How to Do Risk Assessments and Management Plans for Johne's Disease" 2003.

B.   Criteria for Herd Qualified to Enter the Program

1. The herd has been in existence for at least one year; or the herd was assembled with cattle originating directly from test-negative level certified herds only.

2. A herd assembled with cattle originating directly from certified herds only, shall start at the lowest certification level of the herds from which the assembled cattle were acquired. A negative first test will qualify the newly assembled herd for the next certification level.

1. All animals must have permanent identification other than a plastic eartag or neck chain. All cattle must be individually identified using an identification method approved by the State Johne's group. It is recommended that all animals in participating herds should be individually identified using an official identification. Any previous regulations listed in 9 CFR regarding animal identification with other animal health programs still apply.

2. Acceptable means of permanent identification are registration or association numbers accompanied by identification document, ear tattoos, USDA uniform series eartag (metal tags), and electronic tag or microchip identification.

These measures should be in place to reduce exposure to manure or milk from cattle of unknown JD status. Care is needed to prevent exposure to other susceptible species (e.g., sheep, goats, farmed deer, camelids, non-program cattle). The herd management plan should include the following biosecurity measures:

1. Ensure that animals added to the herd come only from low-risk or known test-negative level herds and from known sources (do not purchase from sale yards). Record the source and manage additions as higher risk animals unless you have evidence to the contrary.

2. Minimize exposure of young stock to manure from adult animals, including other susceptible species. How the exposure is minimized will vary depending on management of the cattle located on the premises.

3. Minimize exposure of livestock to susceptible animals that are infected or have been exposed to infected animals.

4. Never feed calves unknown sources of colostrum. Never feed calves unknown sources of milk unless it is pasteurized.

5. Minimize exposure of feed, water, equipment, and vehicles to manure.

1. Keep maternity area clean and dry and separate from other adult animals.

2. Immediately separate each newborn calf from adult animals.

3. Provide colostrums from a single identified cow; do not use pooled colostrum.

4. Feed each calf colostrum from a test-negative or healthy low-risk animal.

5. Feed calves milk replacer or pasteurized milk.

6. Keep calves and heifers free from exposure to the manure of mature cattle and house by age and separate from older animals.

7. Separate clinical suspects from maternity and young stock. Record all clinical suspects.

For beef herds, the herd management plan should include the following biosecurity measures:

1. Keep calving areas as clean and dry as possible.

2. Minimize the density of cow and calf pairs as much as possible.

3. Use feeding practices that reduce manure contamination of feed and feeding areas as much as possible.

4. Provide colostrum from the calf's dam or from another single source that is from a test-negative or healthy low-risk animal.

5. Raise weaned replacements, separated from older animals.

To continue in the program, a herd owner and Johne's certified veterinarian must annually repeat the risk assessment and make appropriate changes to the herd management plan. The updated risk assessment and herd management plan must be submitted to the DJC.

H.   Previously Infected Herds Herds previously culture positive for M. paratuberculosis may enter the program by completing Standard Track entry requirements. Infected (positive on an organism-detection test) and/or test positive (positive on any Johne's test) animals must be removed from the herd or test negative on approved fecal culture test before program entry. These same animals must be included in each yearly herd test sample for a minimum of three years if they are retained in the herd.

I.   Herds Tested Negative Prior to Implementation of this Program

Herds tested negative for Johne's disease prior to implementing this program may be entered at Standard Track level 1 or Fast Track level 2 determined after a review by the DJC. The review must include:

1. Verification that the herd meets the minimum standards for testing (fecal culture results can be used in place of ELISA results). Future program tests must be performed in accredited laboratories.

2. Risk assessment of the number, source, and testing history of herd additions made after the first qualifying test.

3. Previously tested animals identified by other than official means (see C.2 above) will be accepted if owner provides a list of official identification corresponding to the ID listed on test chart.

Initial testing is required to determine the herd's test-negative level, using either a screening test or an official JD test on a minimum of 30 randomly selected animals at 36 months or older. In herds with fewer than 30 animals 36 months old or older, animals 24 months old and older must also be included until 30 animals are tested or until all animals have been tested. Herd owners should be encouraged to test statistical subsets (see Table 1 - Sample Sizes for Subset Testing) or greater numbers of cattle when possible.

All samples must be collected by, or under, the supervision of an accredited veterinarian or a state or federal animal health official. Vaccinated herds are eligible (no vaccine is approved for use in Nebraska). Vaccinated herds must be tested by an organism- detection test. All samples from herds applying for herd classification must be submitted to a laboratory approved by NVSL.

Part III: Program Protocol

A.   Test-Positive Component

The purpose of the test-positive component is for the State to assist producers with approved JD control risk assessment and herd management plans in place. Herds enrolled in this component must use testing protocols approved by the DJC.

Requirements for herds in the test-positive component

1. Application - The herd owner enrolling the herd must sign an agreement to abide by the requirements concerning minimum biosecurity and management, identification, testing, and herd addition strategies. This agreement must be renewed every 10 to 14 months from the anniversary date.

2. Animal Identification - All cattle must be individually identified using an identification method approved by the State Johne's group. Any previous regulations listed in 9 CFR regarding animal identification with other animal health programs still apply.

3. Herd Additions - All purchased animals should be from herds with approved herd management plans. Heifers raised off the premises should be raised with at least the minimum biosecurity and control measures in place.

4. Testing - The herd owners along with the Johne's certified veterinarian must develop a testing protocol as follows:

a. All samples must be collected by, or under, the supervision of an accredited veterinarian or a State or Federal animal health official.

b. Vaccinated animals (no vaccine is approved for use in Nebraska) must be tested using an official JD test.

c. All samples must be submitted to the Nebraska Veterinary Diagnostic Center.

d. All animals specified in the test protocol must be tested within 10 to 14 months of the anniversary date or may split the testing in the 12-month period following the anniversary date.

e. Herds not adhering to the prescribed testing requirements will be placed in the management element.

f. It is recommended that animals with positive results on an official JD test be identified as infected and restricted to the premises. When infected cattle leave the herd, it is recommended that they go directly to slaughter or rendering.

g. A test-positive animal may be appealed using the same appeal process described below for entry into the herd testing and classification element. If an animal is removed from the herd while screening test results are pending, a fecal culture should be collected, submitted, and held at the laboratory. This will allow the owner to appeal the herd level if the animal tests positive to a screening test. Final classification of the animal and herd will be made by the DJC.

The test-negative component must include a herd management plan based on the requirements in the management element. The test-negative component includes a series of levels; owners may improve their herd test-negative level (achieve a higher level) by additional testing and biosecurity measures. Each higher level represents a greater probability that the herd is free of JD. However, this does not certify that a herd is free of JD. Herds in the test-negative component may remain at any given level by doing monitoring testing or may advance to a greater surveillance level with additional testing. Requirements for herds in the test-negative component include:

1. Application

The herd owner enrolling the herd must sign an agreement to abide by the requirements concerning minimum biosecurity and management established in the management element as well as the identification, testing, and herd addition requirements listed below.

2. Herd Advancement - Test negative

Test-negative herds may achieve Level 1, 2, 3, or 4. Each higher level represents a greater probability that the herd is free from JD; however, a higher level does not certify that a herd is free of JD. The level achievement year should also be indicated. For example, a herd completing Level 2 testing in 2001 and electing to remain at Level 2 in 2002 would have a Level 2 2001 test-negative level. The level achievement year should be noted because continued monitoring increases confidence the herd is not infected. To advance from one level to the next, a statistical subset must be tested (Table 1 - Sample Sizes for Subset Testing), and the herd must meet the level requirements listed below for standard track or fast track test-negative component levels. Test-negative level herds should get an official test on clinical suspects when they are culled.

a. Standard Track - The standard track is designed to allow entry into the program with a minimal investment of funds and gradually increase the producer's investment in the program. The standard track will require at least three years and four tests to reach Level 4.

Level 1

A. Negative serum ELISA results on 30 randomly selected animals 36 months of age and older. In herds with fewer than 30 animals 36 months of age or older, animals 24 months of age or older must also be tested;

OR

B. Environmental Sampling - Dairy Farms

From each dairy farm, collect two composite environmental samples tested by an official JD test from each of the following locations on the farm: manure concentration areas (cow housing alleyways or gutters), manure storage areas (lagoons, piles, pits, or manure spreader), and another manure concentration area (sick cow pens or other cow alleyways and travel-ways). A total of six samples should be collected for submission to the diagnostic laboratory.

Each composite environmental sample should contain approximately 20 g of fecal material with bedding or soil from a minimum of four different sites within each sampling location. Collect each sample with a separate disposable latex glove, place in a 95-mL plastic-covered specimen container labeled with the sampling location and herd identifier, and store in a cooler with ice during transport to the laboratory. Be sure to include sufficient manure in the sample when sampling from well-bedded areas such as sick cow pens or loose-housing lots.

In free stall barns, obtain each cow alleyway sample from a minimum of four sites across cow alleyways, collected to represent most of the mature cows (24-36 months of age and older) within the farm. Crossovers (areas around waterers, corners, and ends of scrape lanes) are excellent places to collect well-mingled manure subsamples.

In tie stall barns, obtain samples from all gutters containing manure from mature cows (24-36 months of age and older). Other suitable locations for subsampling are places where the gutter cleaner exits the barn, along the ramp, and in corners.

Sample manure storage lagoons (using personal precautions for safety) from four locations at the perimeter of the lagoon by submerging the sampling container up to 10 cm beneath the water's surface.

Each composite sample from manure piles should be obtained from four different sites up to 10 cm beneath the surface.

Each composite sample from manure pits can be collected using two to three sterile 4 x 4 gauze pads tied to fishing line with fishing weight and soaked at least 10 cm below the manure's surface.

(Modified from Raizman, Wells, et al., 2004. J. Dairy Sci. 87:2959 2966.)

OR

C. Pooled fecal cultures

Test individual fecal samples tested by an official JD test of 50 randomly selected animals, 36 months of age or older, in 10 pools from 5 animals. Submit individual samples to laboratories approved for fecal pooling.

Herds of fewer then 50 adult animals can still use fecal pooling. All animals 24 months or older are to be included until a total of 50 animals has been reached or all 24- month-old animals have been tested. At least 3 pools of 5 (15 animals) should be available to use pooling. If 15 animals are not available, individual samples should be used.

(2) Level 2 - Herds have met the requirements for Level 1 and have had negative screening tests on a statistical subset (see definition) of animals 36 months or older. Level 2 testing must be completed within 10 to 14 months of the anniversary date.

(3) Level 3 - Herds have met the requirements for Level 2 and have had negative official JD test results on fecal samples on a statistical subset (see definition) of 36 months or older herd members. Bulls 24 months of age and older must be included in this testing. The official JD test on fecal samples must be collected within 10 to 14 months of the anniversary date.

OR

B. Pooled fecal samples tested by an official JD test from a statistical subset of animals (see Appendix 1, Sampling sizes for statistical subset testing) 36 months or older may be used to advance a herd from Level 2 of test-negative program to Level 3 of the test-negative program provided that at least one negative whole herd test has been conducted by screening tests or official Johne's disease test on individual samples. Individual samples must be submitted to the laboratories approved for fecal pooling. Each pool will contain no more than five individual animals and the pooling of samples will be done according to the pooling methods documented for Level 1 of the test negative component at the VBJDCP. Bulls 24 months of age or older must be included in this testing as well.

In addition to the pooled fecal cultures taken from the statistical subset of animals, six composite environmental samples also must be taken according to the protocol described for dairy herds for entrance to Level 1.

For beef herds, environmental samples should be taken from areas around feed bunks, waterers and other animal collection points. Any pool of samples found positive for Mycobacterium avium subsp. paratuberculosis will cause the herd to be classified as an infected herd. Individual animals included in the positive pool will be considered suspect until individual fecal samples from all animals in the pool are tested using an official Johne's disease test. Those animals with a negative test result on the individual samples will be classified as test negative and animals found with a positive test result will be classified as infected. In the case that a herd has a positive pool test result but all individual animals are tested negative for the presence of Mycobacterium avium subsp. paratuberculosis on the individual fecal samples, the herd shall remain classified as infected. If the herd owner elects to challenge the test results, they must do so by the appeal process for an official Johne's disease test (see Part III, paragraph C, 3 d) for each animal included in the positive pooled samples. Individual animals classified as infected are subject to reporting and identification and movement restrictions in accordance with Title 9 CFR part 80.

(4) Level 4 - Herds have met the requirements for Level 3 and have had a negative screening test on a statistical subset (see definition) of 36 months or older animals. Level 4 testing must be completed within 10 to 14 months of the anniversary date.

b. In herds with previous documented testing, the DJC, after evaluation of the testing and that introduced cattle are of low risk, may assign a herd a test-negative level up to Level 3 after the herd has the required test-negative testing to get to that level and the owner signs a declaration stating: "No Johne's disease was known or suspected in the herd during the last five years."

1. Negative ELISA test on a random sample every 10 to 14 months.

2. Additions to herd (other than natural additions) since last test must be verified by a Johne's Certified Veterinarian.

a. Purchased heifers and bulls less than 24 months of age may be added to the herd, provided that:

(1) For Levels 1 and 2:

The animal was purchased from a herd with a test-negative level that is equal to or higher than the herd it is entering;

OR

The purchased animal is tested with an official JD test in the next herd test after it reaches 24 months of age. The herd will not lose its test-negative level if additions that are test positive and any progeny are removed from the herd within 30 days after the positive test and the DJC conducts a follow up epidemiological study. The addition animal reaches herd test-negative level and becomes part of the herd after a test-negative result on the herd test.

(2) For Levels 3 and 4:

The animal was purchased from a herd with a test-negative level that is equal to or higher than the herd it is entering;

OR

The purchased animal is from a herd one level below the test-negative level of the purchasing herd and is tested with an official test at 12, 18, and 24 months of age and is tested in the next herd test after it reaches 24 months of age. The herd will not lose its test-negative level if additions that are test positive and any progeny are removed from the herd within 30 days after the positive test and the DJC conducts a follow up epidemiological study. The addition animal reaches herd test-negative level and becomes part of the herd after a test-negative result on the herd test;

OR

If the addition animal is more than one level below the herd's current test-negative level, the herd will be demoted one test-negative level. The addition animal must be tested with an official test at 12, 18, and 24 months of age and tested in the next herd test after it reaches 24 months of age. The herd will not lose its test-negative level if additions that are test positive and any progeny are removed from the herd within 30 days after the positive test and the DJC conducts a follow up epidemiological study. The addition animal reaches herd test-negative level and becomes part of the herd after a test-negative result on the herd test.

b. Purchased or replacement animals 24 months of age or older may be added to the herd provided that:

(1) For test-negative Level 1 or 2 herds:

The animal was purchased from a herd with a test-negative level equal to or higher than the herd it is entering;

OR

The animal has a screening test within 30 days before entering the test-negative level herd with negative results, and fecal samples from each addition animal have been collected and submitted between 30 days before and 30 days after arrival for an official JD test, and the herd addition is tested on the next herd test. The herd will not lose its test-negative level if additions that are test-positive and any progeny are removed from the herd within 30 days after the arrival of the additions and the DJC conducts a follow up epidemiological study. The addition's test-negative level will remain at its entry level until it has tested negative at the next herd test.

(2) For test-negative Levels 3 and 4 herds:

The animal was purchased from a herd with a test-negative level that is equal to or higher than the herd it is entering;

OR

The herd of origin has a test negative level of one level below. The purchased addition has a screening test with negative results within 30 days before entry into the program herd. Fecal samples from each animal added have been collected and submitted between 30 days before and 30 days after arrival for an official JD test. The herd addition is tested on the next herd test. The herd will not lose its test-negative level if additions that are test positive and any progeny are removed from the herd within 30 days after arrival, and the DJC conducts a follow up epidemiological study. The addition's test-negative level will remain at its entry level until it has tested negative at the next herd test;

OR

If the addition is more than one level below the herd's current test-negative level, the herd will be demoted one level, and each addition must have an official test on fecal samples between 30 days before and 30 days after arrival. The herd addition is tested on the next herd test. The herd will not lose its test-negative level if additions that are test positive and any progeny are removed from the herd within 30 days after the arrival of the additions and the DJC conducts a follow up epidemiological study. The addition's test-negative level will remain at its entry level until it has tested negative at the next herd test.

c. Once all herd additions have been granted test-negative level equal to the herd's current test-negative level, the herd may advance in the test-negative level program by following the testing protocol for the next level.

d. Heifers raised off the premises must be raised with the proper biosecurity measures in place and with animals at an equal or greater test-negative level.

e. Test-negative program herds may use semen and embryos from other cattle herds, if the semen used is processed according to Certified Semen Services' standards and the embryos are processed according to International Embryo Transfer Society protocols. Embryo transfer recipient cows must meet herd addition requirements.

1. Herds are to be tested every 10 to 14 months (60 days before to 60 days after anniversary date.

2. Herds will be removed from the program if the testing interval requirements are not met unless an extension has been received from the Johne's epidemiologist. The next negative herd test will qualify the herd for Level 1 certification.

All samples for program testing must be collected by an accredited veterinarian or state or federal animal health official and submitted to an accredited laboratory.

F.   Testing Laboratories

All tests for the program must be performed at an accredited laboratory (a laboratory that has passed an annual check test for Johne's disease administered by the National Veterinary Services Laboratories). All program testing must be conducted by a laboratory approved for the specific test being used.

G.   Changes in Herd Test-Negative Level

1. A positive official Johne's test will cause a herd to lose its test-negative classification.

2. Change of ownership: Herd test-negative level is determined by the test-negative level of the lowest herd member. Therefore, when ownership of a herd or part of the herd changes and no new animals are added to the herd, the herd level remains the same. When ownership of a herd changes and the herd is commingled with another herd or herds, the resulting new herd shall be assigned the lowest level and achievement year of the herds combined to make the new herd.

1. Appealing ELISA positive results: Confirmation of ELISA positive cattle will be done by fecal culture. Fecal culture must be submitted within 45 days of notification of ELISA results. If found to be culture-negative, the herd may advance or retain its test-negative level. The animal(s) must also be included in the next round of program testing if still resident in the program herd. (Test-negative level of herd with ELISA positives is "suspended pending confirmation").

2. Appealing fecal culture positive results: Fecal culture-positive cattle will be assumed to be infected with M. paratuberculosis and the herd will have its test-negative level revoked. Herds may re-enter the program at Level 1 by following program standards. If the animal owner wishes to appeal this decision, the herd is assigned a test-negative level of "suspended pending appeal." Appeal may be made using one of three means of proving the animal in question is not infected:

a. Necropsy of the animal with culture and histopathology of at least the ileum, mesenteric lymph node and ileocecal lymph node; or

b. Biopsy of the ileum (full thickness) and mesenteric or ileocecal lymph node with histopathology and culture of the tissues and culture of a fecal sample taken at the time of biopsy; or

c. Six separate fecal cultures from the animal on samples collected not less than 30 days and not more than 45 days apart. The herd Johne's disease test-negative level will be suspended until all testing is completed. Only negative results on all tests on all samples will allow the herd to advance or retain its program level.

3. Appeal of test-negative level for other reasons: Herd owners may appeal any decision or discuss extenuating circumstances that prevent compliance with the program rules, to the state Johne's Advisory Committee.

A.   Approved Laboratories

1. General

All official JD and screening tests used for the program may be conducted in a private, university, State, or Federal laboratory that has been specifically approved by NVSL for conducting JD testing. The State animal health official has the authority to decide if any laboratory passing the NVSL check test may participate in its State program. States must have the authority to periodically audit the JD diagnostic laboratories participating in the program. If a laboratory lies outside of the State, the State may rely on audits conducted by the animal health officials from the State in which the laboratory is located.

2. Check tests must be passed in order for laboratories to be approved to participate in the VBJDCP. The check test procedures and other requirements are outlined in VS Memorandum 553.7 Laboratory Approval for the Voluntary Bovine Johne's Disease Control Program. Contact NVSL- Diagnostic Bacteriology Laboratory for further information regarding the check test procedures.

1. Official JD tests - An organism detection test used to confirm the presence of M. avium subsp. paratuberculosis infection within a herd or individual animals.

a. Fecal and tissue culture - Culture is the standard for organism-based tests, although culture methods are not currently standardized. Methods include both solid and liquid culture preparations. Protocols for recommended methods can be obtained from NVSL upon request. Sensitivity is estimated at 40 ± 10 percent; specificity is considered to be 99 percent.

b. DNA probe - DNA probes can detect the presence of M. avium subsp. paratuberculosis without having to grow it. The test has the advantage that it takes less than three days but has the disadvantages of higher cost and the potential of missing low shedders. Sensitivity is estimated at 40 percent; specificity is about 99.9 percent.

c. Histology of tissue - No check test is available at this time. Microscopic identification of the characteristic pathological changes and of M. avium subsp. paratuberculosis organisms in tissue is a definitive test for JD. Tissue changes and bacteria can be observed in the intestinal lining and in nearby ileal, mesenteric, and ileocecal lymph nodes in infected animals. Sensitivity depends on the stage of disease and the number and type of specimens collected but is typically considered to be greater than other laboratory tests.

2. Screening test - Screening tests are tools that have been developed to aid in determining the presence or absence of M. avium subsp. paratuberculosis within a herd.

a. USDA approved ELISA - Animals found positive with ELISA tests should be considered suspect until confirmed using an official JD test. ELISA tests are to be used as screening tools or for helping make management decisions. Sensitivity has been estimated at 25 percent for non-clinical cases and approximately 85 percent for clinical cases; specificity is between 98 and 99 percent.

b. Environmental sampling - Fecal material samples collected in areas where a large proportion of the herd is commingled. Samples are tested using organism detection methods in approved laboratories. The sampling protocol is available in Appendix 1.

c. Pooled fecal cultures - Fecal samples collected from individual animals that are pooled together in groups of five. Individual samples should be submitted to laboratories approved for fecal pooling. The pooling protocol is available in Appendix 1.

Current Approved Testing Strategies

Estimated confidence levels take into account the testing in the current level of the program plus testing that has occurred in previous levels.

Level 1 - 85 percent confidence of having a non-infected herd

Negative screening test (enzyme-linked immunosorbent assay (ELISA)) results on 30 randomly selected animals 36 months of age and older. In herds with fewer than 30 animals 36 months of age or older, animals 24 months of age or older must also be tested;

OR

Environmental Sampling - Dairy Farms

From each dairy farm, collect two environmental samples from each of the following locations on the farm: manure concentration areas (cow housing alleyways or gutters), manure storage areas (lagoons, piles, pits, or manure spreader), and another manure concentration area (sick cow pens or other cow alley- and travel-ways). A total of six samples should be collected for submission to the diagnostic laboratory. Each sample should contain approximately 20 g of fecal material with bedding or soil from a minimum of four different sites within each sampling location. Each sample should be collected with a separate disposable latex glove, placed in a 95-mL plastic-covered specimen container labeled with the sampling location and herd identifier, and stored in a cooler with ice during transport to the laboratory. Care should be taken to include sufficient manure in the sample when sampling from well-bedded areas such as sick cow pens or loose-housing lots.

In free stall barns, each cow alleyway sample should be obtained from a minimum of four sites across cow alleyways, collected to represent most of the mature cows (24 to 36 months of age and older) within the farm. Crossovers, areas around waterers, corners and ends of scrape lanes are excellent places to collect well-mingled manure sub- samples. In tie stall barns, samples should be obtained from all gutters containing mature cow (24 to 36 months of age and older) manure. Other suitable locations for sub- sampling are places where the gutter cleaner exits the barn, along the ramp and in corners. Manure storage lagoon samples should be sampled (using personal precautions for safety) from four locations at the perimeter of the lagoon by submerging the sampling container up to 10 cm beneath the water surface. Each sample from manure piles should be obtained from four different sites up to 10 cm beneath the surface. Each sample from manure pits can be collected using two to three sterile 4 x 4 gauze pads tied to fishing line with fishing weight and soaked at least 10 cm below the manure's surface.

(Modified from Raizman, Wells, et al., 2004. J. Dairy Sci. 87:2959 2966).

OR

Pooled fecal culture - Individual fecal samples of 50 randomly selected cows, 36 months of age or older, be tested in 10 pools of five animals. Individual samples should be submitted to laboratories approved for fecal pooling.

Fecal Pooling Procedure to be done in the laboratory - 5 Samples/Pool

I.   Homogenization by stirring

Weigh out 2 grams of each sample to be pooled and place into a sterile 50 ml conical centrifuge tube. Mix samples by stirring with a sterile wooden stick. Vortex samples vigorously for 10 to 15 seconds until the mixture appears homogeneous. Remove 2 g of the resulting mixture for processing and culture by routine method used in laboratory.

II.  Homogenization by stomaching

Weigh out 2 grams of each sample to be pooled and place into a stomacher bag. Be sure to add the samples to the same corner of the bag to ensure even mixing. Stomach the samples on the highest setting for 2 minutes. Inspect the mixture to determine if it appears homogeneous. Additional stomaching may be required to homogenize the mixture. Remove 2 g of the resulting mixture for processing and culture by routine method used in laboratory.

References:
Wells, S. J. et al. 2002. Evaluation of bacteriologic culture of pooled fecal samples for detection of Mycobacterium paratuberculosis. A JVR 63:1207-1211.

Wells, S.J. et al. 2003. Evaluation of bacteriologic culture of individual and pooled fecal samples for detection of Mycobacterium paratuberculosis in dairy cattle herds. JAVMA 223:1022-1025.

Van Schaik, G. et al. 2003. Pooled fecal culture sampling for Mycobacterium avium subsp. paratuberculosis at different herd sizes and prevalence. J Vet Diagn Invest 15:233-241.

Negative screening tests (ELISA) on a statistical subset (see table page 4) of animals 36 months of age or older.

Negative official JD test results on fecal samples on a statistical subset (see table page 4) of herd members 36 months of age or older. Bulls 24 months of age and older must be included in this testing.

Negative screening test (ELISA) on a statistical subset (see table page 4) of animals 36 months of age or older.

Ideally, the veterinarian should get a list of eligible cows and randomly select identification from that list. Then, the veterinarian should collect samples from the animals selected. This works in small herds or in herds where the animals can be easily caught. Animals can be selected by randomly picking animals from the list. For a more scientific method, select animals using a random number generator or random number tables.

If the list is made in random order, a systematic sampling can be used on the list (i.e., selecting every nth individual from a list (or coming through a chute) after choosing a random number from 1 to n as a starting point). Determine n by dividing the number in the herd by 30 (or whatever sample size is needed in the statistical subsets).

In large herds, veterinarians should use a more stratified randomization to collect a more representative sample. The veterinarian should determine how many animals are in each group (e.g., high producers, low producers, and dry cows) and compute the percentage of each type of animal. The percentage of samples collected from each group should equal the percentage of each group of animals in the herd. In other words, if 10 percent of the herd is low producers, then 10 percent of the samples should be collected from low producers. Sampling in each group would be done by simple or systematic sampling.

These will not be perfect randomized sample collections, but they are better than the convenience sampling. Unless the producer is suspicious of infection in the herd, targeting "high-risk" animals is not the method to use. Testing high-risk groups or target testing should be reserved for herds in the management or control programs. All test-negative status program testing should be as close to random as possible.

Uniform Methods For Nebraska VJDCP for Cattle Revision May 2006, based on APHIS 91-45-016