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2006 USAHA Johne's Meeting Notes

Report of the NJWG

Report of the Lab Committee

Thurs Afternoon Session

Fri Morning Session

Fri Afternoon Session

Report of the National Johne’s Working Group by Robert Whitlock & John Adams:

Approximately 125 persons signed the attendance rooster, plus another estimated 50 persons attended parts of the meeting, occasionally standing room only. The two day meeting was split into 4 half day sessions with ½ day on NJWG committee reports with a discussion of the Canadian JD program; ½ day session on the status and findings of the Johne’s demonstration herd project; ½ session of testing methods for Johne’s disease and the last ½ day on milk ELISA testing and implementation by Dairy Herd Improvement Associations (DHIA).

Bob Ehlenfeldt, Chair of the Johne’s Committee reported on the resolutions (# 19 & 20) and the four recommendations passed at last years USAHA meeting in Hershey, PA. Recommendation # 1; USDA-APHIS-VS provided $50,000 to help fund the Johne’s White paper being coordinated through JDIP, Vivek Kapur-PI. Recommendation # 2; USDA-APHIS-VS provided funding to NIAA for the Johne’s Educational Initiative, Ken Olson will be responsible for this effort at NIAA. Recommendation # 3; the proposed topics for inclusion in the recertification Curriculum were approved; will be added to Program standards. Recommendation # 4: the Knowledge Gaps report was approved and included in the minutes of the Johne’s committee report last year.

Ken Olson presented the treasurer’s report for the NJWG. The current balance is $25,941. To date approximately 800 Johne’s and Beyond CD’s have been sold. The USAHA office has a few additional CD’s for sale.

Mike Carter, National Johne’s Coordinator, summary report that indicated as of Oct 3, 2006, 48 states have Johne’s Advisory committees with more than 8441 herds enrolled in program including 1792 status herds. For the past year approximately 784,978 serum ELISA tests and 125,336 fecal cultures, 4,077 pooled fecal samples and 717 composite environmental fecal samples were completed under the National Johne’s program. More than 4,113 Risk Assessments (RA) and Herd Management Plans (HMP) were completed in the last year. Herds enrolled in the Status program included 700 herds at level 1, 600 herds at level 2, 125 herds at level 3, and 242 herds level 4, with 1068 dairy herds and 724 beef herds enrolled. The National Johne’s Demonstration herd project was funded at $1,280, 000.

Frank Garry and Jeannette McDonald, Co-chairs of the Education Group reported that the online Johne’s Disease Veterinary Certificate Program was initiated Feb 2004, currently 45 states accept the online training for veterinarians in their states that wish to become certified for the Johne’s control program. The recertification module was approved at last year’s USAHA meeting and was made available in April 2006. This Johne’s disease update has also been added to the Certificate Program for a total of 7 modules. At the time of the USAHA conference 53 individuals had registered to be recertified. New veterinary modules for other species, including Goats, Sheep, and Cervidae, have been added. Producer modules are also available for Dairy, Beef, Sheep, Goats, Cervidae & Dairy in Spanish. Two virtual dairy farm visits are available for veterinarians to practice risk assessments and management plans. In addition, two virtual beef production visits are currently being developed. Modules currently in progress include “producer stories” funded by JDIP, followed by a 360o evaluation of JDVCP (Certificate Program) and a 3600 evaluation of the Dairy Producer Module. Other projects planned include a Special Topics course to include vaccine usage, Monensin usage, zoonotic issues and a Stakeholders needs assessment tool.

John Adams reported that Congress has not passed a budget for Agriculture. The House has proposed $7.7 million from House while the Senate has requested $10 million for Johne’s disease. Companies such as IDEXX, Prionics, BD, Trek, Tetracore as well as producers and producer organizations are encouraged to contact their Congressional Representatives and Senators in Washington about the proposed cut-backs in the Johne’s program. Legislative contracts in Texas and California would be most helpful. The congressional conference committee will include Senators Kohl from Wisconsin and Specter from PA. OMB‘s budget proposal only included $3 million to go forward for APHIS to the Johne’s program. NMPF continues to have Johne’s disease a major interest and has hired a specialist to lobby for the Johne’s program.

Jason Lombard reported the 2007 Dairy NAHMS study will include 1,000 cattle operations to evaluate calf health & nutrition, BVD, milking procedures and Johne’s disease among other objectives. The Johne’s disease component will include an estimate of the national herd level prevalence (using environmental manure sampling with 6 composite manure samples per farm), bulk milk samples for PCR & ELISA, and will assess producer’s familiarity with Johne’s disease.

Beth Patton, chair of the Program Standards committee reported the Scientific Advisory committee will review the statistical confidence of being free of Johne’s at each level of the status program. Major discussion points on the program standards included: the A, B, C, D categories for infected herds (still used in 13 states); not all tests are the same in the same areas, i.e. Florida vs. Vermont for ELISA for example, beef cattle may not respond to Johne’s infection in the same way as dairy cattle thus influencing test results; bi-annual or annual RA & HMP’s; abbreviated forms for RA & HMP (currently used in MN & WI; PA has a modified follow-up form); what is the science behind renewals for annual testing or RA & HMP? The committee needs to develop a form (RA-HMP) for professional heifer raisers and will seek input from the Professional Dairy Heifer Growers organization.

Report of the Laboratory Committee to Program Standards Committee to the NJWG October 12, 2006 Submitted by Lab Committee Chair, RH Whitlock.

Members (18) of the laboratory committee include: Byrum, Bev; Capsel, Randy; Carter, Mike; Cui, Jing; Glazer, Amy; Harris, Beth; Henderson, Louise; Marquardt, Janet; Lombard, Jason; Payeur, Janet; Stehman, Sue; Tucker-Schroeder, Linda; Rajev, Sree; Stabel, Judy; Tewari, Deep: Whitlock, Bob; Wu, Ching Ching. The committee held 6 separate conference calls between August 11 and September 28, 2006. Each conference call was graciously hosted by Mike Carter, National Johne’s Coordinator.

Item numbered 1, 2 & 3 are forwarded to the program standards committee for incorporation into the Johne’s Program Standards and were approved by the Johne’s Committee. A motion was approved for three items (# 1, 2, and 3). Item # 6 will be implemented this year by NVSL & CEAH staff. Items # 4, 5, 7 & 8 are informational. Near unanimous approval by NJWG on October 12, 2006 with minor edits/clarifications.

1. Preliminary approvals for labs that have passed the NVSL culture or PCR check tests.

Any laboratory that passed the Johne’s organism detection check test outside the normal time sequence (typically February through May each year) should be given “preliminary approval” as an approved laboratory for that specific methodology i.e. solid media, liquid media or PCR testing. Preliminary approval would be given when laboratory results are submitted after NVSL report at the annual USAHA meeting. Additionally, requests for check test kits would be honored from laboratories that are implementing a new test method outside the time when test kits are routinely shipped to participating laboratories. Preliminary approval would be provided following submission of check test results that meet or exceed the test criteria established that year. However, that preliminary approval would not include listing of that laboratory in the approved laboratory list as published in the USAHA proceedings nor would that laboratory be listed on the USDA-APHIS web site of approved laboratories that year. Laboratories that pass the annual organism based proficiency test are officially approved January 1 following the annual USAHA meeting.

2. Response for any laboratories that fails organism detection check test.

Each laboratory would be required to provide a written self-assessment report outlining possible deficiencies or situations as to what factors lead to an inadequate check test. Included would be a plan to enhance the laboratories proficiency to detect MAP in fecal samples. (A template for this report is being developed). If a commercial test kit or test system is being used for organism detection, the company should be contacted to help determine the source of the problem and their findings should be included in the self assessment.
Each laboratory would be encouraged to seek additional training either from another local laboratory considered proficient in organism detection or at NVSL.
Letters from NVSL notifying each laboratory about test results will also be sent to the DJC for that state and to the National Johne’s Coordinator for their information. Labs that do not pass the check test must contact the National Johne’s Coordinator and their Designated Johne’s Coordinator regarding continuation of their opportunity to perform organism detection tests for the Voluntary Bovine Johne’s Disease Control Program.
Labs that fail the organism based check test are encouraged to re-take the check test following submission of their written self-assessment and approval of the National Johne’s Coordinator, if adequate check test kits are available at NVSL.

3. Response for any laboratories that fails organism detection check tests in two sequential years.

Each laboratory would be required to provide a written self-assessment report outlining possible deficiencies or situations as to what factors lead to an inadequate check test. Included would be a plan to enhance the laboratories proficiency to detect MAP in fecal samples. If a commercial test kit or test system is being used for organism detection, the company must be contacted to help determine the source of the problem and their findings should be included in the self assessment.
Laboratories in this category will be required to send the person responsible for the organism detection testing to NVSL or to another laboratory with the necessary experience and expertise (approved by National Johne’s Coordinator) for further training in mycobacterial detection methods.
Laboratory would be required to purchase and submit results from a second check test following mandatory training at NVSL or another laboratory as approved by the National Johne’s Coordinator.
Letters from NVSL notifying each laboratory about test results will also be sent to the DJC for that state and to the National Johne’s Coordinator for their information.

4. Reporting results for fecal culture by shedding category: This issue was discussed at some length but no consensus was reached except that labs should report an interpretation based on their experience. Since heavy shedders represent a spectrum of cfu/gm ranging from 10,000 to 10,000,000 cfu MAP per gram, perhaps laboratories should be required to detect heavy shedders as heavy shedders with less focus of sensitivity of fecal culture. The heavy shedders and perhaps super-shedders represent the greatest source of environmental contamination and thus risk spreading JD on farms to susceptible cattle (calves). SS then pointed out that laboratories should continue to have a sensitive test, especially at the herd level since detection of MAP in status herds remains an important issue.

5. AAVLD standards and protocols for laboratory testing. The AAVLD is in the process of implementing more rigorous standards for approved AAVLD laboratories and that some of their procedures may impact Johne’s testing. 2007 is the target date to implement these validated testing protocols in laboratories. Publication of the organism based MAP detection tests would help in this effort.

6. Z-scores for each lab submitting results for organism based tests will be implemented when NVSL reports this year’s check test results. The Z-scores for all labs reporting for the organism based tests would be included on a Z-score plot. Each lab would know their Z-score and be able to compare to all other labs how they compared. Z-score calculations would include: a) the mean cfu of all tubes for that sample. Some labs use 3 and others use 4 tubes, b) mean cfu for heavy shedders (above 50) would be truncated at 50 cfu to provide a more uniform method to record heavy shedders as one number, rather a wide range. Previously the lab committee reported that counting cfu above 50 was not necessary, thus for the purposes of the Z-score calculation, the truncation of heavy shedder cfu at 50; c) Both critical and non-critical samples required to pass the organism based test would be included in the Z-score calculations.

7. Quality control for organism detection tests. Low positive fecal samples could be utilized as QC samples in laboratories doing organism based testing. Several questions arose including:

Frequency of inclusion of these QC sample?
Ability of NVSL to provide these samples?
Added cost this would represent to the laboratory?
Are we able to implement this on a pilot basis?

8. Milk ELISA testing & check tests:

NVSL will be obtaining milk ELISA test kits for evaluation from Antel Bio
NVSL is requesting milk samples from known positive cows for their use to evaluate the milk ELISA test.
Both Mike Carter & Janet Marquardt indicated the Lab committee’s input on this subject will not be needed at this time.
At the present time, the milk ELISA test has not been licensed by USDA-Biologics.

Sue Stehman provided a brief discussion on the quantification of shedding level based on liquid culture using the Trek ESP system as follows: If time to detection (TTD) was less than 21 days, the sample was categorized as a high shedders, if TTD was between 22-28 days, a moderate shedder and if > 29 days, a low shedder. The Johne’s laboratory at Cornell is collaborating with NVSL (Payeur) on a “quantification study” to compare TTD in liquid culture for both TREK & MGIT liquid culture systems. The samples initially run on TREK-ESP liquid culture system, were frozen up to six months at -700C. The samples were then shipped to NVSL for culture on MGIT and on HEYM using a standard protocol. The samples represent a range of TTD, 25 super-shedders (TTD 7-14 days) , 75 heavy shedders (TTD 15 – 21 days), 75 moderate shedders (TTD 22-28 days), 75 low shedders (TTD 29-33 days and 25 negative samples.

Jason Lombard reported on the ELISA Quality Control sera being used by about 31 laboratories to monitor ELISA variability over time. USDA-APHIS provides the QC at no cost, if the laboratory reports results to USDA-APHIS-CEAH on a regular basis. The present QC sera being supplied by NVSL for the Prionics ELISA needs to be replaced, as the optical density (OD) is too high. Well to well variation is minimal for the Prionics (BioCor) ELISA test. Nearly 40% of variation is due to kit lot for the Prionics ELISA. Idexx well to well variation was 83% compared to 98% for Prionics. IDEXX began distributing an improved kit in July of 2006. CEAH needs to provide at least a quarterly report back to laboratory participants and the overall QC test performance among participating laboratories. This report should be sent to Laboratory directors and the lab quality assurance person. As of October 12, 2006, the data should be available on the website of USDA-APHIS-CEAH. Center for Veterinary Biologics (CVB) would also like to be aware of the QC reports to monitor variation among labs.

Mike Collins reported a meeting of National Advisory Committee of Microbiological Criteria for Foods, subcommittee on Paratuberculosis, David Acheson, chair. The first meeting was in March, 2006 and the second committee meeting was held in October 2006 in Washington, DC. Using published reports; the committee will assess the possible sources of MAP, the levels of MAP, evaluate the methods to detect MAP in foods and will determine what research needs to be done to further define the issue. The committee will not assess the zoonotic potential of MAP. A final report will be completed in 2007.

Steve Hendrick from Saskatoon, Saskatchewan provided an overview of the Canadian Johne’s program entitled the Canadian National Voluntary Johne’s Disease Prevention and Control Program (CJDPCP). A ready reference is available: Canadian Vet Journal 47:539-541, 2006. The program is being supported by the Canadian Animal Health Coalition (CAHC) which includes: Dairy Farmers of Canada (DFC), the dairy industry, the Canadian Federal Government, Provincial Government’s of Ontario and Alberta, academicians and veterinary practitioners. Alberta had previously established (2001) a JD status program with four levels. One of the early objectives of the program was to facilitate easy participation by producers and to focus of infected herds with the goal of reducing herd prevalence over time. Penalties for test positive herds are being kept to a minimum. Tested results are being kept confidential. The impact of the CJDPCP will be periodically assessed by periodically determining the herd prevalence of JD.

The program includes two pathways: Status Pathway (SP) and the Prevention Pathway (PP). Emphasis with both education and financial support for testing will focus on test-positive herds with the goal to minimize spread of disease both within and among herds thus lowering the national herd prevalence. The program includes risk-assessments and herd-specific herd management plans designed to reduce the spread on Johne’s disease.

Prevention Pathway (JD-PP) does not have any specific testing requirements. However a pre-assessment questionnaire is required to be completed by the producer to reduce the time and cost required to do the Risk Assessment (RA) & Herd Management Plan. No self assessment options are available. These herds will be required to complete a follow-up check list and questionnaire 10 months after the initial RA & HMP. Provincial JD coordinators are encouraged to send participation certificates to herds enrolled in the program.

Status Pathway (JD-SP) must complete the RA & HMP in addition to the pre-assessment survey. The testing requirements for level 1 include ELISA testing or culture of strategically planned environmental manure samples from high cow traffic areas. If testing results do not identify JD in the herd, the herd advances to level 1. Owners may elect to remain at this level by re-testing at least once every two years with all organism based tests be negative. In order to remain in the JD-SP, fecal culture positive cows must be culled for level one herds. For level 2, 10 fecal samples from all eligible cows individual cows are pooled and cultured, if negative and with completion of a second RA & HMP, the herd advances to level 2. No other levels are recognized at this time. Producers are recognized for the number of years enrolled in the program in either path in the program. This is a low risk program, not certified free of JD if enrolled in the JD-SP. While implementing the program, knowledge gaps will be identified and investigated if funds become available. At some point a national Johne’s coordinator will be appointed. Implementation of program is being lead at the provincial level with participation by industry/ government partnership. Canadian producers also have concerns about being identified as a positive herd.

Mark Kinsel, DJC for Washington State reported that Northwest Dairyman’s Association (NDA- Anton Mickleson & John Bosma) have adopted a resolution strongly recommending their producers (more than 500 herds) each have a RA & HMP in place. NDA believes this is important for sustainability of their membership to maintain markets. This is similar to the resolution adopted by Tillamook County Creamery Association (Mark Wurtenberg) in 2003 (about 150 dairies) that requires their producers to have a RA & HMP in place in order to sell milk to the association. Mark Kinsel will provide free software that allows the RA & HMP to be computerized and facilitates data retrieval to state Veterinarians and DJC’s. Contact Mark by E-mail at mkinsel@agr.wa.gov if you would like a copy of the software.

Janet Marquardt at NVSL provided an update on the PPD Johnin project that was result of a 2005 USAHA resolution. Four batches of Johnin PPD have been prepared to date; each one has been different from the other despite very similar growing conditions for the mycobacteria. NADC has and will continue to collaborate with NVSL to characterize the proteins in each lot of Johnin produced to date.

Thursday Afternoon Session of the NJWG Meeting: Johne’s Demonstration Herd Project

Scott Wells provided the background for the National Johne’s Demonstration Herd Project that was part of Goal II of Johne’s disease strategic plan approved by USAHA in 2002. Johne’s demonstration herds are critical and have the highest priority for funding for the National Johne’s Program. The demonstration herd committee of NJWG developed four objectives for this project. That included 1. effectiveness and feasibility, 2. a mechanism to provide information and materials for educational purposes, 3. to develop and evaluate the JD program and 4. to create a mechanism for add-on projects.

Jason Lombard reported on the National Johne’s Demonstration herd project. In the third year of the study, 17 states are participating in the Demo project involving 70 dairy herds with 74,000 milk cows from 16 states and 27 beef herds with 6,400 beef cows from 11 states. The forms used for RA & HMP have been modified from the standard form for National program to allow better data collection and analysis. Two meetings of investigators were held; Denver, CO on October 11-12, 2005 and in St Paul, MN on September 20, 2006 in conjunction with the AABP meeting. Many investigators also presented research abstracts during public sessions and poster sessions. The first manuscript outlining the National Demonstration herd project is being planned. A CD was made of the research presentations in St. Paul. As with the national program there is a greater focus on dairy herds with an overall fecal culture prevalence up to 10% positive cultures in some herds. The seroprevalence of JD has decreased from 9 to 6% over three years in 26 of the dairy herds. One specific question critically evaluated was associations with testing culture positive using a logistic regression model. Heifer calves born to culture positive dams were twice the risk to be culture positive compared to calves born to culture negative dams. Other lessons learned include the necessity to cull cows with clinical signs from herd as quickly as possible as well as removing calves from dams prior to nursing. Data from the project will be shared with all demo herd owners and their veterinarians. Proposed studies include prevalence & risk areas for transmission on the farm, cost benefits for participation in the project and gamma interferon study.

Ching Ching Wu reported data on 7 Indiana herds with two of the herds practicing Johne’s vaccination. First herd receiving vaccine was an Amish herd which started vaccinating since 2001. Whole herd fecal cultures are done annually and ELISA testing quarterly. They cull clinical suspects quickly. In 2003, 13/42 (30.9%) were vaccinated, 19/42 (45.2%) fecal culture positive which 6/19 were vaccinated, 6/13 (46.2%) vaccinates were fecal positive; in 2005, 26/40 (65.0%) were vaccinated, 3/40 (7.5%) fecal culture positive which 1/3 (33.3%) was vaccinated, 14/40 (35.0%) ELISA positive. The second vaccinated herd has 900 cows and has been vaccinating since 2003. Fecal positive culture rate changed from 58% in 2003 to 17% in 2006.

Roxanne Pillars for Michigan (team members included JB Kaneene and DL Grooms) reported that 7 dairy herds have been enrolled for 4 years. Environmental monitoring of manure samples is being compared to reduction in herd fecal culture prevalence. Of 547 environmental samples 59 were culture positive. MAP cfu’s decreased as prevalence decreased. MAP has been consistently detected in the manure storage areas and or cow housing area 79% of the time when the herd prevalence is greater than 2%. When herd prevalence exceeds 5%, MAP cultured from many other areas, most commonly the maternity pen floor. The number of MAP positive environmental samples and number of cfu’s in those samples increased as number of cows shedding MAP in the herd increased. Thus, cleanliness and sanitation of maternity pen must be emphasized in JD control program.

In an ongoing calf study-27/1,500 (2%) of manure samples were culture positive from calves, of the 27 positive calves, 8 were repeat culture positive. Fecal culture test positive calves are 8.6 times more likely to be born to a test positive dam as from test negative dams. Their group is also estimating the costs of the Johne’s control programs on these herds. An annual questionnaire (2003-2005) has been administered to herds participating in the Johne’s control program assessing four categories (supplies/testing, management, capital investment and labor). Preliminary estimates indicate the average cost is $61 per cow with a range of $30 to $102/cow/year.

Frank Garry with colleagues JE Lombard, HL Hirst, MM Dennis, MC Antognoli and MD Salman reported on antemortem identification of cattle with disseminated Mycobacterium avium ssp paratuberculosis infection. If a food safety risk for MAP is presumed to exist, which cows should be excluded from human consumption? How should cattle be screened prior to slaughter? Current antemortem tests available to screen for MAP infection include physical exam/ clinical signs, ELISA, fecal culture, tissue biopsy (histopathology or culture), PCR and CMI testing. Their objective was to determine the association of antemortem test results with presence of disseminated MAP infection (DI) in cattle at slaughter.

To date 40 cows with complete BACTEC results from 4 gastro-intestinal samples (feces, mesenteric lymph nodes, ileum, and ileo-cecal lymph node) and 36 cows with conventional culture results on multiple organs. Histopathology has been completed on 36 cows. Animals were classified as infected if at least one tissue or fecal culture positive for MAP and animals had disseminated infection (DI) if intestinal and extra intestinal involvement of MAP as identified by culture. Results found 12 of 36 not infected, 8 of 36 were localized intestinal infections only and 16 of 36 cows had disseminated infection.

The liver was positive in 6 of 16 cases while the hepatic lymph node was positive in 13 of 36 cows.

Preliminary conclusions indicated almost 50% of the MAP infected cows had DI. Combined ELISA results detected 100% DI cows (needs further verification). Fecal culture detected high proportion of DI cows. Preferred tissues to collect to detect DI are intestinal related but kidney and suprammamary lymph nodes are important tissues to collect to detect DI. MAP was never found in skeletal muscle. Heart muscle was found to be colonized. Peripheral lymph nodes could be included in ground meat. DI was common in serum-ELISA-positive cows without clinical signs of Johne’s disease. If food safety policy moves toward exclusion of cows with MAP from the food chain, then a clearer understanding of the occurrence and identification of DI would be necessary.

Scott Wells, C. Ferrouillet and SM Godden reported for Minnesota report one of their goals was to determine if farm management factors were effective to reduce JD transmission. Three management factors included limiting transmission during the perinatal period, limiting contamination of the environment and limit introduction of animal from infected herds. The tools used to make these assessments included: annual risk assessment, annual herd control plan and testing of adult cows (serum ELISA and fecal culture). Their study includes 6 herds enrolled between February 2000 and January 2001. Two herds range from 45 to 50 cows while four herds range between 220 and 330 cows. Herds are sampled once annually and at time of confirmed pregnancy in two herds. The mean herd ELISA prevalence was 13.5% (5.6% to 28.7%) and culture prevalence was 12.9% (3.85 to 34%) with a clinical case prevalence of 18.3% (0 – 24.8%) of all culled cows. Survival analysis by Kaplan-Meier curves and Cox proportional hazards analysis will be used. Cohorts are defined by birth date of cows and it is assumed that animals are negative until first positive test. The maximum follow-up for all cows is 45 months. Results from the Cox model suggested a decrease in seroconversion and fecal shedding over time. Birth cohorts are different in regards to when in the lives of animals the management improvements are implemented.

Sue Stehman reported for the New York program. Team members included P Leids, R Scrafford, R Ellis, C Johnson, J Huntley. NY program includes 4 herds with 750 to 1200 cows per herd.

These four herds used different control strategies including strategic testing and integrated management, rolling herd testing (120-150 days in calf) with culling of fecal culture positive prior to dry off; rolling herd fresh cow testing prior to breeding management with minimal testing and vaccine and management. Management plans are assessed on a quarterly basis in each herd.

The diagnostic lab switched to TREK-ESP system in June 2004 and more recently the Trek media may have changed with an increased number of culture positive samples (16%). One herd began vaccinating for JD in 1997, with no fecal culture positive samples since birth cohort in 2001. This herd is now vaccinating only 50% of newborn calves. This herd has lowest % positive environmental samples. Identification and removal of highest shedding cattle at or before critical management decision points, especially maternity pen management. Determination of the best method to monitor herd prevalence once low prevalence is reached remains an unanswered question.

Ernest Hoving with colleagues D Wolfgang, R Whitlock and D Tewari reported on the PA demonstration herd project. Depending on the producer’s goals, most laboratory tests used can identify infection at the herd level. Economic considerations will be important in future for determining a testing plan. Herds relying on testing 30 random ELISA tests on second lactation or older cattle as screening method may not be truly capturing the herd status. Individual animal culture is still gold standard in diagnosis.

Mike Collins reported on one of the two Wisconsin demonstration herd projects. The primary hypothesis: Will management changes together with ELISA testing control Johne’s disease? The outcome evaluation will be comparison of rate of infection in animals born before and after start of the Johne’s disease control program. Only ELISA test results will be given to producer to manage cows. Fecal culture results not given to producer; but will be used to judge infection rate independent of ELISA testing. Results will be assessed by Herd: Pre- Post- Control Program, first lactation cows ~ incidence before and after implementation of the control Program. Conclusions at this time indicate the program has been successful with a decreased apparent prevalence (p<0.001), cessation of clinical cases of JD and satisfied dairy producers. One side light has been the use of zip-ties in cow’s ears to facilitate identification as ELISA test positive.

Some herd controlled JD faster than others. Factors affecting rate of JD control included 1) initial prevalence as a high prevalence reduces rate of progress, 2) ability to aggressively cull ELISA-positive cows, 3) quicker culling results in faster control, 4) affected by the number of heifers successfully raised and ready to enter the milking herd, 5) diligence in following herd management plan and 6) herd owners most able (labor and money) to strictly follow recommendations, without exception, had the fastest success. Next steps include the creation of educational programs and products based on our study herds; online delivery (audio & video), lay publications, and analysis of herd data for statistical evidence in support of our clinical observations about factors affecting success of our program.

Beth Patton reported for the second Wisconsin demonstration herd study. More than 500 herds have used Johne’s vaccine in Wisconsin. Three herds of these herds, each with over 300 head have enrolled in the demonstration herd project. The initial herd prevalence must exceed 7% and both the herd owner and herd veterinarian must agree to use the vaccine. A farm RA & HMP must be completed along with a whole herd TB test. Every other calf was vaccinated until a cohort of 50 head or 10% of the herd is vaccinated. After initial cohorts were established, every heifer calf is vaccinated. First lactation heifers are tested at calving time, thereafter at 90 days pregnant. Annual environmental samples are obtained from maternity pens, cow alleys, manure storage areas, hospital pens and flush systems. Preliminary results indicate there is a significant difference in infection prevalence, lower in vaccinates. Several more years will be needed to critically assess the effectiveness of the program.

Friday Morning Session of the NJWG Meeting: Testing Strategies

Mike Collins provided a detailed report on the USDA-VS supported project: Best Tests-Recommendations followed by comments by DJC’s: Andy Schwartz-Texas, Tom Schomer-Nebraska, Beth Patton-Wisconsin and Boyd Parr-South Carolina. Comments to improve the document included: all ELISAs are not the same (regional differences), producers are always in charge of operation and will make the final decision despite the best test recommendations, and seedstock producers must be able to sell animals, among others. All agreed the final document needed to widely distributed and made available on the web.

Sue Stehman reported that ELISA testing does not work in low JD prevalence herds. As a result demand for fecal cultures exceeds ELISA demand in NY State. NY has tested 7,100 pools (1:5) to date.

Scott Wells with Saraya Tavornpanich and Ian Gardner, reported on identification of Best Herd Testing Strategies for Detection of M. paratuberculosis (Map). Map detection using individual cow testing is costly and imperfect. Cows shed variable concentrations of Map in feces, partly based on stage of infection, and those shedding high concentrations are easiest to detect. Culture of pools of fecal samples from individual cows detects most pools with culture-positive cows. Culture of environmental fecal pools detects most (90%) of infected dairy herds. The stochastic simulation model was developed to compare the herd sensitivity (HSe) of testing strategies for detection of Map in Midwestern US dairies with no previous testing and culling related to paratuberculosis. ELISA serologic testing by 2 different assays (EA and EB), ELISA testing with follow-up fecal culture (EAIFC and EBIFC) Individual fecal culture (IFC) and Pooled fecal culture (PFC) and culture of environmental samples (ENV).

Disease structure modeled on the basis of within-herd prevalence, proportion of infected cows in the herd that shed no, low, moderate, or high numbers of Map in feces, and number of Map as colony forming units per gram of feces (CPG) corresponding to Map-shedding level of infected cows. Misclassification probability of ELISAs in herds was included. Comparison of herd-level sensitivity (HSe) with culture of 5 environmental samples per herd from model to NAHMS 2002 estimates.

Summary: The magnitude of HSe was strongly associated with within-herd prevalence, amount of Map organisms shed in feces by infected cows, and number of samples tested. ELISA alone was a sensitive and low cost testing method; however, without confirmatory fecal culture testing 30 cows per herd in non-infected herds yielded HSp of 21% and 91% for EA and EB, respectively. Testing all cows using ELISA testing with follow-up fecal culture (EAIFC and EBIFC) as commonly done in paratuberculosis-screening programs is unlikely to achieve 95% HSe in low prevalence herds.

Among evaluated testing methods with 100% herd specificity, ENV was the most cost-effective method for low (5%), moderate (16%) and high (35%) prevalence herds followed by PFC, IFC, and EAIFC and EBIFC, respectively. Culture of 6 environmental samples per herd yielded > 99% HSe in herds with > 16% within-herd prevalence, but not sufficient to achieve 95% HSe in low prevalence herds (5%). This model can be used to evaluate the impact of factors influencing the HSe of different testing strategies and provide decision makers with information about the cost-effectiveness of testing strategies for particular situations. Culture of pooled fecal samples (environment or cow samples) is efficient method of detecting infected dairy herds. Further work needed to evaluate efficient methods for detecting infected beef cattle herds and to evaluate efficient methods for herds after years of testing (Level 3).

Paul Anderson from Minnesota reported on fecal PCR compared to fecal culture. The PCR test detects most high shedders in a timely manner, less than a week. Thus producers are able to cull these animals more quickly. When only fecal cultures are used, the time required for producers to cull high shedding cattle ranges from 7-12 months based on Minnesota experience (Scott Wells). Minnesota is recognizing more pass-through or passive shedding cattle. In a study of 91 fecal tests that were culture positive and PCR negative, 76 samples were low positive with 1-10 cfu, most may be pass-through cows. In Minnesota, 1900 herds are enrolled in the Johne’s program. The MN program focuses more resources on infected herds than the 600 herds in the status program. 200 herds are at status level 1, 200 herds at status level 2, 100 herds at status level 3, and 100 herds at status level 4.

Jeff Nelson from NVSL reported on the “Validation Project” to validate NVSL decontamination protocol for Johne’s disease in other laboratories, to compare BBL-HEYM media with BBL-HEYM flasks and to compare 8 weeks to 16 weeks incubation time. To date, 11 laboratories have participated in this project. Each sample (20 total replicated samples to each lab) was to be processed in the same manner and plated on one flask of BD-HEY and two tubes of BD-HEYM with mycobactin. The fecal sample size was 2.0 gms, placed in 35 ml of sterile distilled water in a 50 ml conical centrifuge tube. The tube was to be shaken vigorously to break up the large clumps then placed on a horizontal rocker for 30 minutes. 5 mls of liquid are removed from the upper 1/3 of the original centrifuge tube and placed into a new 50 ml centrifuge tube containing 25 ml of 0.9% HPC in ½ x BHI broth. Centrifuge tube incubated at 37º ± 2ºC for 18-24 hours (overnight). Incubated sample is centrifuged at 900 x g for 30 ± 2 minutes. Supernatant is discarded and the pellet is re-suspended in 1ml of BHI broth containing 100µg/ml naladixic acid, 100µg/ml vancomycin and 50µg/ml amphotericin B (Antibiotic Brew). Sample is shaken or vortexed for 15 seconds. Incubate at 37º ± 2ºC overnight. On day 3, Shake or vortex inoculum for 15 seconds prior to inoculating the media. Inoculate each HEY tube and HEY flask with 200µl of inoculum. Inoculum is rolled to ensure that the surfaces are covered. Media is incubated at 37º ± 2ºC for 8 wks.

All low, medium, and high samples were from three single sources, HEY media without Mycobactin J was not used since there were no “trick samples”. More than 50 colonies observed are called TNTC.

Cattle feces were used only. Findings included: Flasks have greater colony counts than tubes for the same inoculum; most labs have similar results but there are lab to lab variations for the same protocol. All labs had no growth on negative samples, 2 labs had no growth on samples that should have been positive, 1 lab had no growth on all HEY tubes (except one tube and 1 colony), flask colony counts were similar to what other labs counted, all labs except two had growth on at least one of the two HEY tubes for this inoculation volume when growth was expected. 8 Week Results included; Most labs had between 2-3X the amount of growth on flasks vs. tubes for the same inoculum; most labs cultured about the same amount of MAP for the low, medium, and high count feces; High count feces was not as high as was originally, but shows how effective the flasks were at allowing more MAP to grow. The 16 week results indicated most samples did not have more growth after allowing 8 more weeks and more contamination was noted after 16 weeks. Other results included; High count feces form heavy shedders was lower than previously tested; for quantifying these results, 60 colonies was used for TNTC. Results include colony counts from all of the tubes and flasks used in this study

Some variability is noted in the amount of colonies counted. Flasks were easier to read overall than the tubes; glare and condensation made the flasks a bit difficult to read; the flask was superior to the tube(s) in the reduced time to spot visible MAP colonies, greater number of colonies, and ease of handling and observation; some found it hard to read through the glass flasks. Overall results indicated flasks are too thick to view under a microscope; Color change is indicative of egg yolk consumption by MAP growth; flasks are a great space saver in the incubator; the flask was superior to the tube(s) in the reduced time to spot visible MAP colonies, greater number of colonies, and ease of handling and observation, more difficult to cover surface of flask with 200µl of inoculum than the tubes, overall flasks were preferred over the tubes.

Lisa Espey from IDEXX reported the company had improved their ELISA test kit to give a much better and consistent specificity and with a sensitivity of 27% to detect fecal culture positive cattle.

Tom Kellner from Prionics indicated the company would be submitting data for the milk ELISA to USDA-CVB in the near future to seek licensure of the milk ELISA test.

Shiga Eda at the University of Tennessee reported on their new and more sensitive ELISA test. For additional information see: New method of serological testing for Mycobacterium avium subsp. paratuberculosis (Johne's disease) by flow cytometry. Foodborne Pathog Dis 2:250-62, 2005; A novel enzyme-linked immunosorbent assay for diagnosis of Mycobacterium avium subsp. paratuberculosis infections (Johne's Disease) in cattle. Clin Vaccine Immunol. 13:535-540, 2006; A highly sensitive and subspecies-specific surface antigen enzyme- linked immunosorbent assay for diagnosis of Johne's disease. Clin Vaccine Immunol 13:837-844, 2006.

Bev Byrum from Ohio reported on their experience with the TREK-ESP liquid culture system. Their’s is a high volume laboratory processing over 20,000 samples per year with 9 ESP units. In order to detect all MAP positive samples, an AFB stain is done on every signal positive liquid culture bottle at the end of protocol (EOP). An automated shaker is used to dislodge MAP from the sponge in the tube before staining. Their laboratory prefer the Auromine O/Rhodamine stain with brilliant green or methylene blue counter stain as that gives faster visual assessment. They use an automated slide stainer. If the tube is AFB pos, then they do PCR ( IS 900), if negative then the tube is re-returned to the incubator. If PCR positive on IS-900, then reconfirmed on PCR using 251. If positive for both IS 900 & 251 they are reported out as positive. If the sample is IS900 positive but 251 negative it is sub cultured. A MAP positive control is set up each week to detect any variation in the detection system. All liquid culture bottles left in the incubator for 42 days. Approximately 17-60% of over all positives are signaling negative. As many as 53-63% of the negative tubes will be AFB positive at the end of protocol (ave 36%. Not all of the signal negative tubes are low shedders. The machine does detect high/heavy shedders and doing PCR on 20,000 samples would be too expensive.

Sue Stehman reported that Cornell now has 16 TREK-ESP liquid culture units. They first began using TREK systems in January 2001 with about 1,000 cultures per month. Tubes are removed after 35 days of incubation. Each liquid culture bottle has an AFB stain done. For the first few years the average positivity rate was 10% to 12%. In the fall of 2004 they saw an increase in prevalence 14 to 18% when they added more AFB staining of non-signal tubes. The majority of these new positives are detected before 35 days. Individual cow results are reported back to the herd owner as soon as it is confirmed. Negative samples report after AF staining. False positive rate may increase due to line voltage change but using the associated graphs can help identify most of those samples. The report to practitioners included time to detection (TTD) and the relative cfu reported as many, moderate and few. Each positive sample is confirmed by PCR.

Deep Tewari from Pennsylvania reported on a robotics system he has been using for both ELISA and for Real Time (RT)-PCR (Tetracore). Their annual Johne’s testing has been at 60,000 ELISAs and about 25,000 cultures per year. They use a TecanTM Bio-Robotics for IDEXX ELISA. Four plates are processed in three hours; the results to date suggest a very consistent performance of the assay. The Tecan system reduces operator error, increases throughput and improves turnaround time. Following their excellent experience with the automated ELISA assay they then considered automation of the TetracoreTM RT-PCR assay for Johne’s disease. Their initial experience with the Tetracore assay was excellent. They chose the Bio-Robot by Qiagen BioRobot model M 48 which uses paramagnetic beads for nucleic acid extractions. In the 2005 fecal check test the Bio-Robot gave 23/25 correct while the Tetracore processed manually gave 21/25 samples correctly. Concern exists about potential PCR inhibitors, but the automated system looks promising.

Friday Afternoon Session of the NJWG Meeting: Milk ELISA Testing for Johne’s Disease

Bruce Dokkebakken of the Minnesota Dairy Herd Improvement Association (DHIA) gave an overview of the organizational aspects of DHIA. Basic service of DHIA is to measure and test milk samples from individual cows at the farm level and report individual animal management data. Data is summarized to provide individual animal and herd management reports for producers. 80 DHIA technicians in Minnesota evaluate milk samples from 270,000 cows per month. Sample identification and integrity is a critical factor for all DHIA organizations. A national Quality Certification program is in place to verify laboratory, field service and DRPC performance. Minnesota DHIA, working in cooperation with the University of Minnesota and the MN Board of Animal Health, has recently made Johne's Milk ELISA testing available for producers in the state. This is added to the range of services provided including milk analysis, somatic cell counts, mastitis culturing, milk urea nitrogen, water testing, manure testing and forage analysis.

Ken Olson also gave an overview of DHIA activities from a consultant’s perspective. DHIA operates through multiple organizations and is available in all states and has international links with ICAR. Data on individual animal records from birth to culling from the herd are available. The data is also used to facilitate genetic improvements; body type, body condition scoring, calving ease. Participation in DHIA has facilitated movement of cattle to Mexico. DHIA is one of the leaders in moving the National Animal Identification System forward.

Mike Collins reported on a large study he conducted to evaluate the milk ELISA compared to several serum ELISA tests and compared those results to fecal culture tests and PCR tests. Of more than 2,145 cows tested, 443 fecal samples were positive on at least one organism based test. The study also included 412 non infected cows from status level IV herds in Minnesota. Overall the milk ELISA test performed better than some blood ELISA tests and equal to the best serum ELISA test. For further information, see Evaluation of five antibody detection tests for diagnosis of bovine paratuberculosis. Clinical Diagnostic Laboratory & Medicine 12:685-692, 2005.

Steve Hendrick from the Western College of Veterinary Medicine in Saskatoon reported on the Canadian Dairy Industry (CanWest DHI) implementation of the AntelBio Milk ELISA, “Milk ELISA Project”. The Canadian Johne’s program has focused on education and awareness with involvement of producers, veterinarians and government. The Canadian dairy industry has about 1 million dairy cows in 20,000 herds. Milk supply is based on supply management (quota) system. CanWest DHI was originally Ontario Dairy Herd Improvement that expanded to western Canada in 2003 to include BC, AB, SK, MB and ON servicing 4,900 herds (378,500 cows) as a traditional milk recording agency with value-added services: return-over feed and herd management clubs and Johne’s milk ELISA testing.

Ontario DHI (2002) Initially compared AntelBio Milk ELISA to fecal culture results in 6 herds from 2002-2004 and then screened 126 herds to compare to serum ELISA in 82 herds and compared to fecal culture (HEYM) in 9 herds. This study randomly surveyed 50 dairy herds in Ontario; 18% and 30% of herds had 2 or more milk or serum enzyme-linked immunosorbent assay (ELISA)-positive cows, respectively. The apparent cow level prevalence was 1.7% and 2.6% on the milk and serum ELISA, respectively. The serum and milk assays agreed moderately. For further information see “The prevalence of milk and serum antibodies to Mycobacterium avium subspecies paratuberculosis in dairy herds in Ontario”. Can Vet J. 46(12):1126-9, 2005. CanWest DHI began to offer the milk ELISA as a regular service to Ontario producers in June 2005 and expanded to western Canada in December 2005. The “Milk ELISA Project” received federal funding (ACAAF) as an extension project incorporating the milk ELISA, training veterinarians and producers about Johne’s disease. The milk ELISA project was initiated in Ontario (June 2005) with 80 producer-veterinarian pairs to focus on education and JD awareness, Risk assessment and implementation of BMPs. Milk ELISA testing was subsidized ($400) and compensation to vet for RA and HMP training ($200), a one time payment. The project was expanded to western Canada (Dec 2005). The provincial coordinator keep paper work in order, collects risk assessments and consent forms and arranges for veterinary training sessions. Provincial veterinary trainer facilitates completed on-farm training with veterinarians as one-on-one or in small groups.

The second phase of the project includes a revisit to a sub-set of herds in each province and a re-test in subsequent years, assesses changes in management and re-administer risk assessments. This will serve as the basis for a PhD research project. From producers perspectives on the milk ELISA include: 1) the project is convenient, relatively cheap and provides quick turn-around time, 2) has a risk of false-positive results, 3) culling decisions may be impacted and 4) some producers just don’t want to know if they have JD. For veterinarians: 1) profit made from diagnostic testing is not significant, 2) more income from consulting doing the herd plan and completing risk assessments, 3) this becomes part of the “herd health” program and 4) education is important, explaining how to interpret the test results, completing RAs and making management plans. From the Government perspective: 1) extension veterinarians see value in the test and participate in the project and 2) regulatory veterinarians accept the test as a herd screening tool not a certification test. There is no standardization for Johne’s disease testing in Canada and commercial labs may participate in the NVSL check-test program. In summary, the “Milk ELISA Project” is an extension program provincially administered, provides education and awareness about JD to veterinarians and producers with a focus on management. The opinions of producers, veterinarians and regulators are quite favorable to this point. For further information on the milk ELISA project, see http://www.canwestdhi.com/johnes.htm.

Jason Lombard reported on the results of the milk ELISA study as one part of the 2002 dairy NAHMS study. Milk and serum samples from 35 dairy herds in 17 states were evaluated for cow- and herd-level Mycobacterium avium subspecies paratuberculosis (MAP) antibody test agreement. Evaluation of 6,349 samples suggested moderate agreement between milk and serum enzyme-linked immunosorbent assay (ELISA) results, with a kappa value of 0.50. Cow-level sensitivity (Se) for 18 dairy operations with 1,921 animals was evaluated relative to fecal culture results. At the cow level, the milk ELISA relative Se was not significantly different from that of the serum ELISA (21.2 and 23.5%, respectively). Logistic regression models revealed a positive association between lactation number and milk ELISA status. Non-Holstein cows were more likely to test milk ELISA positive than Holstein cows. Cows in the first 2 weeks of lactation and after week 45 of lactation were more likely to test milk ELISA positive than cows between 3 and 12 weeks of lactation. Milk production > 80% of herd average was negatively associated with testing milk ELISA positive. Animals in the West and Midwest regions were less likely than animals in the Southeast region to test ELISA positive by either test. Estimates for herd-level sensitivity for the milk and serum ELISA, relative to fecal culture results, ranged from 56 to 83%. At the cow and herd levels, milk ELISA performed equivalent to serum ELISA using fecal culture as a reference for MAP infection and has the advantage of decreased labor costs on farms that use Dairy Herd Improvement Association testing. For further information see the Journal of Veterinary Diagnostic Investigation 18(5):448-58, 2006.

Todd Bryem at Antel Bio reported on the milk ELISA that was developed at their laboratory in Lansing, Michigan. Five laboratories now run Milk ELISA tests (Waldo Wisconsin, Minnesota).

Next year Antel Bio will do about 100,000 milk ELISA tests. The DHIA technician is able to order JD Milk ELISA. DHIA technicians are offered a monetary incentive to have producers run the milk ELISA tests. Milk ELISA testing at the time of “dry off” seems to offer one of the best times to test.

In an effort to make herd veterinarians aware of the milk ELISA test data, post cards were sent to vets for a number of months with only one response to request copies of report. The state veterinarians from Wisconsin and Michigan have requested copies of the data.

From the herd veterinarian’s perspective, the milk ELISA is another tool to use in the war against JD.

Milk ELISA testing is a viable alternative to official program. Very few herds that use the milk ELISA have done Risk Assessment (RA) and developed a herd management plan (HMP). Confidentiality of test results is a major issue for some farms, even though an un-official test and the majority of herd owners do not see JD as a problem (low cost benefit). Very few herds using the milk ELISA have entered status program, even if eligible. For further information see: http://www.antelbio.com/.

Janet Marquardt from NVSL reported on some of the issues involved in establishing a check test for a milk ELISA. Issues include: 1) long term storage of milk samples, 2) acquisition of animals to provide regular availability of milk samples (presently only have room for 4 animals) 3) milking these cows on a regular basis at a federal facility, and 4) logistical issues. Since the primary difference between milk and serum ELISA is the dilution rate used, 1:1 for milk vs. 1:20 for milk, could serum be used as a surrogate for milk? Staff at NVSL has discussed these issues and are planning to implement a milk ELISA check test in the near future.

Louise Henderson from USDA-Center for Veterinary Biologics (CVB) reported on Licensing Veterinary Diagnostic Test Kits for Johne’s disease. To qualify for CVB licensure, diagnostic test kits should with reasonable certainty yield the results intended when used according to label (insert) instructions. The design, architecture, claims, recommendations, target disease, target animal, sample source, and intended uses determine specific requirements. CVB goal is to license tools of value to users.

Different standards are often appropriate for a specific test. Each serial of all licensed kits must pass NVSL proficiency panel (firm and CVB); kits licensed for milk samples will have to pass milk proficiency panel. USDA licenses kits for diagnosis of disease in animals, not food safety. Each sample type must be validated independently. Data must support performance characteristics of the test. Variations need to be assessed between plate, assay, run, day and laboratory. The analytical sensitivity and specificity for each sample type needs to be estimated. The test must be able to distinguish target from non-target.

The test must have the ability to correctly identify samples from positive animals (definition of pos animal) and from negative animals. The dynamic range and test ruggedness need to be defined. Prior to licensure, the firms must report how they make the test (Outline of Production), how they have established the cutoff (data) points and how they have established performance (data) and how the test performs in field on each sample type (data). Serials of production for consistency of performance (Outline testing) must be provided.

Pre-license validation should be done on a large number of “known” positive and “known” negative animal samples covering a range of reactivity and all sample types. They need to determine and justify cutoff values, determine performance in different populations, address matrix effects, cross reactivity and estimate performance characteristics. The test needs to be evaluated in the field in expert labs (2 serials in 3 labs); the labs must have expertise in disease testing and be cooperative in the evaluation. These evaluations need to assess suitability of test kit, adequacy of instructions (insert) and confirm performance characteristics. A rigorous evaluation at this stage is critical. USDA licensed Kits are validated for recommended purposes prior to licensure.

Claims supported with data are independently evaluated. Each test kit is documented and must have controlled manufacturing protocols of all kits for consistency. CVB monitors field performance and investigates variations. Independent testing of performance characteristics for each serial prior to release for sale is required. For further information call CVB-Inspection and Compliance, 1-800-752-6255.

A cooperative effort of the National Institute for Animal Agriculture and USDA, APHIS, Veterinary Services,
in association with the National Johne's Working Group & United States Animal Health Association