Title 2-DEPARTMENT OF AGRICULTURE

Division 30-Animal Health

Chapter 2-Health Requirements for Movement of Livestock, Poultry, and Exotic Animals

ORDER OF RULEMAKING

By the authority vested in the director of Agriculture under section 267.645, RSMo 2000, the director amends a rule as follows:

2 CSR 30-2.020 is amended.

A notice of proposed rulemaking containing the text of the proposed amendment was published in the Missouri Register on December 15, 2010 (35 MoReg 1846-1848). Those sections with changes are reprinted here. This proposed amendment becomes effective thirty (30) days after publication in the Code of State Regulations.

SUMMARY OF COMMENTS: Five (5) comments were received.

COMMENT #1: Dale Ridder, Hermann, MO, states absolute objection to the proposed rule as currently written and urges the Department of Agriculture to put a hold on the proposed amendment until the technology to better predict false-positives becomes available or to at least allow for a procedure to remove the "death sentence" of a potential false-positive Trich test from bulls and Missouri breeders. The producer agrees with the intent of curtailing the spread of Trichomoniasis in Missouri but it is absolutely unfair to do so based on one (1) positive test with no collaborative test, and no chances of ever reversing a test which current research indicates can and does have false positives. The real private cost in time and dollars would exceed $500 (five hundred dollars) and has already exceeded this producer $20,000 (twenty thousand dollars). This does not take into consideration potential future sales of semen and breeding animals. In an article published in JAVMA, Vol. 237, No. 9, November 1, 2010 pages 1068-1073, authored by Ondrak et al., states "This provides further evidence that sporadic false-positive results detected by the use of culture, gel PCR assay, and real=time PCR assay can occur." Other false-positives were reported by Cobo et al. in their 2007 research. Ondrak indicates that the unnecessary sale and slaughter of false-positive bulls would substantially increase the financial impact of T foetus outbreaks.

RESPONSE: The research conducted by E.R. Cobo, et al., was attempting to determine the specificity and sensitivity of the PCR and culture utilized in different testing protocols to establish the most efficient and accurate method of testing bulls for Tritrichomonas foetus. The current gold-standard testing protocol consists of six (6) weekly cultures, which is expensive and time consuming for the producers. The study concluded PCR or both tests applied in parallel on three (3) consecutive weeks may be as sensitive and specific as the gold-standard. The research concluded the sensitivity and specificity of the gold-standard test was 87.7% (Se) and 97.5% (Sp) compared to PCR and culture combined on a single sample the sensitivity was 78.3% and specificity was 98.5%. The sensitivity of the PCR was found to be 78%, which would indicate the test was not able to identify 22/100 positive bulls and would identify their status as negative. The specificity of the combination (98.5%) would predict approximately 1.5 bulls tested out of 100 would be false positives, given the prevalence of the disease in the general population is comparable to the animals tested. The prevalence of Trichomoniasis in the general population is predicted to be between three percent to five percent (3%-5%), this corresponds to the current prevalence of disease in our sample submissions.

The objective of the research published in JAVMA, Vol 237, No. 9, November 1, 2010, was to determine whether the percentage of nonpregnant cows was associated with the percentage of bulls infected with Tritrichomonas foetus. The results cite a similar conclusion to the aforementioned research conducted by E.R. Cobo, which determined a combination of culture and PCR (this study utilizes gel PCR) was comparable to the gold-standard testing protocol. The study does not discuss how it was determined the RT-PCR yielded three (3) false-positives out of one hundred twenty-one (121) bulls.

Research conducted by Michael S.Y. Ho, et al. in 1993, published in the Journal of Clinical Microbiology, Jan. 1994, evaluated a method to increase the accuracy of the diagnosis of Tritrichomonas foetus by developing a more sensitive testing protocol through the utilization of PCR technology. The need for the technology was due to the low sensitivity ten percent to forty-eight percent (10%-48%) of the traditional method of culturing and microscopic examination to identify the parasite, thus several positive bulls were classified as negative. The study concluded the PCR protocol was able to detect as few as one (1) parasite in culture media or ten (10) parasites in bovine preputial smegma. The analysis of fifty-two (52) samples showed that forty-seven (47) (90.4%) were correctly identified, with no false-positive results. In comparison, the culture method detected 44/52 (84.6%), thus classifying three (3) positive bulls as negative.

In 2002, the PCR assay was improved to increase the ability of the test to identify positive bulls, as described in the research conducted by D. Douglas Nickel, et al. The improved assay identified four (4) positive bulls out of eight hundred forty-seven (847) samples, compared to three (3) positive bulls utilizing the culture method. The increased sensitivity of the PCR decreases the possibility of misdiagnosis of a positive animal. The results were repeated and the positive samples were verified.

Dr. James A. Kennedy, a co-author on the research conducted by E. R. Cobo, has published research advocating the utilization of pooled samples utilizing PCR to detect Tritrichomonas foetus to decrease the cost to producers. He identifies a "pitfall" of the culture method is the low specificity, thus high number of false-positives.

The standard protocol to verify culture positive animals utilizes the PCR to differentiate the Tritrichomonas foetus parasite from other trichomonad organisms. The PCR assay has the ability to identify a particular sequence of DNA of the Tritrichomonas foetus, this sequence is unique to this specific Tritrichomonas spp. Dr. Kennedy's research states the PCR identified sixteen (16) out of sixty-one (61) pools (five (5) samples combined), identifying two (2) pools containing samples that had previously been considered negative by culture.

Research has proven the PCR assay has the ability to detect the Tritrichomonas foetus parasite when only a few organisms are present. The ability to obtain and maintain the quality of a sample prior to arrival at a diagnostic laboratory and the inability to accurately detect and diagnosis Trichomoniasis in infected bulls has been detrimental to the control and eradication of the disease. The incidence of false-positive results is extremely low, especially in comparison to other diagnostic tests we currently or have utilized in previous years to eradicate financially devastating livestock diseases, i.e., brucellosis, tuberculosis. The financial impact to producers by inaccurately diagnosing a negative bull can be tremendous.

Positive bulls remain carriers for life, except for the five percent (5%) of bulls less than thirty (30) months of age which one (1) research study has indicated may clear the parasite due to the lack of depth in the crypts of their sheath. However, this theory is controversial among the experts in the field of Trichomoniasis and additional research is needed to validate the findings. This represents a very small number of bulls.

Research has proven virgin bulls may become infected if co-mingled with positive bulls due to naturally occurring homosexual activity (Sarah Parker, et al., 2003).

The Texas and UMC laboratory utilize the same PCR assay protocol to analyze the samples for Tritrichomonas foetus. The Ct value determines the presence or absence of the parasite, 35-40 (TX) or 35-37 (UMC) have been established as the range for "suspect" samples. The owners/veterinarians are contacted and the lab recommends the animal is retested. The animals below thirty-five (35) are classified as positive and samples above thirty-seven (37) (UMC) or forty (40) (TX) are designated as negative. The UMC laboratory has identified a trichomonad organism in the samples that had been classified as "suspect" upon analysis of results and attempting to determine the reason of the "suspect" category. Since the discovery of this organism, all the samples with Ct values of 32-41 have been sequenced out and were all identified as Tritrichomonas foetus, thus eliminating the possibility of any false-positive results being reported.

The Texas Animal Health Commission classifies any RT-PCR positive bull as infected with Trichomoniasis. Positive bulls are quarantined and transported directly to slaughter on a VS 1-27 permit or to a livestock market for slaughter only on a VS 1-27 permit.

The department has considered this comment and will not make a change to the proposed regulations.

COMMENT #2: Neal Hendrix commented that setting up rules that could result in the slaughter of bulls is premature. Given the economic damage that trich can do to an operation, unbiased education of producers may be the first step along with using proven, reliable, and verifiable testing procedures. After these steps are accomplished, it will be possible to get a handle on the scope of this problem within the state.

RESPONSE: I am unaware of the seven (7) false-positives or how Mr. Hendrix has determined the bulls are not infected with Tritrichomonas foetus. However, regarding the possibilities of false-positives animals, please refer to the response to Mr. Ridder's comments. The Department of Agriculture (MDA) has been proactive in utilizing opportunities to educate and enlighten livestock producers and veterinarians throughout the state regarding the need to implement biosecurity to protect their herds from this financially devastating disease. We have provided continuing education to veterinarians to enhance their ability to obtain quality samples and implement prevention and control management protocols. The MDA veterinarians have spoke to several producer groups, including a purebred production sale, to increase the awareness of trichomoniasis to livestock producers. The department has considered this comment and will not make a change.

COMMENT #3: Carroll Craig commented in favor of testing of bulls for trich beginning at age one (1) and if a bull tested positive, he should be branded and the herd should be quarantined until they have cleaned up or sold for slaughter. Bulls should be tested and found negative before sold through a sale barn; if positive, they should be sent to slaughter.

RESPONSE: The department appreciates this comment. This disease can be the most financially devastating to the Missouri cattlemen since brucellosis.

COMMENT #4: Dr. Chuck Massengill presented several comments on the proposed changes:

Comment #1: Subsection (1)(D) and the exclusion of exotic bovids. Through several of the definitions, reference is made to "bovines." If the intent was to address only cattle and bison, why not refer to cattle and bison? Comment #2: Subparagraph (1)(D)5.C. addresses female bovines from a T.foetus herd. There is not a provision for release of quarantine for a herd that does not have bulls. Possibly there needs to be a clarification on how a herd can be released from quarantine. Comment #3: Paragraph (1)(D)2. should not the statement read "the absence of" rather than "the presence of" both permanent central incisor teeth in wear. The presence and in wear of the central incisors is an indication that the animal is at least twenty-four (24) months old in our livestock market regulations. Comment #4: Part (1)(D)5.B.(I) that the department would reconsider the requirement of identifying positive T.foetus with a "V" brand on the left jaw by an accredited veterinarian. Comment #5: Questioned the use of the undefined stand alone term "T.foetus" to assign a status requiring regulatory action might need attention. Comment #6: Regarding feral swine proposed regulations, paragraph (2)(D)1. that swine moving within Missouri required an entry permit and that owners of pot bellied pigs in Missouri have a feral swine permit number and individuals that keep pot bellied pigs in their houses and yards will meet the requirements for that facility permit. Also, how can two tests be sixty (60) days apart and occur within thirty to sixty (30-60) days prior to movement.

RESPONSE AND EXPLANATION OF CHANGE: Comment #1-exclusion of exotic bovids. The department has reviewed and considered this comment and is convinced that the title in the proposed rulemaking provides clarity and the rule will remain as is. Comment #2-In response to 2 CSR 30-2.020(1)(D)5.C., the department has reviewed and considered this comment and agrees that the proposed rule lacks clarity. The department has rewritten this section to address clarity issues. Comment #3-the presence or absence of the central incisiors. The department has reviewed and considered this comment and will not make a change. The current language is taken from and in compliance with federal regulations. Comment #4-The department has reviewed and considered this comment and is in agreement with the commenter. The department has rewritten this section to address identification of positive animals. Comment #5-the department has reviewed this comment and agrees for clarification that "T.foetus" will be spelled out. Comment #6-The department has reviewed and considered this comment. A change with the proposed rule will be made accordingly.

COMMENT #5: Upon departmental review, Dr. Hagler recommended that positive Tritrichomonas foetus test results be reported to the state veterinarian within seventy-two (72) hours. Additional departmental review of proposed paragraph (1)(D)5. regarding quarantine requirements needed further clarification. Also, section (11) regarding large carnivores needs further guidance than what is noted.

RESPONSE AND EXPLANATION OF CHANGE: The state veterinarian agrees that positive Tritrichomonas foetus test results should be reported within seventy-two (72) hours and will include a reporting period in the regulations and agrees with the change to the quarantine requirements. The department agrees with the comment regarding section (11) and will remove this section at this time.

2 CSR 30-2.020 Movement of Livestock, Poultry, and Exotic Animals Within Missouri

(1) Cattle, Bison, and Exotic Bovids.

(D) Trichomoniasis (Excluding Exotic Bovids).

1. Definitions.

A. Official laboratory-Veterinary Diagnostic Laboratory operated and under the direction of the state veterinarian, University of Missouri Veterinary Medical Diagnostic Laboratory, or other diagnostic laboratories approved by the state veterinarian.

B. Positive Trichomoniasis (Tritrichomonas foetus) bull-male bovine which has ever tested positive for Trichomoniasis (Tritrichomonas foetus).

C. Trichomoniasis-venereal disease of cattle caused by the protozoan parasite species of Tritrichomonas foetus.

D. Positive Trichomoniasis (Tritrichomonas foetus) herd-group of bovines that have commingled in the previous breeding season and in which an animal (male or female) has had a positive diagnosis for Tritrichomonas foetus.

E. Negative Trichomoniasis (Tritrichomonas foetus) herd-a group of bovines that have been commingled in the previous breeding season and all test-eligible bulls have tested negative for Tritrichomonas foetus within the previous twelve (12) months.

F. Test-eligible animal-any bull at least thirty (30) months of age or any non-virgin bull that is sold, leased, bartered, or traded in Missouri.

G. Negative Trichomoniasis (Tritrichomonas foetus) bull-a bull from a negative Trichomoniasis herd with a series of three (3) negative cultures at least one (1) week apart or one (1) negative PCR test for Trichomoniasis foetus or two (2) negative PCR if commingled with a positive herd.

2. All breeding bulls (excluding exotic bovids) sold, bartered, leased, or traded within the state shall be-

A. Virgin bulls not more than twenty-four (24) months of age as determined by the presence of both permanent central incisor teeth in wear, or by breed registry papers; or

B. Tested negative for Trichomoniasis with an official culture test or official Polymerase Chain Reaction (PCR) test by an approved diagnostic laboratory within thirty (30) days prior to change in ownership or possession within the state.

(I) Bulls shall be tested three (3) times not less than one (1) week apart by an official culture test or one (1) time by an official PCR test.

(II) Shall be identified by official identification at the time the initial test sample is collected and the official identification recorded on the test documents.

(III) Bulls that have had contact with female cattle subsequent to or at the time of testing must be retested prior to movement.

C. The official identification, test results, date of test, test performed, and laboratory where test was performed should be included on the certificate of veterinary inspection.

3. If the breeding bulls are virgin bulls and less than thirty (30) months of age, they shall be-

A. Individually identified by official identification; and

B. Accompanied with a breeder's certification of virgin status signed by the breeder or his representative attesting that they are virgin bulls.

C. The official identification number shall be written on the breeder's certificate.

4. Bulls going directly to slaughter are exempt from Trichomoniasis testing.

5. Tritrichomonas foetus positive herd-

A. Shall be quarantined or sold directly to slaughter or to a licensed livestock market for slaughter only and shipped on a VS 1-27 permit.

(I) Any non-virgin female or female twelve (12) months of age or older may be sold directly to slaughter and move on a VS 1-27 or remain quarantined.

(II) Positive bulls shall be sent directly to slaughter or to a licensed livestock market for slaughter only and shipped on a VS 1-27 permit.

(III) Positive animals shall be identified by a state-issued temper-evident eartag; and

B. The quarantine shall be released upon the following:

(I) All bulls in a positive Tritrichomonas foetus herd shall have tested negative to three (3) consecutive official Tritrichomonas foetus culture tests or two (2) consecutive official Tritrichomonas foetus PCR tests at least one (1) week apart. The initial negative test is included in the series of negative tests required; and

(II) Female(s) has a calf at side (with no exposure to other than known negative Tritrichomonas foetus bulls since parturition), has one hundred twenty (120) days of sexual isolation, or is determined by an accredited veterinarian to be at least one hundred twenty (120) days pregnant.

6. All positive Tritrichomonas foetus test results must be reported to the state veterinarian within seventy-two (72) hours of confirmation.

(2) Swine.

(A) Swine in Missouri are classified as follows:

1. Commercial swine-swine that are continuously managed and have adequate facilities and practices to prevent exposures to feral swine;

2. Feral swine-swine that are free roaming or Russian and Eurasian that are confined. This includes javelinas and peccaries; and

3. Transitional swine-swine raised on dirt or that have reasonable opportunities to be exposed to feral swine.

(D) All feral swine (including Eurasian and Russian) moving within Missouri must:

1. Obtain an entry permit;

2. Be officially identified;

3. Be listed individually on a Certificate of Veterinary Inspection, in addition to age, gender, and permit number of feral swine facility of destination;

4. Be from a validated and qualified herd, last test date, and herd numbers must be listed on the Certificate of Veterinary Inspection; or

5. Have two (2) negative tests sixty (60) days apart for brucellosis and pseudorabies within thirty to sixty (30-60) days prior to movement. The laboratory and test date must be listed on the Certificate of Veterinary Inspection.

6. Feral swine moving directly from the farm-of-origin to an approved processing facility or to an approved slaughter-only facility will be exempt from required testing.