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 30-2.010 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 1845-1846). 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: Four (4) comments were received on the proposed amendment.

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 five hundred dollars ($500) and has already exceeded this producer twenty thousand dollars ($20,000). 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 Polymerase Chain Reaction (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 seventy-eight percent (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 diagnose 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 comingled 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: Dr. Charles T. Winslow, Lamar Animal Clinic, commented that the importation of feral swine into Missouri is unneeded and unnecessary, and therefore, should be restricted to the same entry allowance as breeding bulls testing positive for Trichomoniasis. Questioned what penalties would be given people who either import Trich positive bulls or feral swine without OCVs, etc. but suspected few if any feral swine importers will find the time to abide by these rules and that feral swine importation may be mostly done with disregard to the revised law so perhaps a figure of more than five hundred dollars ($500) should be included in the cost of the change to enforce the new regulation and/or educate the citizens who may import feral swine. If these revisions are accepted, it would be interesting to know how many entry permits are granted by the state of Missouri for importation of feral swine each year and is this information available to the public.

RESPONSE: Currently feral swine are not allowed to enter into Missouri; however, during the last legislative session legislation was introduced and passed enabling feral swine to move into Missouri. The new regulations reflect the change and require the owners to obtain a permit prior to movement. The legislation did not give us the authority to assess a fee to those not adhering to the regulations. No changes were made as a result of this comment.

COMMENT #3: Dr. Charles Massengill commented that in 2 CSR 30-2.010(4)(D)1.B. that references is made to "approved laboratory." How can a veterinarian in another state determine if the laboratory they rely on is "approved" by the state veterinarian? He suggested the requirement be changed to "laboratory approved by the AAVLD." This way laboratories will already know if they are approved and can share this information with their client. Also, in 2 CSR 30-2.010(15), reference is made to "all aquaculture entering Missouri…" and that the intent is to address all aquatic animals entering Missouri. However, aquaculture is a process or practice.

RESPONSE: The department has reviewed the comment regarding the approved laboratory and feels that the current language includes any laboratory approved by the AAVLD and the language will remain the same. In response to the comment on section (15) Aquaculture, the department has reviewed and according to statute 277.024, RSMo 2000, aquaculture is classified as livestock and therefore referred to as aquaculture. No changes were made to this section.

COMMENT #4: Upon further administrative review, potbelly pigs are covered by current regulations and will be removed from the section regarding feral swine, and section (16) regarding large carnivores needs further guidance than what is noted.

RESPONSE AND EXPLANATION OF CHANGE: The department agrees and will remove this section at this time to make the suggested change.

2 CSR 30-2.010 Health Requirements Governing the Admission of Livestock, Poultry, and Exotic Animals Entering Missouri

(5) Swine.

(A) Swine are classified as the following:

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

2. Feral swine-any swine that are free roaming or Russian and Eurasian that are confined. This also 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 swine) entering 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. Must 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 any required testing.

(16) Miscellaneous and Exotic Animals. All exotic animals must be accompanied by an official Certificate of Veterinary Inspection showing an individual listing of the common name(s) of the animal(s) and appropriate descriptions of animal(s) such as sex, age, weight, coloration, and the permanent identification.

(A) Elephants (Asiatic, African) must test negative for tuberculosis within one (1) year prior to entry.

(B) Importation of skunks and raccoons into Missouri is prohibited by the Missouri Wildlife Code, 3 CSR 10-9.

(C) No tests are required for animals moving between publicly-owned American Zoos and Aquariums (AZA)-accredited zoos but must be accompanied by a Certificate of Veterinary Inspection. Cervids moving between publicly-owned AZA-accredited zoos must meet the chronic wasting disease monitoring requirements as outlined in subsection (10)(E). An entry permit is required on all animals moving between publicly-owned American Zoos and Aquariums (AZA)-accredited zoos.