In 1994, the Rabies Steering Committee which met at the Centers for Disease Control and Prevention (CDC) in Atlanta recognized the utility of molecular typing of virus variants in rabies surveillance. The committee suggested an expansion of typing capabilities at both national and regional facilities. This knowledge allows those who survey rabies to recognize when established reservoirs expand into new areas or when different animal species become involved in cycles of rabies virus transmission. Key elements in this type of disease surveillance include a broad-based sample collection system and the ability to process a large number of samples in a timely fashion. Though methods of sample collection and rabies diagnostic testing already exist through public health laboratories in all 50 states, few of these laboratories have the personnel or operating budget to extend their laboratory testing to virus typing. The rabies laboratory at CDC types several hundred samples each year. CDC does not have the staff to characterize the thousands of rabies-positive animals identified each year in state laboratories. It is in this latter area that regional typing laboratories such as Texas' have expanded to complement the flow of national surveillance data by increasing their surveillance activities to include molecular typing of virus samples, including samples from surrounding states.

Three major categories of the rabies virus are found in Texas: bat, skunk, and canine. Within each category are several subcategories or variants. In the canine category there are three variants: Domestic dog/coyote (DDC), Texas fox (TF) and Sonora dog (SD). The DDC variant was responsible for the outbreak of rabies in South Texas that began in 1988 in Starr County. The variant spread to 21 contiguous counties primarily in coyotes and dogs and was eventually contained at the U.S./Mexico Border. A West-Central Texas epizootic, caused by the TF variant, began in Sutton County in 1988 and has spread to 50 contiguous counties primarily in gray foxes. (The Sonora dog variant is rare in Texas, but health officials are concerned about a potential outbreak along the border with Mexico.)

As the two epizootic zones expanded and grew closer to one another, it became more critical that the Texas Department of Health be able to accurately distinguish between the DDC and TF variants, especially when testing samples from areas along the boundaries of the two zones. Formerly, TDH laboratory methods could only determine if a rabies test specimen was positive or negative and could identify antigenic variants with monoclonal antibodies. In most samples from the skunk and bat categories, the exact variant could be identified. However, no method was available to identify specific variants within the canine strain.

In December 1994, TDH began using a PCR (polymerase chain reaction) process with specific primers, which gave scientists the ability to differentiate the variants. In late 1995, a general primer was substituted and the restriction enzyme analysis step was added. The PCR produces an amplified "picture" of the virus' genetic makeup. The restriction enzyme analysis step cuts the "amplicon" into unique DNA fragment patterns (sometimes referred to as DNA fingerprints). This allows for specific variant identification. These methods empowered the laboratory to identify virtually all variants in the three major Texas rabies categories.

The laboratory methods described above were developed by the CDC in Atlanta and brought to TDH through the efforts of TDH's laboratory and Zoonosis Control Division (ZCD). The molecular epidemiologist with the Zoonosis Control Division is responsible for genetic typing of rabies virus variants. Technical assistance is provided by the CDC. The project is funded through federal grants. The methodology gives TDH the capability to accurately track the spread of the DDC and TF variants and improve the effectiveness and cost efficiency of TDH's Oral Rabies Vaccination Program (ORVP). The ORVP program utilizes geographically strategic airdrops of oral vaccine baits to immunize the primary carriers (coyotes and gray foxes respectively) of the disease.

In 2002, TDH began using DNA sequencing techniques to further advance rabies variant typing. Prior to 2002, all sequencing was performed at the CDC. This advanced procedure can distinguish between rabies variants based solely on their genetic code. Genetic sequences of the virus can be quickly and accurately identified within 2-3 days, no longer requiring a referral to the CDC. This procedure is especially useful for typing bat rabies variants, which are often difficult to identify through other molecular methods.

Abstract. Public health laboratories are facing critical issues, not the least of which is the problem of accurate surveillance of all of the rabies virus variants throughout the U.S. and other countries. The Rabies Steering Committee, which met at the CDC in Atlanta in 1994, recognized the utility of antigenic and molecular typing of virus variants in rabies surveillance and suggested an expansion of typing capabilities. The successful collaborations between the CDC and the Texas rabies laboratory over the last 15 years* produced the typing data to identify rabies variants common to animal reservoirs in the southwestern U.S. and Mexico and mapped their geographic distribution. Appropriately used, this knowledge should allow those who survey rabies to recognize when established reservoirs enlarge or expand into new areas or when different animal species become involved in cycles of rabies virus transmission. Key elements in this type of disease surveillance are a broad based sample collection system and the ability to process a large number of samples in a timely fashion. Methods of sample collection and rabies diagnostic testing already exist through public health laboratories in all 50 states; however, few of these laboratories have the expertise or operating budget to extend their laboratory testing to virus typing. The rabies laboratory at the CDC types several hundred samples each year, but does not have the staff to characterize the thousands of rabies positive animals identified each year in state laboratories. It is in this latter area that regional laboratories can expand and complement the flow of national surveillance data by increasing their surveillance activities to include antigenic and molecular typing of virus samples from the surrounding states.

This presentation will describe historical perspectives of rabies virus typing; discuss the process of program implementation; illustrate examples of various identified rabies virus variants; and assess the benefits associated with a regional reference laboratory. It is critical that all of the states function and work together if we are to augment a more vigilant surveillance system and the Texas Department of Health is in the unique position to lead the way.

*Smith, J.S., et al. 1986. Demonstration of antigenic variation among rabies virus isolates by using monoclonal antibodies to nucleocapsid proteins. J. Clinical Micro. 24:573-580.

Smith, J.S., D.B. Fishbein, C.E. Rupprecht, and K. Clark. 1991. Unexplained rabies in three immigrants in the United States. A virologic investigation. N. Engl. J. Med. 324:205-211.

Clark, K.A.,, S.U. Neill, J.S. Smith, et al. 1994. Epizootic canine rabies transmitted by coyotes in south Texas. J. Am. Vet. Med. Assoc. 204:536-540.

Rohde R.E., S.U.Neill, K.A. Clark, and J.S. Smith. 1997. Molecular epidemiology of rabies epizootics in Texas. Clin Diagn Virol. 8:209-217.

Fearneyhough, M.G. , et al. 1998. Results of an oral rabies vaccination program for coyotes. J Am Vet Med Assoc. 212, No. 4: 498-502.

Rohde R.E. and C. Hodges. 1998. Healthy Texans: The Veterinary Perspective. Texas Veterinarian 60, No 2: p. 16.

Sabouraud, A., J.S. Smith, L.A. Orciari, C. de Mattos, C. de Mattos, and R.E. Rohde. 1999. Typing of rabies virus isolates by DNA enzyme immunoassay. J Clin Virol. 12: 9-19.

Current contact information for Senior Author:

Rodney E. Rohde, Ph.D, MS, SV/SM/MB (ASCP)^CM
Assistant Professor
Texas State University-San Marcos
Clinical Laboratory Science, HSC 361
601 University Drive
San Marcos, TX 78666-4616
512-245-2562
512-245-7860 (fax)
Email: rrohde@txstate.edu
Texas State website: https://rodneyerohde.wp.txstate.edu/

This abstract was presented at the Rabies Committee Meeting, United States Animal Health Association Conference, on October 13, 1999, and at the 10th Annual Rabies in the Americas Meeting, on November 15, 1999.