Molecular epidemiology of rabies epizootics in Texas

Background: Texas is in the midst of two independent epizootics of rabies, involving coyotes (Canis latrans) and domestic dogs (Canis familiaris) in southern Texas and grey foxes (Urocyon cinereoargenteus) in west central Texas. The domestic dog/coyote (DDC) and grey fox (TF) rabies virus variants cannot be differentiated by antigenic typing with currently available monoclonal antibodies. These two variants also cannot be distinguished from a third variant, Sonora dog (SD) rabies, that is not enzootic in Texas, but occasionally occurs in animals along the western border with Mexico.

Objectives: To determine a method for the differentiation of the DDC, TF and SD variants, which is essential for epidemiologic monitoring of the Oral Rabies Vaccination Program (ORVP), a program instituted to control rabies in coyotes and grey foxes in Texas.

Study Design: Primers complementary to nucleoprotein sequence of either the DDC or TF rabies virus permit specific reverse transcription and amplification by polymerase chain reaction. In addition, general primers, which recognize a broad range of rabies variants, used in conjunction with a restriction digest for the differentiation of DDC, TF or SD rabies virus were investigated.

Results and Conclusions: Of 122 specimens tested with specific primers, 111 (91%) were specifically identified as either DDC (33 samples) or TF (78 samples). Overly stringent conditions, enzyme inhibitors, or limiting RNA may account for the 11 non-amplifications. Amplification of RNA under less stringent conditions, with primers recognizing a broad range of rabies variants followed by digestion with either restriction enzyme Desulfovibrio desulfuricans I (DdeI) or Haemophilus influenzae Rf. (HinfI), was used to identify the 11 isolates that did not amplify with specific primers (6 DDC, 4 TF and 1 SD). In addition to these 11 isolates, the less stringent method of amplification, followed by enzyme digestion has identified a total of 125 additional specimens (26 DDC, 94 TF and 5 SD) that were not tested by variant-specific amplification. These data provide a means to track the spread of the different rabies virus variants and allow the ORVP to plan its vaccine disbursement by defining the two epizootic boundaries.