Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, there is a need to validate additional diagnostic techniques to improve rabies surveillance, particularly in developing countries. Here, we present a standard protocol for the DRIT as an alternative, laboratory or field-based testing option that uses light microscopy as compared to the DFA. Touch impressions of brain tissue collected from suspect animals are fixed in 10% buffered formalin. The DRIT uses rabies virusspecific monoclonal or polyclonal antibodies (conjugated to biotin), a streptavidin-peroxidase enzyme, and a chromogen reporter (such as acetyl 3-amino-9-ethylcarbazole) to detect viral inclusions within infected tissue. In approximately 1 h, a brain tissue sample can be tested and interpreted by the DRIT. Evaluation of suspect animal brains tested from a variety of species in North America, Asia, Africa, and Europe have illustrated high sensitivity and specificity by the DRIT approaching 100% with results compared to DFA. Since 2005, the United States Department of Agriculture's Wildlife Services (USDA WS) program has conducted large-scale enhanced rabies surveillance efforts using the DRIT to test >94,000 samples collected from wildlife in strategic rabies management areas. The DRIT provides a powerful, economical tool for rabies diagnosis that can be used by laboratorians and field biologists to improve current rabies surveillance, prevention and control programs globally.

Rabies remains a globally significant zoonotic disease, but rabies control is achievable under certain circumstances.Canine rabies has been eliminated from the U.S.; however, approximately 55,000 humans die annually worldwide from the disease. In the U.S., economic losses continue to be substantial and the risk to humans and domestic animals has not been eliminated. As an example of the complexity of rabies management, we describe a local rabies control program and efforts to restore Cape Cod, MA to terrestrial rabies-free status, after a 2004 oral rabies vaccination (ORV) barrier breach following 10 years of rabies-free status. The emergence of raccoon rabies in southeastern New England in 1992 prompted the U.S. Centers for Disease Control and Prevention, the Tufts Cummings School of Veterinary Medicine, and the Massachusetts Department of Public Health to begin an ORV program to reduce the occurrence of carnivore rabies in an area directly adjacent to the Cape Cod Canal. In 2001, USDA APHIS Wildlife Services began full-time collaboration on the Cape Cod Oral Rabies Vaccination Program (CCORVP) as part of national wildlife rabies control efforts. The primary objective of the CCORVP was to use ORV in tandem with the physical barrier created by the Canal to prevent the spread of rabies to peninsular Cape Cod, a heavily-populated tourist destination southeast of Boston. After an increase in rabies cases within the traditional Cape Cod ORV zone, ORV bait distribution efforts were modified to reduce the risk of rabies spread onto the Cape. In spite of these modifications, raccoon rabies was detected for the first time on peninsular Cape Cod in March 2004. A trap-vaccinate-release campaign, removal of suspect raccoons and skunks, and expanded ORV efforts were unsuccessful in preventing the spread of the virus. Rabies surveillance became the priority of the Cape Cod Rabies Task Force. In 2006, rabies was finally detected at the eastern extremity of the peninsula. In this paper, we summarize ORV efforts, explore possible causes for the spread of raccoon rabies onto the Cape, summarize several small-scale Cape Cod rabies research projects, and suggest a 5-year plan for future Cape Cod rabies controls efforts.

Knowledge of the rate and extent of spread of epizootic diseases is critical to facilitate effective management. Terrestrial rabies was first detected in spring 2004 on Cape Cod Massachusetts, compromising a long-standing ORV zone established from the west side of the Cape Cod Canal to serve as a barrier to raccoon rabies spread onto the Cape. In March 2004, USDA Wildlife Services and local and state cooperators implemented a surveillance program to track the spread of rabies on Cape Cod for planning contingency action strategies aimed at containment and elimination. During 13 months of enhanced rabies surveillance, 198 (167 raccoons and 29 skunks) out of 942 specimens tested positive for rabies. We discuss management implications of these results to the Cape Cod Oral Rabies Vaccination program and to other integrated rabies control programs.

Efforts to eliminate the raccoon variant of the rabies virus (raccoon rabies) in the eastern United States by USDA, APHIS, Wildlife Services and cooperators have included the distribution of oral rabies vaccine baits from polyvinyl chloride (PVC) bait stations in west-central Florida from 2009 to 2015. Achieving sufficient vaccine bait uptake among urban raccoons is problematic, given limitations on aerial and vehicle-based bait distribution for safety and other reasons. One or three bait stations/km2 were deployed across four 9-km2 sites within rural and urban sites in Pasco and Pinellas Counties, Florida. Based on tetracycline biomarker analysis, bait uptake was only significantly different among the urban (Pinellas County) high and low bait station densities in 2012 (p = 0.0133). Significant differences in RVNA were found between the two bait station densities for both urban 2011 and 2012 samples (p = 0.0054 and p = 0.0031). Landscape differences in terms of urban structure and human population density may modify raccoon travel routes and behavior enough for these differences to emerge in highly urbanized Pinellas County, but not in rural Pasco County. The results suggest that, in urban settings, bait stations deployed at densities of >1/km2 are likely to achieve higher seroprevalence as an index of population immunity critical to successful raccoon rabies control.