The authors would like to acknowledge Coconino County Health Services, especially Sabrina Kelley, for facilitating the implementation of the rabies quarantine. Additionally, the authors would like to recognize Dennis Slate and Richard Chipman for facilitating the TVR program and Tad Theimer for advice on trapping. The project was entirely funded by the US Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife Services, and the National Rabies Program.

The safe handling and care of animals demonstrated in this protocol comply with the standards put in place by APHIS Wildlife Services.
1. Vaccination protocol
<ol>
	<li>Preparation
	<ol>
		<li>Don PPE, including face shield/goggles, nitrile gloves, and coveralls.</li>
		<li>Remove a pair of numbered ear tags from the package to mark the captured animal. Record the ear tag numbers on the datasheet and place them in a resealable plastic bag labeled with the ear tag number to keep them safe and organized until they are ready to be deployed.</li>
		<li>Quietly and calmly approach the trap. If a skunk has been captured, use a piece of tarp, burlap, or plastic sheeting to cover the trap and shield oneself from any skunk spray.</li>
		<li>Carefully wrap the cover around the trap and use a steel spring clamp to secure it to the trap. Cover is not required for a raccoon or fox.</li>
		<li>Carefully position the trap on its end with the door of the trap facing up (towards the sky).</li>
	</ol>
	</li>
	<li>Vaccination
	<ol>
		<li>Once the captured animal has moved to the bottom of the trap, open the door and place the wooden plunger inside the trap on the animal&#39;s body.</li>
		<li>Apply gentle pressure on the animal to prevent the animal from moving freely. Use mechanical immobilization to safely vaccinate and mark the animal.</li>
		<li>Retrieve the rabies vaccine from the cooler and draw the correct dosage (1 mL) into the 3 mL syringe. Place the ear tags in the sandwich bag for the animal in an easily accessible location.</li>
		<li>Carefully pull back the cover around the trap to locate a region to administer the vaccine.</li>
		<li>Carefully insert the needle into the animal&#39;s muscle tissue in its hind leg and smoothly depress the plunger to administer the rabies vaccine.</li>
		<li>Dispose of the used syringe in a sharps container.</li>
		<li>Position the trap so the ears are accessible, and place one metal ear tag into the locating groove on the pliers.</li>
		<li>Position the open end of the ear tag over the ear and apply even and gentle pressure until the plier handles stop. Repeat the sequence on the other ear.</li>
		<li>With the trap on its end, remove the plunger, close the trap door, and attach the hook end of the handheld scale to the trap. Gently lift the trap and hold steady. Record the weight of the trap and animal on the datasheet.</li>
		<li>Lay the trap down flat and position the cover so that it is no longer wrapped around the trap but lying over the top of the trap.</li>
		<li>Gently lift the trap off the ground using the handle and position the handheld mirror under the trap. Using the animal&#39;s reflection in the mirror reflection, determine the sex of the animal. Record on the datasheet. 
		&#8203;NOTE: A mirror is not necessary to sex a fox or a raccoon.</li>
		<li>Estimate the age of the animal as either adult or juvenile. Record on the datasheet.</li>
		<li>Finish filling out the datasheet. A metal clipboard with a storage area best protects the sheets in inclement weather and keeps them in one place during the trapping session.</li>
		<li>Use a metal clamp to hold the trap door in the open position. Slowly back away and let the animal exit the trap unpressured.</li>
		<li>Once the animal has exited, reweigh the empty trap, ensuring that the clips and protective cover remain with it.&#160;Subtract this weight from the weight recorded earlier to calculate the animal&#39;s weight.</li>
	</ol>
	</li>
</ol>

<ol><li>Rosatte, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+elimination+of+raccoon+rabies+from+Wolfe+Island%2C+Ontario%3A+animal+density+and+movements%5BTitle%5D)+AND+%22Journal+of+Wildlife+Diseases%22%5BJournal%5D)">The elimination of raccoon rabies from Wolfe Island, Ontario: animal density and movements.</a> Journal of Wildlife Diseases. 43 (2), 242-250 (2007).</li>
<li>Marston, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetic+analysis+of+a+rabies+virus+host+shift+event+reveals+within-host+viral+dynamics+in+a+new+host%5BTitle%5D)+AND+%22Virus+Evolution%22%5BJournal%5D)">Genetic analysis of a rabies virus host shift event reveals within-host viral dynamics in a new host.</a> Virus Evolution. 3 (2), vex038(2017).</li>
<li>Freuling, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+elimination+of+fox+rabies+from+Europe%3A+determinants+of+success+and+lessons+for+the+future%5BTitle%5D)+AND+%22Philosophical+Transactions+of+the+Royal+Society+B%3A+Biological+Sciences%22%5BJournal%5D)">The elimination of fox rabies from Europe: determinants of success and lessons for the future.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 368, 20120142(2013).</li>
<li>Slate, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oral+rabies+vaccination+in+North+America%3A+opportunities%2C+complexities%2C+and+challenges%5BTitle%5D)+AND+%22PLoS+Neglected+Tropical+Diseases%22%5BJournal%5D)">Oral rabies vaccination in North America: opportunities, complexities, and challenges.</a> PLoS Neglected Tropical Diseases. 3 (12), e549-e549 (2009).</li>
<li>Wohlers, A., Lankau, E. W., Oertli, E. H., Maki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Challenges+to+controlling+rabies+in+skunk+populations+using+oral+rabies+vaccination%3A+A+review%5BTitle%5D)+AND+%22Zoonoses+and+Public+Health%22%5BJournal%5D)">Challenges to controlling rabies in skunk populations using oral rabies vaccination: A review.</a> Zoonoses and Public Health. 65 (4), 373-385 (2018).</li>
<li>Sterner, R. T., Meltzer, M. I., Shwiff, S. A., Slate, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tactics+and+economics+of+wildlife+oral+rabies+vaccination%2C+Canada+and+the+United+States%5BTitle%5D)+AND+%22Emerging+Infectious+Diseases%22%5BJournal%5D)">Tactics and economics of wildlife oral rabies vaccination, Canada and the United States.</a> Emerging Infectious Diseases. 15 (8), 1176-1184 (2009).</li>
<li>Elmore, S. A., Chipman, R. B., Slate, D., Huyvaert, K. P., VerCauteren, K. C., Gilbert, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Management+and+modeling+approaches+for+controlling+raccoon+rabies%3A+The+road+to+elimination%5BTitle%5D)+AND+%22PLOS+Neglected+Tropical+Diseases%22%5BJournal%5D)">Management and modeling approaches for controlling raccoon rabies: The road to elimination.</a> PLOS Neglected Tropical Diseases. 11, e0005579(2017).</li>
<li>Kuzmin, I. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+inferences+suggest+multiple+host+shifts+of+rabies+viruses+from+bats+to+mesocarnivores+in+Arizona+during+2001-2009%5BTitle%5D)+AND+%22PLoS+Pathogens%22%5BJournal%5D)">Molecular inferences suggest multiple host shifts of rabies viruses from bats to mesocarnivores in Arizona during 2001-2009.</a> PLoS Pathogens. 8 (6), e1002786(2012).</li>
<li>Shwiff, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+and+indirect+costs+of+rabies+exposure%3A+a+retrospective+study+in+southern+California+%281998-2002%29%5BTitle%5D)+AND+%22Journal+of+Wildlife+Diseases%22%5BJournal%5D)">Direct and indirect costs of rabies exposure: a retrospective study in southern California (1998-2002).</a> Journal of Wildlife Diseases. 43 (2), 251-257 (2007).</li>
<li>Shwiff, S. A., Elser, J. L., Ernst, K. H., Shwiff, S. S., Anderson, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cost-benefit+analysis+of+controlling+rabies%3A+placing+economics+at+the+heart+of+rabies+control+to+focus+political+will%5BTitle%5D)+AND+%22OIE+Scientific+and+Technical+Review%22%5BJournal%5D)">Cost-benefit analysis of controlling rabies: placing economics at the heart of rabies control to focus political will.</a> OIE Scientific and Technical Review. 37 (2), 681-689 (2018).</li>
<li>Rosatte, R. C., Power, M. J., Machines, C. D., Campbell, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trap-vaccinate-release+and+oral+vaccination+for+rabies+control+in+urban+skunks%2C+raccoons+and+foxes%5BTitle%5D)+AND+%22Journal+of+Wildlife+Diseases%22%5BJournal%5D)">Trap-vaccinate-release and oral vaccination for rabies control in urban skunks, raccoons and foxes.</a> Journal of Wildlife Diseases. 28 (4), 562-571 (1992).</li>
<li>Slavinski, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trap-vaccinate-release+program+to+control+raccoon+rabies%2C+New+York%2C+USA%5BTitle%5D)+AND+%22Emerging+Infectious+Diseases%22%5BJournal%5D)">Trap-vaccinate-release program to control raccoon rabies, New York, USA.</a> Emerging Infectious Diseases. 18 (7), 1170-1172 (2012).</li>
<li>Rosatte, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+control+of+raccoon+rabies+in+Ontario+Canada%3A+Proactive+and+reactive+tactics%2C+1994-2007%5BTitle%5D)+AND+%22Journal+of+Wildlife+Diseases%22%5BJournal%5D)">The control of raccoon rabies in Ontario Canada: Proactive and reactive tactics, 1994-2007.</a> Journal of Wildlife Diseases. 45 (3), 772-784 (2009).</li>
<li>Rosatte, R. C., Howard, D. R., Campbell, J. B., Maclnnes, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intramuscular+vaccination+of+skunks+and+raccoons+against+rabies%5BTitle%5D)+AND+%22Journal+of+Wildlife+Diseases%22%5BJournal%5D)">Intramuscular vaccination of skunks and raccoons against rabies.</a> Journal of Wildlife Diseases. 26 (2), 225-230 (1990).</li>
</ol>

The authors do not have any conflicts of interest to disclose. Reference in this site to any specific commercial product, process, or service, or the use of any trade, firm, or corporation name is for the information and convenience of the public and does not constitute endorsement, recommendation, or favoring by the US Department of Agriculture.

The Centers for Disease Control and Prevention estimates that approximately 55,000 Americans receive post-exposure prophylaxis (PEP) each year, with the average cost for each exposure around $3,6009. The main economic impacts associated with rabies are the cost of managing the disease and the treatment for preventing human fatalities after exposure. The key to reducing health issues and economic impacts is the successful vaccination of terrestrial reservoir species10. A TVR program is a management tool for intervention in outbreaks threatening human health, domestic animals, and wildlife. TVR is most effective in addressing small-scale rabies outbreaks as it is very costly and labor-intensive to employ at larger landscape scales6. Despite these drawbacks, TVR is a valuable rabies management tool that has repeatedly been shown to be effective at stopping the spread of the disease by vaccinating reservoir species11,12. The TVR protocol demonstrated in this manuscript is in response to rabies outbreaks in the city of Flagstaff, Arizona, which has experienced repeated big brown RABV outbreaks in striped skunk populations since 20018. This TVR program was based upon a TVR study conducted by Rosatte in which skunks and raccoons were intramuscularly vaccinated against rabies in metropolitan Ontario13. This protocol addressed how to safely and successfully vaccinate captured striped skunks and other mesocarnivores.
One of the most important steps in this protocol is to securely immobilize the animal. The animal needs to be secure, but the force of immobilization should not harm the animal in any way. Proper immobilization ensures the animal receives a full dose of the vaccine (1 mL) by preventing the animal from pulling away during the injection. Additionally, a secure animal is unable to bite and claw at the tools used to install the ear tags. This helps reduce the stress put on each animal by shortening the time required to process each animal.
One challenge we encountered was vaccinating and ear-tagging skunks captured in the large (10 inches x 12 inches x 32 inches) traps. This process can be difficult, as it gives the skunks more room to pull away from the sides of the trap and out of reach of the tools. This issue was even more apparent when juvenile skunks were caught. These situations required some modification of our processing technique. In these situations, we would use the wooden plunger for the small traps to trap the skunk in a corner of the trap and process them from above through the door opening. This made it possible to process the skunks, but it still proved to be challenging.
We documented skunk captures in all three-sized traps. Raccoon captures were documented in medium and large traps, and grey foxes were captured exclusively in the large traps. There is a tendency for some animals to become accustomed to the traps and be recaptured frequently. The constant movement of traps to new locations helps minimize recaptures and gives the opportunity to capture and vaccinate new individuals on the landscape.
One limitation of TVR is the size of the area that can be effectively treated. In large-scale rabies management operations, TVR is often used in conjunction with ORV to cover large areas of the landscape. TVR, by itself, covers a much smaller area and is best utilized in urban areas as a fast emergency response, or in our case, addressing rabies outbreaks in skunk populations. TVR efforts can prove to be costly as well, with estimates of $625.00 per km2 treated12. These costs can vary significantly as larger areas require more personnel and equipment, subsequently increasing costs.
Previous work determined that inoculation with 1 mL of rabies vaccine was effective at stimulating rabies antibody production in mesocarnivores14. There are several papers based on TVR that mention the vaccination of animals1,12,13,14, but we could not find any published articles offering a detailed explanation of the protocol for administering the vaccine. Therefore, we could not compare the protocol offered in this manuscript to existing methods. The materials and methods shared in this manuscript will allow field crews to deliver precise, effective vaccinations to wildlife populations. This protocol has proven effective and will continue to be used in ongoing and future TVR efforts.

The rabies virus (RABV) is a deadly zoonotic disease that impacts human health on a global scale. The World Health Organization (WHO) reports that approximately 59,000 people are killed each year, predominantly in Asian and African countries. Additionally, 29 million people received a post-exposure vaccination each year due to being bitten by an animal. Impacts on wildlife populations cannot be accurately measured, but wildlife populations can experience high rates of mortality during outbreak events when geospatial conditions are favorable. Rosatte reported that the raccoon (Procyon lotor) population on 124 sq km (48 sq mi) declined about 71% in the months following a reported RABV outbreak1.
The control and management of RABV are essential for protecting human health, domestic animals, and vulnerable wildlife populations. Since all mammals are susceptible to the RABV, over time, RABV has adapted to several distinct mammalian species, which now act as reservoir hosts. These host species and their associated variants typically inhabit specific geographic regions. Within these regions, spillover into non-reservoir hosts does occur (cross-species transmission), but in most cases, the infected non-reservoir species do not carry the infection to other individuals in the population. A host shift is a rare event in which the virus has successfully adapted to a new host. These events are the most dangerous, as they can influence the spread of RABV and undermine any RABV management efforts put in place2.
In North America, RABV management efforts are focused on several specific terrestrial wildlife reservoir species. Oral rabies vaccination (ORV) programs have proven to be effective at managing rabies at large landscape scales. ORV utilizes aerial and hand deployment of small baits that contain an oral RABV vaccine. These baits are distributed on the landscape; any targeted wildlife that encounters these packets receives RABV vaccine orally when they bite into them. The use of ORV has been essential in the control of red fox (Vulpes vulpes) RABV in Europe3, raccoon RABV&#160;in Canada, and coyotes (Canis latrans)&#160;and raccoons in the United States4. ORV baits have proven to be a valuable tool in RABV management programs, as they can be implemented over vast regions of the landscape and reduce the number of personnel needed in management programs. One drawback of the ORV program is its ineffectiveness in successfully vaccinating skunk species. Inadequate vaccine uptake from current ORV packets seems to be the current setback for ORV effectiveness in wild skunk populations in the field5.
Trap-vaccinate-release (TVR) is an alternative rabies management tool, that is occasionally used simultaneously with ORV. The goal of TVR is to increase the number of rabies vaccinated individuals in a population. Because it requires labor-intensive trapping of animals and is more expensive than ORV per square kilometer6, TVR is done on a much smaller scale than ORV and is often applied in urban areas where many challenges to ORV exist7.
Trap-vaccinate-release of skunks in the United States has been effective in Flagstaff, Arizona, on several occasions. Since 2001, Flagstaff has experienced repeated outbreaks of big brown bat (Eptesicus fuscus) RABV variant in striped skunk (Mephitis mephitis) populations8. From 2001-2011 (in multiple distinct TVR efforts led by the US Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife Services), 842 animals were trapped, vaccinated, and released, including 753 striped skunks (Wildlife Services Capture Data). Each effort stopped the bat rabies outbreaks in skunks. In 2021, this cross-species phenomenon happened again when rabid striped skunks were confirmed in two heavily urbanized regions of Flagstaff. Wildlife Services immediately coordinated and launched a TVR program in these areas to contain the outbreak in cooperation with the Coconino Department of Health Services and several state agencies. The goal of this manuscript is to safely demonstrate the steps in a TVR program targeting skunks.
The equipment required for trapping and vaccination are as follows:
TVR field equipment 
A detailed list of all the equipment used can be found in the accompanying Table of Materials file. However, there needs to be some clarification on the &#34;wooden plungers&#34; utilized. These are handmade devices comprised of a wooden dowel for the handle. Fastened to the end of the dowl is a piece of plywood cut to the dimensions of the trap opening. Since we utilized three different trap sizes, we constructed plungers of the following dimensions (6 inches x 6 inches, 8 inches x 8 inches, 10 inches x 10 inches) (Figure 1). Additionally, trap covers are only required when working up a skunk. Trap cover material can be canvas, an old tarp, or disposable plastic sheeting like we used.
Personal protective equipment (PPE) 
Field crews are required to have received pre-exposure rabies vaccinations prior to participating in any TVR program. PPE necessary for TVR work includes eye protection such as a disposable safety shield, lab goggles, or safety glasses. A disposable coverall suit is the best protection from the many different volatile compounds contained in a skunk&#39;s spray. (Note: suit not required when working up a raccoon or fox). Disposable nitrile gloves protect from various chemicals and liquids and offer protection from saliva and body fluids containing pathogens from captured animals.
Trap preparation and deployment 
We utilized three sizes of live capture traps: small (7 inches x 7 inches x 24 inches), medium (9 inches x 9 inches x 26 inches), and large (10 inches x 12 inches x 32 inches) (Figure 1). Prior to traps being deployed in the field, they should be washed thoroughly with soap and water. Traps should be left out in the open air for several weeks to months to allow for odor elimination. Triggering mechanisms are tested and repaired when required prior to setting. Warning labels stating &#34;Rabies Vector&#34; are installed on each trap with trap wire. An additional rabies warning sign is posted in the general vicinity of each trap; this secondary warning provides general trapping information and provides program contact information. Traps are placed in or near animal travel routes and feeding locations. Locations should be selected that minimize stress to the animal, including weather-related events and disturbance by human activities. An established trapping location receives a unique name, and the location is logged in a handheld GPS unit.

<table><tbody><tr><td>#8 x 1-1/2 in. R4 Multi-Purpose Star Drive Flat Head Screw (110 per Pack)</td><td>Home Depot</td><td>96085</td><td>Used to attach base to plunger push rod</td></tr><tr><td>#8 x 1-1/4 in. #2 Phillips, Wafer-Head Wood Screw (100-Pack)</td><td>Home Depot</td><td>SD8X1.25-R</td><td>Used to attach trap sign to stake</td></tr><tr><td>2&amp;nbsp; in. x 2 in. x 8 ft. Furring Strip Board Lumber</td><td>Home Depot</td><td>75800593</td><td>Push rod for plunger</td></tr><tr><td>20 G&amp;nbsp; x 1.5 &quot; needle</td><td>Kendall monoject</td><td>8881</td><td>Used to vaccinate animal</td></tr><tr><td>3 mL syringe</td><td>Kendall monoject</td><td>513934</td><td>Used to vaccinate animal</td></tr><tr><td>48 oz. Steel Drilling Hammer</td><td>Home Depot</td><td>MRF3LB</td><td>Used to put trap sign stake into the ground</td></tr><tr><td>7-1/4 in. x 140-Tooth OSB/Plywood Circular Saw Blade</td><td>Home Depot</td><td>A07140A</td><td>Blade needed for circular saw to cut plywood</td></tr><tr><td>Aluminum Clipboard - Top Opening, Letter</td><td>ULINE</td><td>S-20173</td><td>Used to record and keep datasheets</td></tr><tr><td>Circular saw</td><td>Home Depot</td><td>CSB144LZK</td><td>Used to cut wood to make plunger</td></tr><tr><td>Cold Packs - 6 oz</td><td>ULINE</td><td>S-9902</td><td>Used to regulate temperature of the vaccine</td></tr><tr><td>Dish soap</td><td>ULINE</td><td>S-9975</td><td>Used to clean traps</td></tr><tr><td>Dupont Tyvek&amp;nbsp;protective clothing</td><td>ULINE</td><td>S-11495</td><td>Used to protect personnel from skunk spray. Size will be dependent upon size of personnel. You can also use the non-hooded coverall S-9636.</td></tr><tr><td>Handheld GPS unit</td><td>Garmin</td><td>ETrex 32X</td><td>Used to record location of trap and captured animals</td></tr><tr><td>Handheld mirror 6 in. x 7 in.</td><td>Walmart</td><td>Equate</td><td>Used to sex animal</td></tr><tr><td>Headlamp</td><td>Home Depot</td><td>HDB32EH</td><td>To check traps before sunup</td></tr><tr><td>Jar of peanut butter</td><td>Walmart</td><td>40 oz jar</td><td>Bait for trapping skunks.&amp;nbsp;</td></tr><tr><td>Live trap for skunks</td><td>Tomahawk</td><td>103</td><td>Used to catch skunks</td></tr><tr><td>Live trap for skunks and other carnivores</td><td>Tomahawk</td><td>205; 602</td><td>Used to catch skunks and other carnivores</td></tr><tr><td>Nitrile gloves</td><td>ULINE</td><td>S-14179</td><td>Used to protect personnel from pathogens. Size and use of powdered or non-powdered will be dependent on personnel choice.</td></tr><tr><td>ONE+ 18 V Cordless 3/8 in. Drill/Driver Kit with 1.5 Ah Battery, Charger, Hand Tools, Accessories, and Tool Bag</td><td>Home Depot</td><td>PCL201K1SB</td><td>Used to assemble signs on stakes</td></tr><tr><td>Paper Mate Profile 8 pk Ballpoint Pens 1.4 mm Bold Tip Black</td><td>Staples</td><td>2095460</td><td>Used to write on data sheets</td></tr><tr><td>Rabies trapping sign 9 in. x 12 in.</td><td>FedEx Office</td><td>NA</td><td>Attach to stake near trap location</td></tr><tr><td>Rabies vaccine</td><td>Boehringer Ingelheim</td><td>Imrab&amp;reg;</td><td>1 mL per animal; https://www.boehringer-ingelheim.com/animal-health/companion-animals-products/rabisin-imrab#:~:text=Rabisin%C2%AE%20%2F%20Imrab%C2%AE%20is,signs%20due%20to%20rabies%20infection.</td></tr><tr><td>Rabies warning sign 4 in. x 6 in.</td><td>FedEx Office</td><td>NA</td><td>Attach to top of trap</td></tr><tr><td>Raccoon and fox ear tag</td><td>National Band and Tag Company</td><td>1005-3</td><td>Ear tags for raccoons and foxes</td></tr><tr><td>Raccoon and fox ear tag</td><td>National Band and Tag Company</td><td>1005s3</td><td>Ear tap pliers to attach ear tags to raccoons and foxes</td></tr><tr><td>Refresh 6-Pack Lunch Cooler</td><td>4imprint.com</td><td>155149</td><td>Used to carry vaccine in the field</td></tr><tr><td>Roll of plastic sheeting 12 ft. x 400 ft.</td><td>3M</td><td>6723</td><td>Used to calm animal and protect personnel</td></tr><tr><td>Safety goggles or disposible face shield</td><td>ULINE</td><td>S-24607</td><td>Used to protect personnel face from skunk spray and pathogens.</td></tr><tr><td>Sanded Plywood (Common: 1/2 in. x 2 ft. x 4 ft.; Actual: 0.451 in. x 23.75 in. x 47.75 in.)</td><td>Home Depot</td><td>300896</td><td>Base for plunger</td></tr><tr><td>Scale 55 lb x 0.5 oz</td><td>Brecknell</td><td>Model 816965007486</td><td>Used to weigh cage and animal</td></tr><tr><td>Sharpie Black Fine Tip Permanent Marker 1 pk</td><td>Ace Hardware</td><td>35010</td><td>Used to label sandwich bags</td></tr><tr><td>Sharps Container - 1 Quart</td><td>ULINE</td><td>S-15307</td><td>Holding used syringes</td></tr><tr><td>Skunk ear tag</td><td>National Band and Tag Company</td><td>1005-1</td><td>Ear tags for skunks</td></tr><tr><td>Skunk ear tag applicator</td><td>National Band and Tag Company</td><td>1005s1&amp;nbsp;</td><td>Ear tag pliers to attach ear tags to skunks</td></tr><tr><td>Tap water</td><td>NA</td><td></td><td>Used to clean traps</td></tr><tr><td>Trap line wire</td><td>Minnesota Trap Line Products, INC</td><td>11GA-01</td><td>Trap wire to attach sign to trap https://www.minntrapprod.com/Snares/departments/577/</td></tr><tr><td>Universal Forest 48 in. x 1 in. x 2 in. Wood Grade Stake 24 pk</td><td>Ace Hardware</td><td>5606413</td><td>Stake for trap sign&amp;nbsp;</td></tr><tr><td>XM Series 3 in. Capacity Steel Spring Clamp with Handles and Tips, 3-1/3 in. Throat Depth</td><td>Home Depot</td><td>XM7</td><td>Used to attach plastic sheeting to trap or to hold trap door open</td></tr><tr><td>Ziploc Sandwich Bags</td><td>ULINE</td><td>S-23777</td><td>Used to hold ear tags</td></tr></tbody></table>

a trap-vaccinate-release protocol for immunization of skunks and additional rabies vectors against rabies

Oral vaccination of wildlife against rabies via the distribution of vaccine-laden baits is used widely as a management tool in Europe and North America. Over the past several decades, successful programs have targeted important reservoirs, including coyotes, foxes, raccoon dogs, and raccoons, for prevention and control. However, other species (e.g., skunks) or certain habitats (e.g., urban) may be less amenable to oral vaccination, requiring consideration of additional strategies. Trap-vaccinate-release (TVR) is one alternate method that entails the use of live traps to capture targeted wildlife. Traps are distributed along grids in suitable, accessible habitats. Captured animals are identified by tagging, vaccinated parenterally with an inactivated commercial vaccine, sexed, aged, weighed, and released at the site of capture. Since 2001, cross-species transmission of rabies virus from bats to mesocarnivores, such as skunks, has been detected in Flagstaff, Arizona. During the past two decades, TVR has been used as a safe and effective method to prevent and control rabies during such events in this region.

We implemented the ground-based RABV management technique called TVR to address RABV outbreaks in the city of Flagstaff, Arizona (Figure 2). The goal of this project was not to assess TVR effectiveness but rather an emergency response to vaccinate mesocarnivores inside the RABV quarantine areas and minimize any threats to public health. From August 2021 to December 2021, several sick-acting striped skunks were reported to the Flagstaff Police Department and Flagstaff Animal Control; these skunks were subsequently euthanized and tested for RABV. Dead skunks reported by the public during this period were also submitted for testing. In total, 16 striped skunks tested positive for big brown bat RABV, 4 in west Flagstaff and 12 in east Flagstaff. In response, we initiated TVR efforts in early December 2021 and documented the following vaccination success. In west Flagstaff, we vaccinated 27 striped skunks, 3 grey foxes, and 4 raccoons. Additionally, we recaptured striped skunks on 37 occasions. In east Flagstaff, we vaccinated 106 striped skunks and 2 raccoons. Additionally, we recaptured striped skunks on 110 occasions. Trapping efforts were sustained throughout the entirety of 2022. During this period, no RABV-positive skunks were detected in either trapping area. In July and August of 2022, 3 rabid skunks were detected in a neighborhood in south Flagstaff (Figure 3). We immediately coordinated and initiated TVR in this new area, where we vaccinated 22 striped skunks, 1 hog-nosed skunk, and 10 raccoons. Recaptured animals included 34 striped skunks and 1 hog-nosed skunk. Trapping in south Flagstaff began in late July and continued throughout the remainder of 2022. In the subsequent months, no additional rabid skunks were detected in south Flagstaff. An additional 15 skunks were captured by local pest control companies outside of the TVR areas. We coordinated with the pest control companies to vaccinate the animals before they were released.
Management programs such as TVR are effective for controlling RABV because they decrease the number of susceptible individuals present in a wildlife population. The results presented here (Table 1) demonstrate how effective and successful the protocol is at vaccinating wildlife against RABV. As noted, we observed no new RABV cases after TVR was implemented. (Figure 4) TVR has a distinct advantage of direct inoculation of captured animals. By contrast, ORV cannot guarantee that targeted wildlife will uptake randomly distributed baits. TVR guarantees that every captured animal will be inoculated with the vaccine. Assessing the effectiveness of these vaccinated animals at stopping the spread of the RABV is difficult, but when we attempt to analyze the results subjectively, we can determine that any threats to public health are reduced by having 190 fully vaccinated mesocarnivores in these urban areas.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Year</td>
			<td rowspan="2">Trap-nights</td>
			<td rowspan="2">Total captures</td>
			<td>Striped Skunks</td>
			<td>Raccoons</td>
			<td>Grey Foxes</td>
			<td>Hog-nosed Skunk</td>
		</tr>
		<tr>
			<td>T (V)</td>
			<td>T (V)</td>
			<td>T (V)</td>
			<td>T (V)</td>
		</tr>
		<tr>
			<td>2021 (Dec)</td>
			<td>180</td>
			<td>4</td>
			<td>4 (4)</td>
			<td>0 (0)</td>
			<td>0 (0)</td>
			<td>0(0)</td>
		</tr>
		<tr>
			<td>2022</td>
			<td>5,343</td>
			<td>367</td>
			<td>346 (166)</td>
			<td>16 (16)</td>
			<td>3 (3)</td>
			<td>2 (1)</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>5,523</td>
			<td>371</td>
			<td>350 (170)</td>
			<td>16 (16)</td>
			<td>3 (3)</td>
			<td>2 (1)</td>
		</tr>
	</tbody>
</table>
Table 1: Mesocarnivores vaccinated against RABV during TVR operations in Flagstaff, Arizona, during 2021-2022. T = total number of animals captured, V = number of different individuals successfully vaccinated and released.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65117/65117fig01.jpg" /> 
Figure 1: Trap and plunger sizes. (Left) Smallest trap 7 inches x 7 inches x 24 inches, (Middle) medium 9 inches x 9 inches x 26 inches, (Right) and large 10 inches x 12 inches x 32 inches. <a href="https://www.jove.com/files/ftp_upload/65117/65117fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65117/65117fig02.jpg" /> 
Figure 2: RABV positive skunks Flagstaff Arizona, USA 2021-2022. Map of RABV-positive skunks detected in the city of Flagstaff, Arizona, from 2021-2022. The red dots show the locations where RABV-positive skunks were detected. The blue polygons show the TVR trapping areas inside the city limits. <a href="https://www.jove.com/files/ftp_upload/65117/65117fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65117/65117fig03.jpg" /> 
Figure 3: South Flagstaff TVR area. Aerial map of an established trapping line in south Flagstaff, Arizona, during 2021. The red dots show where rabies-positive animals were detected. The green dots show the locations of traps that have not captured any animals. The blue dots show trap locations where an animal has been successfully captured, vaccinated, and released. The white numbers near the blue dots indicate how many animals have been vaccinated at that trap site. The orange dots show trap sites that have only recaptured previously vaccinated animals. The data table shows how many species have been captured and vaccinated on this trapline. <a href="https://www.jove.com/files/ftp_upload/65117/65117fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65117/65117fig04.jpg" /> 
Figure 4: RABV-positive skunks and TVR vaccinations per month in Flagstaff 2021-2022. The red line represents RABV-positive animals detected in Flagstaff from July 2021 through Dec 2022. The blue line shows the number of animals vaccinated from Dec 2021 through Dec 2022. The numbers above the blue line indicate how many mesocarnivores were vaccinated for that particular month. <a href="https://www.jove.com/files/ftp_upload/65117/65117fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Here, we present a protocol to demonstrate safe, trap-vaccinate-release (TVR) techniques for carnivores susceptible to rabies.

A Trap-Vaccinate-Release Protocol for Immunization of Skunks and Additional Rabies Vectors Against Rabies

Oral vaccination of wildlife against rabies via the distribution of vaccine-laden baits is used widely as a management tool in Europe and North America. ...

a-trap-vaccinate-release-protocol-for-immunization-skunks-additional

Research

JoVE Journal

Environment

844 Views.  US Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife Services. Here, we present a protocol to demonstrate safe, trap-vaccinate-release (TVR) techniques for carnivores susceptible to rabies.

Video: A Trap-Vaccinate-Release Protocol for Immunization of Skunks and Additional Rabies Vectors Against Rabies

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

Immunology and Infection

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

Genetics

Standard Operating Procedure for Lyssavirus Surveillance of the Bat Population in Taiwan

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are natural reservoirs of most lyssaviruses, a lyssavirus surveillance program of bats has been conducted in Taiwan since 2008 to understand the ecology of these viruses in bats. In this program, non-governmental bat conservation organizations and local animal disease control centers cooperated to collect dead bats or bats dying of weakness or illness. Brain tissues of bats were obtained through necropsy and subjected to direct fluorescent antibody test (FAT) and reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus antigens and nucleic acids. For the FAT, at least two different rabies diagnosis conjugates are recommended. For the RT-PCR, two sets of primers (JW12/N165-146, N113F/N304R) are used to amplify a partial sequence of the lyssavirus nucleoprotein gene. This surveillance program monitors lyssaviruses and other zoonotic agents in bats. Taiwan bat lyssavirus is found in two cases of the Japanese pipistrelle (Pipistrellus abramus) in 2016&#8211;2017. These findings should inform the public, health professionals, and scientists of the potential risks of contacting bats and other wildlife.

This protocol introduces a standard laboratory operating procedure for diagnostic testing of lyssavirus antigens in bats in Taiwan.

Viruses within the genus Lyssavirus are zoonotic pathogens, and at least seven lyssavirus species are associated with human cases. Because bats are ...

Transsynaptic Tracing from Peripheral Targets with Pseudorabies Virus Followed by Cholera Toxin and Biotinylated Dextran Amines Double Labeling

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This approach has been most extensively used in the brain by utilizing the swine pathogen pseudorabies virus (PRV)1. PRV does not infect great apes, including humans, so it is most commonly used in studies on small mammals, especially rodents. The pseudorabies strain PRV152 expresses the enhanced green fluorescent protein (eGFP) reporter gene and only crosses functional synapses retrogradely through the hierarchical sequence of synaptic connections away from the infection site2,3. Other PRV strains have distinct microbiological properties and may be transported in both directions (PRV-Becker and PRV-Kaplan)4,5&#160;.&#160;This protocol will deal exclusively with PRV152. By delivering the virus at a peripheral site, such as muscle, it is possible to limit the entry of the virus into the brain through a specific set of neurons. The resulting pattern of eGFP signal throughout the brain then resolves the neurons that are connected to the initially infected cells. As the distributed nature of transsynaptic tracing with pseudorabies virus makes interpreting specific connections within an identified network difficult, we present a sensitive and reliable method employing biotinylated dextran amines (BDA) and cholera toxin subunit b (CTb) for confirming the connections between cells identified using PRV152. Immunochemical detection of BDA and CTb with peroxidase and DAB (3, 3&#39;-diaminobenzidine) was chosen because they are effective at revealing cellular processes including distal dendrites6-11.

Transsynaptic tracing has become a powerful tool for analyzing central efferents regulating peripheral targets through multi-synaptic circuits. Here we present a protocol that exploits the transsynaptic pseudorabies virus to identify and localize a functional brain circuit, followed by classical tract tracing techniques to validate specific connections in the circuit between identified groups of neurons.

Transsynaptic tracing has become a powerful tool used to analyze central efferents that regulate peripheral targets through multi-synaptic circuits. This ...

Neuroscience

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose (Herpestes Auropunctatus) Sera for Use as an Oral Rabies Vaccination Biological Marker

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases reported annually. The control of RABV circulation in wildlife reservoirs is typically accomplished by a strategy of oral rabies vaccination (ORV). Currently no wildlife ORV program exists in Puerto Rico. Research into oral rabies vaccines and various bait types for mongooses has been conducted with promising results. Monitoring the success of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RABV neutralizing antibodies (RVNA) post vaccination. This strategy may be difficult to interpret in areas with an active wildlife ORV program or in areas where RABV is enzootic and background levels of RVNA are present in reservoir species. In such situations, a biomarker incorporated with the vaccine or the bait matrix may be useful. We offered 16 captive mongooses placebo ORV baits containing ethyl-iophenoxic acid (et-IPA) in concentrations of 0.4% and 1% inside the bait and 0.14% in the external bait matrix. We also offered 12 captive mongooses ORV baits containing methyl-iophenoxic acid (me-IPA) in concentrations of 0.035%, 0.07% and 0.14% in the external bait matrix. We collected a serum sample prior to bait offering and then weekly for up to eight weeks post offering. We extracted Iophenoxic acids from sera into acetonitrile and quantified using liquid chromatography/mass spectrometry. We analyzed sera for et-IPA or me-IPA by liquid chromatography-mass spectrometry. We found adequate marking ability for at least eight and four weeks for et- and me-IPA, respectively. Both IPA derivatives could be suitable for field evaluation of ORV bait uptake in mongooses. Due to the longevity of the marker in mongoose sera, care must be taken to not confound results by using the same IPA derivative during consecutive evaluations.

Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker

We offered captive mongooses placebo oral rabies vaccine baits with ethyl or methyl iophenoxic acid as a biomarker and verified bait uptake using a novel liquid chromatography with tandem mass spectrometry (LC-MS/MS) method.

The small Indian mongoose (Herpestes auropunctatus) is a reservoir of rabies virus (RABV) in Puerto Rico and comprises over 70% of animal rabies cases ...

Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test

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 virus-specific 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&#8217;s Wildlife Services (USDA WS) program has conducted large-scale enhanced rabies surveillance efforts using the DRIT to test &#62;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.

The direct rapid immunohistochemical test (DRIT) offers a World Organization for Animal Health and World Health Organization (OIE/WHO) recognized alternative to the direct fluorescent antibody (DFA) test for rabies diagnosis. This test allows for field-based applications that can be performed in approximately 1 h upon brain impressions using light microscopy.

Laboratory-based surveillance is integral for rabies prevention, control and management efforts. While the DFA is the gold standard for rabies diagnosis, ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Field Postmortem Rabies Rapid Immunochromatographic Diagnostic Test for Resource-Limited Settings with Further Molecular Applications

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, animals suspected as rabies-positive must be submitted to a postmortem confirmation using classical or molecular laboratory methods. However, most endemic areas are in low- and middle-income countries where animal rabies diagnosis is restricted to central veterinary laboratories. Poor availability of surveillance infrastructure leads to serious disease underreporting from remote areas. Several diagnostic protocols requiring low technical expertise have been recently developed, providing opportunity to establish rabies diagnosis in decentralized laboratories. We present here a complete protocol for field postmortem diagnosis of animal rabies using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to the final interpretation. We complete the protocol by describing a further use of the device for molecular analysis and viral genotyping. RIDT easily detects rabies virus and other lyssaviruses in brain samples. The principle of such tests is simple: brain material is applied on a test strip where gold conjugated antibodies bind specifically to rabies antigens. The antigen-antibody complexes bind further to fixed antibodies on the test line, resulting in a clearly visible purple line. The virus is inactivated in the test strip, but viral RNA can be subsequently extracted. This allows the test strip, rather than the infectious brain sample, to be safely and easily sent to an equipped laboratory for confirmation and molecular typing. Based on a modification of the manufacturer&#39;s protocol, we found increased test sensitivity, reaching 98% compared to the gold standard reference method, the direct immunofluorescence antibody test. The advantages of the test are numerous: rapid, easy-to-use, low cost and no requirement for laboratory infrastructure, such as microscopy or cold-chain compliance. RIDTs represent a useful alternative for areas where reference diagnostic methods are not available.

We present a complete protocol for postmortem diagnosis of animal rabies under field conditions using a rapid immunochromatographic diagnostic test (RIDT), from brain biopsy sampling to final interpretation. We also describe further applications using the device for molecular analysis and viral genotyping.

Functional rabies surveillance systems are crucial to provide reliable data and increase the political commitment necessary for disease control. To date, ...

Fabrication of Pulsatile Polymeric Microparticles Encapsulating Rabies Antigen

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately burdensome to those living in low- and middle-income countries (LMICs), where the majority of deadly exposures to rabies occur. Different drug delivery strategies have been explored to condense vaccine regimens to a single injection by encapsulating antigens into polymeric particles. However, harsh stressors during the encapsulation process can cause denaturation of the encapsulated antigen. This article describes a method for encapsulating the rabies virus (RABV) antigen into polymeric microparticles that exhibit tunable pulsatile release. This method, termed Particles Uniformly Liquified and Sealed to Encapsulate Drugs (PULSED), generates microparticles using soft lithography to create inverse polydimethylsiloxane (PDMS) molds from a multi-photon, 3D-printed master mold. Poly(lactic-co-glycolic acid) (PLGA) films are then compression-molded into the PDMS molds to generate open-faced cylinders that are filled with concentrated RABV using a piezoelectric dispensing robot. These microstructures are then sealed by heating the top of the particles, allowing the material to flow and form a continuous, nonporous polymeric barrier. Post-fabrication, an enzyme-linked immunosorbent assay (ELISA) specific to the detection of intact trimeric rabies virus glycoprotein is used to confirm the high recovery of immunogenic antigen from the microparticles.

This method describes the encapsulation of the rabies antigen into biodegradable polymeric microparticles with structural and material properties that enable pulsatile release after a predetermined delay. Enzyme-linked immunosorbent assay (ELISA) assessment of the antigen retrieved from the particle core confirms the presence of intact trimeric rabies virus glycoprotein through particle fabrication.

The current guidelines for rabies post-exposure prophylaxis require multiple injections administered over several weeks. This can be disproportionately ...

Bioengineering

A Nanopore-Based Genomic Approach to Control Rabies in Low-Income Countries

Whole Genome Sequencing for Rapid Characterization of Rabies Virus Using Nanopore Technology

Genomic data can be used to track the transmission and geographic spread of infectious diseases. However, the sequencing capacity required for genomic surveillance remains limited in many low- and middle-income countries (LMICs), where dog-mediated rabies and/or rabies transmitted by wildlife such as vampire bats pose major public health and economic concerns. We present here a rapid and affordable sample-to-sequence-to-interpretation workflow using nanopore technology. Protocols for sample collection and the diagnosis of rabies are briefly described, followed by details of the optimized whole genome sequencing workflow, including primer design and optimization for multiplex polymerase chain reaction (PCR), a modified, low-cost sequencing library preparation, sequencing with live and offline base calling, genetic lineage designation, and phylogenetic analysis. Implementation of the workflow is demonstrated, and critical steps are highlighted for local deployment, such as pipeline validation, primer optimization, inclusion of negative controls, and the use of publicly available data and genomic tools (GLUE, MADDOG) for classification and placement within regional and global phylogenies. The turnaround time for the workflow is 2-3 days, and the cost ranges from $25 per sample for a 96 sample run to $80 per sample for a 12 sample run. We conclude that setting up rabies virus genomic surveillance in LMICs is feasible and can support progress toward the global goal of zero dog-mediated human rabies deaths by 2030, as well as enhanced monitoring of wildlife rabies spread. Moreover, the platform can be adapted for other pathogens, helping to build a versatile genomic capacity that contributes to epidemic and pandemic preparedness.

Author Spotlight: A Cost-Effective Genomic Workflow for Advancing Rabies Control in Resource-Limited Settings 

Here, we present a rapid and cost-effective workflow for characterizing rabies virus (RABV) genomes using nanopore technology. The workflow is intended to support genomics-informed surveillance at a local level, providing information on circulating RABV lineages and their placement within regional phylogenies to guide rabies control measures.

Genomic data can be used to track the transmission and geographic spread of infectious diseases. However, the sequencing capacity required for genomic ...