This research was supported in part by the intramural research program of the US Department of Agriculture, Animal and Plant Health Inspection Service, Wildlife Services, National Rabies Management Program and IDT Biologika (Dessau-Rosslau, Germany).

All procedures were approved by the USDA National Wildlife Research Center&#39;s institutional Animal Care and Use Committee under approved research protocol QA-2597.
NOTE: The following protocol describes the analysis procedure to detect methyl-iophenoxic acid in mongoose serum. This method is the final version of an iterative process that began with analysis of ethyl-iophenoxic acid in mongoose serum. During the initial evaluation of ethyl-iophenoxic acid minor modifications were made to the methods, resulting in the final protocol presented below. Representative results include those obtained during both iterations.
1. Preparation of solutions and standards
<ol>
	<li>Purchase me-IPA and et-IPA.</li>
	<li>For mobile phase A, prepare 1 L of 0.1% (v/v) formic acid in water by combining 1 mL of formic acid with 1 L of ultrapure water (&#8805; 18 M&#8486;). For mobile phase B, prepare 1 L of 0.1% (v/v) formic acid in acetonitrile (ACN) by combining 1 mL of formic acid with 1 L of ACN.</li>
	<li>For diluent, prepare 200 mL of 0.5% (v/v) trifluoroacetic acid (TFA) in ACN by combining 1 mL of TFA with 200 mL of ACN.</li>
	<li>Prepare concentrated IPA stock solutions of me-IPA and et-IPA in ACN at concentrations of approximately 1,000 &#181;g/mL.
	<ol>
		<li>Weigh approximately 10 mg of me-IPA on a microbalance and record the mass to &#177; 0.0001 mg. Quantitatively transfer the me-IPA to a 10 mL Class A volumetric flask using 45 mL ACN. Sonicate 1 min to dissolve all solids, and then bring to volume with ACN.</li>
		<li>Transfer ~8 mL of each stock to amber 8 mL glass vials with poly-tetrafluoroethylene (PTFE)-lined caps. Store at room temperature (RT). Transfer the remaining stock to hazardous waste.</li>
	</ol>
	</li>
	<li>For the 25x-7 me-IPA stock (Table 1), prepare a stock of me-IPA in ACN at approximately 200 &#181;g/mL. Example: Transfer 1 mL of the me-IPA concentrated stock from step 1.4.2 to a 5 mL Class A volumetric flask using a 1,000 &#181;L glass syringe. Dilute to volume with ACN. Transfer the stock to an amber 8 mL glass vial with PTFE-lined cap. Store at RT.</li>
	<li>Prepare the six additional 25x me-IPA Stocks described in Table 1. For each stock, combine the volumes indicated using a repeat pipettor in an amber 8 mL amber glass vial with PTFE-lined cap. Store each stock at RT.</li>
	<li>For the 25x surrogate stock, prepare a surrogate stock of me-IPA in ACN at approximately 10 &#181;g/mL from the concentrated stock prepared in step 1.4.2. Transfer 0.100 mL of the concentrated me-IPA stock to a 10 mL Class A volumetric flask using a 100 &#181;L glass syringe, and then dilute to volume with ACN.
	<ol>
		<li>Transfer ~8 mL to an amber 8-mL glass vial with PTFE-lined cap. Store at RT. Transfer the remaining stock to hazardous waste.</li>
	</ol>
	</li>
	<li>Prepare 4x stocks containing both analytes in 2 mL screw-top glass autosampler vials as described in Table 2.
	<ol>
		<li>For example, to prepare stock 4x-7, to a 2 mL vial, add 0.20 mL of the 25x-7 me-IPA stock from step 1.5 using a repeat pipettor with 0.5 mL capacity tip. Add 0.20 mL of the 25x surrogate et-IPA stock from step 1.7 using a repeat pipettor with 0.5 mL capacity tip.</li>
		<li>Add 0.85 mL of ACN using a repeat pipettor with 1 mL capacity tip. Cap the vial securely and invert 5x to mix.</li>
	</ol>
	</li>
	<li>Prepare the standard curve in 2 mL screw-top autosampler vials as described in Table 3.
	<ol>
		<li>For example, to prepare standard 7 (Std 7), to a 2-mL vial, add 0.20 mL of the 4x-7 Stock from step 1.8.2 using a repeat pipettor with 0.5 mL capacity tip. Add 0.60 mL of ultrapure DI water using a repeat pipettor with 1 mL capacity tip. Cap the vial securely and invert 5x to mix.</li>
	</ol>
	</li>
</ol>
2. Sample preparation
CAUTION: Personnel performing this procedure must have received the full series of rabies pre-exposure prophylaxis and have a documented rabies antibody titer above 0.5 IU from a Federal Occupational Health designated medical facility. Personnel must wear lab coats and eye protection at all times while performing the extraction. CAUTION: Perform steps 2.3&#8722;2.6 in a class II biosafety cabinet.
<ol>
	<li>For each sample, prepare a 1.5 mL microcentrifuge tube containing 200&#8722;300 mg of NaCl.Arrange the tubes in an 80-position plastic rack. Set aside for use in step 2.6. 
	NOTE: A micro scoop (or other small measuring device) is recommended for large numbers of samples.</li>
	<li>For each sample, label two 1.5 mL microcentrifuge tubes: one as &#34;A&#34; and the other as &#34;B&#34;. Arrange the tubes in an 80-position plastic rack.</li>
	<li>Place the following materials and equipment needed for serum extraction in a class II biosafety cabinet: microcentrifuge tubes (in racks) prepared in steps 2.1 and 2.2, a vortex mixer, repeat pipettor with 0.5 mL and 5 mL capacity tips, 100&#8722;1,000 &#181;L air displacement pipette with 1,000 &#181;L tips, containers with approximately 100 mL each of diluent and ultrapure DI water, and a biohazard waste container.</li>
	<li>Remove serum samples from frozen storage and warm to RT in the biosafety cabinet. Vortex mix each serum sample prior to sampling.</li>
	<li>Using a repeat pipettor with 0.5 mL capacity tip, dispense 0.050 mL of mongoose serum into tube &#34;A&#34; and add 0.050 mL of 25x surrogate stock. Then add 0.950 mL of diluent to tube &#34;A&#34; using a repeat pipettor with 5 mL capacity tip. Cap securely and vortex mix for 10&#8722;15 s.</li>
	<li>Dispense the pre-weighed NaCl from step 2.1 into tube &#34;A&#34; and vortex mix 3x for 8&#8722;12 s. Wipe down the outside surfaces of the vial rack containing tube &#34;A&#34; using 70% (v/v) isopropanol. 
	NOTE: The rack of samples may now be removed from the class II biosafety cabinet.</li>
	<li>Centrifuge tube &#34;A&#34; at 12,000 x g for 1 min to separate the aqueous and ACN phases. Pipette 0.80 mL of the upper ACN phase to tube &#34;B&#34; using a 100&#8722;1,000 &#181;L air displacement pipette. Transfer the remaining solution in tube &#34;A&#34; to hazardous waste and discard the empty tube in a biohazardous waste container.</li>
	<li>Remove ACN and TFA from tube &#34;B&#34; with a gentle flow of N2 gas in a 45 &#176;C water bath.</li>
	<li>Add 0.250 mL of ACN to tube &#34;B&#34; using a repeat pipettor, vortex mix for 4&#8722;5 s, and then centrifuge briefly (2&#8722;4 s) at 12,000 x g to collect the liquid in the bottom of the tube.</li>
	<li>Add 0.750 mL of ultrapure DI water to tube &#34;B&#34; using a repeat pipettor with 5 mL capacity tip, vortex mix for 4&#8722;5 s, and then centrifuge for 1 min at 12,000 x g to clarify the sample.</li>
	<li>Transfer 0.75 mL of the supernatant to an autosampler vial using a 1,000 &#181;L air displacement pipette. Discard pipette tips in biohazard waste container.</li>
	<li>Cap autosampler vials securely and analyze by LC-MS/MS (section 4). Transfer the remaining solution in tube &#34;B&#34; to hazardous waste and discard the empty tube to a biohazardous waste container. Dispose of all biohazardous waste by autoclaving or incineration.</li>
</ol>
3. Quality control samples
CAUTION: Follow the cautionary statements described in section 2.
NOTE:The following procedure describes the minimum number of quality control (QC) samples required for an analysis. Replicates at each level are recommended if sufficient control mongoose serum is available.
<ol>
	<li>Prepare four 1.5 mL microcentrifuge tubes containing 200&#8722;300 mg of NaCl. Arrange the tubes in an 80-position plastic rack.</li>
	<li>For each QC sample, label two 1.5 mL microcentrifuge tubes: one as &#34;A&#34; and the other as &#34;B&#34;. Arrange the tubes in an 80-position plastic rack.</li>
	<li>Repeat step 2.3.</li>
	<li>Remove control mongoose serum from frozen storage and warm to RT in the biosafety cabinet. Vortex mix the control serum prior to sampling.</li>
	<li>Dispense 0.050 mL of control mongoose serum into the four 1.5-mL &#34;A&#34; tubes using a repeat pipettor with 0.5 mL capacity tip.</li>
	<li>Fortify each of the four QC samples as specified in Table 4 using a repeat pipettor with 0.5 mL capacity tip. Cap each QC sample securely and vortex mix for 10&#8722;15 s.</li>
	<li>Perform the extraction procedure as described in steps 2.6&#8722;2.12.</li>
</ol>
4. LC-MS/MS analysis
<ol>
	<li>Configure the LC-MS/MS with all parameters described in Table 5. Power on the LC-MS/MS and allow the column to reach 70 &#176;C before setting the flow rate to 0.800 mL/min.</li>
	<li>Set up a sequence in the data acquisition software (Table of Materials) to inject the standard curve before and after each batch consisting of quality control samples and unknown samples.</li>
	<li>Inject all standards and samples and acquire MRM ion chromatograms using parameters listed in Table 5.</li>
	<li>After sequence completion, turn off the LC-MS/MS and dispose of all autosampler vials as hazardous waste.</li>
</ol>
5. Quantification
<ol>
	<li>Use the data analysis software to generate a calibration curve of relative responses versus relative concentrations for me-IPA using et-IPA as the internal standard. Calculate the relative responses from the quantifier MRM transition for me-IPA (556.6 &#8594; 428.7) divided by the MRM transition for et-IPA (570.7 &#8594; 442.7). Construct a 7-level calibration curve using a second order quadratic function that is weighted 1/x and ignores the origin.</li>
	<li>Calculate the serum concentration (Cserum) of me-IPA using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/59373/59373eq1.jpg" style="opacity: 0.9;" /> 
	where cinstrument is the concentration determined by the instrument from the calibration curve in units of &#181;g/mL, 1.25 is the dilution factor <img alt="Equation 2" src="/files/ftp_upload/59373/59373eq2.jpg" style="opacity: 0.9;" />, Vfinal is the final sample volume (1.0 mL), and Vserum is the serum volume in mL (0.050 mL nominal).</li>
</ol>

<ol>
	<li>Nel, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mongoose+rabies+in+southern+Africa:+a+re-evaluation+based+on+molecular+epidemiology.">Mongoose rabies in southern Africa: a re-evaluation based on molecular epidemiology.</a> Virus Research. 109 (2), 165-173 (2005).</li><li>Zieger, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phylogeography+of+rabies+in+Grenada,+West+Indies,+and+implications+for+control.">The phylogeography of rabies in Grenada, West Indies, and implications for control.</a> PLOS Neglected Tropical Diseases. 8 (10), e3251 (2004).</li><li>Monroe, B. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabies+surveillance+in+the+United+States+during+2014.">Rabies surveillance in the United States during 2014.</a> Journal of the American Veterinary Medical Association. 248 (7), 777-788 (2015).</li><li>Slate, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Status+of+oral+rabies+vaccination+in+wild+carnivores+in+the+United+States.">Status of oral rabies vaccination in wild carnivores in the United States.</a> Virus Research. 111, 68-76 (2005).</li><li>Blanton, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vaccination+of+small+Asian+mongoose+(Herpestes+javanicus)+against+rabies.">Vaccination of small Asian mongoose (Herpestes javanicus) against rabies.</a> Journal of Wildlife Diseases. 42 (3), 663-666 (2006).</li><li>Vos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+vaccination+of+captive+small+Indian+mongoose+(Herpestes+auropunctatus)+against+rabies.">Oral vaccination of captive small Indian mongoose (Herpestes auropunctatus) against rabies.</a> Journal of Wildlife Diseases. 49 (4), 1033-1036 (2013).</li><li>Berentsen, A. R., Johnson, S. R., VerCauteren, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+ONRAB&#174;+bait+matrix+flavor+preference+by+mongoose+(Herpestes+auropunctatus)+in+Puerto+Rico:+Implications+for+Oral+Rabies+Vaccination.">Evaluation of ONRAB&#174; bait matrix flavor preference by mongoose (Herpestes auropunctatus) in Puerto Rico: Implications for Oral Rabies Vaccination.</a> Caribbean Journal of Science. 48 (1), 52-58 (2014).</li><li>Ortmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+studies+with+the+oral+rabies+virus+vaccine+strain+SPBN+GASGAS+in+the+small+Indian+mongoose+(Herpestes+auropunctatus).">Safety studies with the oral rabies virus vaccine strain SPBN GASGAS in the small Indian mongoose (Herpestes auropunctatus).</a> BMC Veterinary Research. 14 (1), 90 (2018).</li><li>Linhart, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+field+evaluation+of+baits+for+delivering+oral+rabies+vaccines+to+raccoons+(Procyon+lotor).">A field evaluation of baits for delivering oral rabies vaccines to raccoons (Procyon lotor).</a> Journal of Wildlife Diseases. 30 (2), 185-194 (1994).</li><li>Fry, T. L., Atwood, T., Dunbar, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+rhodamine+B+as+a+biomarker+for+raccoons.">Evaluation of rhodamine B as a biomarker for raccoons.</a> Human Wildlife Interactions. 4 (2), 275-280 (2010).</li><li>Jones, C., Moller, H., Hamilton, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+potential+techniques+for+identifying+individual+stoats+(Mustela+erminea)+visiting+control+or+monitoring+stations.">A review of potential techniques for identifying individual stoats (Mustela erminea) visiting control or monitoring stations.</a> New Zealand Journal of Zoology. 31 (3), 193-203 (2004).</li><li>de Leeuw, A. N. S., Smith, G. C., Woods, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+iophenoxic+acid+to+assess+bait+uptake+by+European+badgers.">Use of iophenoxic acid to assess bait uptake by European badgers.</a> Advances in Vertebrate Pest Management. 4, 243-254 (2006).</li><li>Southey, A. K., Sleeman, D. P., Gormley, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfadimethoxine+and+rhodamine+B+as+oral+biomarkers+for+European+badgers+(Meles+meles).">Sulfadimethoxine and rhodamine B as oral biomarkers for European badgers (Meles meles).</a> Journal of Wildlife Diseases. 38 (2), 378-384 (2002).</li><li>Massei, G., Jones, A., Platt, T., Cowan, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iophenoxic+Acid+as+a+Long-Term+Marker+for+Wild+Boar.">Iophenoxic Acid as a Long-Term Marker for Wild Boar.</a> Journal of Wildlife Management. 73 (3), 458-461 (2009).</li><li>Creekmore, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+evaluation+of+baits+and+baiting+strategies+for+delivering+oral+vaccine+to+mongooses+in+Antigua,+West+Indies.">Field evaluation of baits and baiting strategies for delivering oral vaccine to mongooses in Antigua, West Indies.</a> Journal of Wildlife Diseases. 30 (4), 497-505 (1994).</li><li>Creekmore, T. E., Rock, R. E., Hurley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+baiting+system+for+delivery+of+an+oral+plague+vaccine+to+black-tailed+prairie+dogs.">A baiting system for delivery of an oral plague vaccine to black-tailed prairie dogs.</a> Journal of Wildlife Diseases. 38 (1), 32-39 (2002).</li><li>Fernandez, J. R. R., Rocke, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Rhodamine+B+as+a+biomarker+for+oral+plague+vaccination+of+prairie+dogs.">Use of Rhodamine B as a biomarker for oral plague vaccination of prairie dogs.</a> Journal of Wildlife Diseases. 47 (3), 765-768 (2011).</li><li>Johnston, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+significance+of+tetracycline+stability+in+rabies+vaccine+baits.">Evaluation and significance of tetracycline stability in rabies vaccine baits.</a> Journal of Wildlife Diseases. 41 (3), 549-558 (2005).</li><li>Algeo, T. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+rabies+vaccination+variation+in+tetracycline+biomarking+among+Ohio+Raccoons.">Oral rabies vaccination variation in tetracycline biomarking among Ohio Raccoons.</a> Journal of Wildlife Diseases. 49 (2), 332-337 (2013).</li><li>Crier, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetracyclines+as+a+fluorescent+marker+in+bones+and+teeth+of+rodents.">Tetracyclines as a fluorescent marker in bones and teeth of rodents.</a> Journal of Wildlife Management. 34 (4), 829-834 (1970).</li><li>Fisher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+using+Rhodamine+B+as+a+marker+for+wildlife+studies.">Review of using Rhodamine B as a marker for wildlife studies.</a> Wildlife Society Bulletin. 27 (2), 318-329 (1999).</li><li>Knowlton, F. K., Savarie, P. J., Wahlgren, C. E., Hayes, D. J., Shumake, S. A., Bullard, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+marks+by+coyotes+ingesting+baits+containing+iophenoxic+acid,+Mirex+and+Rhodamine+B.">Physiological marks by coyotes ingesting baits containing iophenoxic acid, Mirex and Rhodamine B.</a> Vertebrate Pest Control and Management Materials. , 141-147 (1987).</li><li>Follmann, E. H., Savarie, P. J., Ritter, D. G., Baer, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+marking+of+arctic+foxes+with+iophenoxic+acid.">Plasma marking of arctic foxes with iophenoxic acid.</a> Journal of Wildlife Diseases. 23 (4), 709-712 (1987).</li><li>Saunders, G., Harris, S., Eason, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iophenoxic+acid+as+a+quantitative+bait+marker+for+foxes.">Iophenoxic acid as a quantitative bait marker for foxes.</a> Wildlife Research. 20, 297-302 (1993).</li><li>Hadidian, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acceptance+of+simulated+oral+rabies+vaccine+baits+by+urban+raccoons.">Acceptance of simulated oral rabies vaccine baits by urban raccoons.</a> Journal of Wildlife Diseases. 25 (1), 1-9 (1989).</li><li>Sweetapple, P. J., Nugent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iophenoxic+acid+as+a+serum+marker+for+red+deer+(Cervus+elaphus+scoticus).">Iophenoxic acid as a serum marker for red deer (Cervus elaphus scoticus).</a> Wildlife Research. 25, 649-654 (1998).</li><li>Ogilvie, S. C., Eason, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+iophenoxic+acid+and+rhodamine+B+for+marking+feral+ferrets+(Mustela+furo).">Evaluation of iophenoxic acid and rhodamine B for marking feral ferrets (Mustela furo).</a> New Zealand Journal of Zoology. 25 (2), 105-108 (1998).</li><li>Eason, C. T., Batcheler, D., Frampton, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+pharmacokinetics+of+iophenoxic+acid+in+cats+and+brushtail+possums.">Comparative pharmacokinetics of iophenoxic acid in cats and brushtail possums.</a> Wildlife Research. 21, 377-380 (1994).</li><li>Fisher, P. M., Marks, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+iophenoxic+acid+as+a+biomarker+for+swamp+wallabies+(Wallabia+bicolor).">Evaluation of iophenoxic acid as a biomarker for swamp wallabies (Wallabia bicolor).</a> Wildlife Research. 24, 97-103 (1997).</li><li>Baer, G. M., Shaddock, J. H., Hayes, D. J., Savarie, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iophenoxic+acid+as+a+serum+marker+in+carnivores.">Iophenoxic acid as a serum marker in carnivores.</a> Journal of Wildlife Management. 49 (1), 49-51 (1985).</li><li>Spurr, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iophenoxic+acid+as+a+systemic+blood+marker+for+assessment+of+bait+acceptance+by+stoats+(Mustela+ermine)+and+weasels+(Mustela+nivalis).">Iophenoxic acid as a systemic blood marker for assessment of bait acceptance by stoats (Mustela ermine) and weasels (Mustela nivalis).</a> New Zealand Journal of Zoology. 29 (2), 135-142 (2002).</li><li>Mudge, G. H., Strewler, G. J., Desbiens, N., Berndt, W. O., Wade, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excretion+and+distribution+of+iophenoxic+acid.">Excretion and distribution of iophenoxic acid.</a> Journal of Pharmacology and Experimental Therapeutics. 178 (1), 159-172 (1971).</li><li>Jones, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+liquid+chromatographic+determination+of+iophenoxic+acid+in+serum.">High-performance liquid chromatographic determination of iophenoxic acid in serum.</a> Journal of Chromatography B: Biomedical Sciences and Applications. 654 (2), 293-296 (1994).</li><li>Wiles, M. C., Campbell, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+chromatography-electrospray+ionization+mass+spectrometry+for+direct+identification+and+quantification+of+iophenoxic+acid+in+serum.">Liquid chromatography-electrospray ionization mass spectrometry for direct identification and quantification of iophenoxic acid in serum.</a> Journal of Chromatography B. 832 (1), 144-157 (2006).</li><li>Ballesteros, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+by+LC/ESI-MS+of+iophenoxic+acid+derivatives+and+evaluation+as+markers+of+oral+baits+to+deliver+pharmaceuticals+to+wildlife.">Analysis by LC/ESI-MS of iophenoxic acid derivatives and evaluation as markers of oral baits to deliver pharmaceuticals to wildlife.</a> Journal of Chromatography B. 878 (22), 1997-2002 (2010).</li><li>Purdey, D. C., Petcu, M., King, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+protocol+for+detecting+two+systemic+bait+markers+(Rhodamine+B+and+iophenoxic+acid)+in+small+mammals.">A simplified protocol for detecting two systemic bait markers (Rhodamine B and iophenoxic acid) in small mammals.</a> New Zealand Journal of Zoology. 30 (3), 175-184 (2003).</li><li>Tobin, M. E., Koehler, A. E., Sugihara, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetracyclines+as+a+fluorescent+marker+in+bones+and+teeth+of+rodents.">Tetracyclines as a fluorescent marker in bones and teeth of rodents.</a> Journal of Wildlife Management. 60 (1), 202-207 (1996).</li><li>Briscoe, J. A., Syring, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+emergency+airway+and+vascular+access+in+special+species.">Techniques for emergency airway and vascular access in special species.</a> Seminars in Avian and Exotic Pet Medicine. 13 (3), 118-131 (2004).</li><li>Eason, C. T., Frampton, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plasma+pharmacokinetics+of+iophenoxic+and+iopanoic+acids+in+goat.">The plasma pharmacokinetics of iophenoxic and iopanoic acids in goat.</a> Xenobiotica. 2 (2), 185-189 (1992).</li><li>Hilton, H. E., Dunn, A. M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mongooses: their natural history. , (1967).</li><li>Stevens, C. E., Hume, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comparative physiology of the vertebrate digestive system. Second edition. , (1995).</li><li>National Rabies Management Program (NRMP). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Oral rabies vaccination draft operations manual. , (2009).</li></ol>

Authors AV and SO are fulltime employees of an oral rabies vaccine bait manufacturer.

The LC-MS/MS method developed for the studies utilized the selectivity of multiple reaction monitoring to accurately quantify me-IPA and et-IPA in mongoose serum. The selectivity of MS/MS detection also allowed for a simple clean-up protocol relying solely on acetonitrile to precipitate proteins from serum prior to analysis.
Iophenoxic acids are soluble in ACN but are practically insoluble in water. To exclude water from the ACN extraction, sodium chloride was added to force a clear water:ACN phase separation by increasing the ionic strength of the aqueous (serum) phase. The volatile acid trifluoracetic acid (TFA) was also added to ensure that iophenoxic acids were protonated during the extraction and would be more readily solubilized in the ACN phase. TFA is removed during the dry-down step prior to LC-MS/MS analysis.
Blood draws from mongoose were approximately 1 mL, yielding 0.5 mL or less of sera. To perform replicate analyses of each sample, a micro-scale sample preparation procedure was required that used microcentrifuge tubes rather than typical analytical laboratory glassware such as Class A volumetric pipettes and flasks. To achieve accurate and precise results it is necessary that analysts be proficient in the use of glass microliter syringes and positive-displacement repeat pipettors with microliter-size tips.
A limitation of this method is that it requires costly LC-MS/MS instrumentation and analysts trained in its use and maintenance. However, because me-IPA and et-IPA are baseline resolved using the HPLC conditions described, the method could potentially be adapted to a single-quadrupole LCMS or HPLC with UV detector provided no interferences were observed.
The difference in mean serum concentrations between mongooses that consumed baits with et-IPA in the blister pack and bait matrix at the 2.8 mg dose suggests spillage when the marker is contained in the blister pack, rather than incorporated into the bait matrix. This may have implications for the purpose of estimating vaccine as opposed to bait uptake. For example, incorporating the marker into the external bait matrix may inflate the estimation of vaccine coverage if the animal eats the matrix but vaccine spills out. However, regulatory restrictions may preclude the ability of mixing a biological marker directly with a vaccine within the blister pack. In the cases in which &#60;100% consumption was recorded, three cases were with baits containing et-IPA in the blister pack, two of which were the higher 1% concentration. This suggests potential taste aversion when et-IPA is at higher concentrations. When baits containing 1% (20.0 mg) et-IPA incorporated into the bait matrix were offered to mongooses, all animals rejected the bait.
Mean IPA concentrations for et- and me-IPA at day 14 were approximately 17 and 19 &#181;g/mL, respectively. Similar research performed at Instituto de Investigaci&#243;n en Recursos Cineg&#233;ticos, Ciudad Real, Spain, using butyl and pentyl-IPA found day 14 concentrations of approximately 45 and 10 &#181;g/mL in mongoose sera when delivered at the same concentration and bait formulation as in our study. These differences suggest that different derivatives of IPA may metabolize at different rates in mongooses, which can be useful when marker retention (either short or long-term retention) is a concern.
Differences in the physiology of the gastrointestinal system may affect absorption and excretion of IPA in different species. The plasma elimination half-life of IPA in domestic cats (Felis catus) when delivered at 1.5 mg/kg was 107 days whereas the rate in brushtail possums at the same dose rate was approximately one day28. When IPA was given to domestic goats (Capra aegagrus hircus) at a dose rate of 1.5 mg/kg the terminal elimination half-life of IPA was 81 days, although investigators continued to find elevated iodine concentrations up to 160 days following administration39. The gastrointestinal system of members of Vivveridae (the family to which mongooses had previously been assigned40) is described as similar to that of the domestic cat41, which may explain the retention time of IPA in mongooses. Research also suggests IPA may be metabolized differently in marsupials, allowing for more rapid excretion than in eutherian species29.
Both derivatives of IPA evaluated in this study provided long-term (4&#8722;8 weeks) marking ability in mongooses. The use of IPA as a biological marker in mongooses should take into consideration the study objectives and the desired duration of marking. In cases where animals are to be marked from the same study sites during consecutive time periods, different derivatives of IPA could be used to ensure results from one marking event are not confounded by marking during previous events. As part of operational ORV for wildlife in the US, sampling the target species and testing for the presence of RVNA and biomarker is typically conducted 4&#8722;6 weeks following ORV distribution42. From a practical standpoint, it appears either et- or me-IPA can be readily detected during this time period. Future research to evaluate the residue decay function in mongooses offered various concentrations of both congeners of IPA evaluated in this study would be useful.

Rabies virus (RABV) is a negative sense single stranded lyssavirus, and circulates among diverse wildlife reservoir species within the orders Carnivora and Chiroptera. Multiple species of mongoose are reservoirs of RABV, and the small Indian mongoose (Herpestes auropunctatus) is the only reservoir in Puerto Rico and other Caribbean islands in the Western Hemisphere1,2,3. The control of RABV circulation in wildlife reservoirs is typically accomplished through a strategy of oral rabies vaccination (ORV). In the United States (US), this management activity is coordinated by the USDA/APHIS/Wildlife Services National Rabies Management Program (NRMP)4. Currently no wildlife ORV program exists in Puerto Rico. Research into rabies vaccines and various bait types for mongooses has been conducted with promising results suggesting an ORV program for mongooses is possible5,6,7,8.
Monitoring the impact of ORV relies on estimating bait uptake by target species, which typically involves evaluating a change in RV antibody seroprevalence. However, this strategy may be challenging in areas with an active wildlife ORV programs or in areas where RV is enzootic and background levels of RABV neutralizing antibodies (RVNA) are present in reservoir species. In such situations, a biomarker included in the bait or the external bait matrix may be useful.
Various biological markers have been used to monitor bait uptake in numerous species, including raccoons (Procyon lotor)9,10,stoats (Mustela ermine)11,12, European badgers (Meles meles)13, wild boars (Sus scrofa)14, small Indian mongooses15 and prairie dogs (Cynomysludovicianus)16,17, among others. In the US, operational ORV baits often include a 1% tetracycline biomarker in the bait matrix to monitor bait uptake18,19. However, drawbacks to the use of tetracycline include a growing concern over the distribution of antibiotics into the environment and that detection of tetracycline is typically invasive, requiring tooth extraction or destruction of the animal to obtain bone samples20. Rhodamine B has been evaluated as a marker in a variety of tissues and can be detected using ultraviolet (UV) light and fluorescence in hair and whiskers10,21.
Iophenoxic acid (IPA) is a white, crystalline powder that has been used to evaluate bait consumption in coyotes (Canis latrans)22, arctic fox (Vulpes lagopus)23, red fox (Vulpes vulpes)24, raccoons9,25, wild boar14, red deer (Cervus elaphus scoticus)26, European badgers12 and ferrets (M. furo)27, among several other mammalian species. Retention times of IPA varies by species from less than two weeks in some marsupials28,29, to at least 26 weeks in ungulates26 and over 52 weeks in domestic dogs (Canis lupus familiaris)30. Retention times may also be dose-dependent31. Iophenoxic acid binds strongly to serum albumin and was historically detected by measuring blood iodine levels32. This indirect approach was supplanted by high-performance liquid chromatography (HPLC) methods to directly measure iophenoxic acid concentrations with UV detection33, and eventually with liquid chromatography and mass spectrometry (LCMS)34,35. For this study, a highly sensitive and selective liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed that utilizes multiple reaction monitoring (MRM) to quantify two analogues of iophenoxic acid. Our objective was to use this LC-MS/MS method to evaluate the marking ability of 2-(3-hydroxy-2,4,6-triiodobenzyl)propanoic acid (methyl-IPA or me-IPA) and 2-(3-hydroxy-2,4,6-triiodobenzyl)butanoic acid (ethyl-IPA or et-IPA) and when delivered in an ORV bait to captive mongooses.
Mongooses were live captured in cage traps baited with commercially available smoked sausages and fish oil. Mongooses were housed in individual 60 cm x 60 cm x 40 cm stainless steel cages and fed a daily ration of ~50 g commercial dry cat food, supplemented twice per week with a commercially available chicken thigh. Water was available ad libitum. We delivered two derivatives of IPA, ethyl-IPA and methyl-IPA, to captive mongooses in placebo ORV baits. All baits were composed of a 28 mm x 20 mm x 9 mm foil blister pack with an external coating (hereafter &#34;bait matrix&#34;) containing powdered chicken egg and gelatin (Table of Materials). Baits contained 0.7 mL of water or IPA derivative and weighed approximately 3 g, of which ~2 g was the external bait matrix.
We offered 16 captive mongooses et-IPA in three concentrations: 0.14% (2.8 mg et-IPA in ~2 g bait matrix; 3 males [m], 3 females [f]), 0.4% (2.8 mg et-IPA in 0.7 mL blister pack volume; 3m, 3f), and 1.0% (7.0 mg ethyl-IPA in 0.7 mL blister pack volume; 2m, 2f). The overall dose of 2.8 mg corresponds to a dose rate of 5 mg/kg27,36 and is based on an average mongoose weight of 560 g in Puerto Rico. We selected 1% as the highest concentration as research suggests taste aversion to some biomarkers may occur at concentrations &#62;1% in some species37. We only offered the 1% dose in the blister pack as flocculation prevented the solute from dissolving in the solvent sufficiently to be evenly incorporated into the bait matrix. One control group (2m, 1f) received baits filled with sterile water and no IPA. We offered baits to mongooses in the morning (~8 a.m.) during or prior to feeding of their daily maintenance ration. Bait remains were removed after approximately 24 hours. We collected blood samples prior to treatment, one day post-treatment and then weekly up to 8 weeks post-treatment. We anesthetized mongooses by inhalation of isoflurane gas and collected up to 1.0 mL of whole blood by venipuncture of the cranial vena cava as described for ferrets38. We centrifuged whole blood samples, transferred sera to cryovials and stored them at -80 &#176;C until analysis. Not all animals were sampled during all time periods to minimize the impacts of repeated blood draws on the health of the animals. Control animals were sampled on day 0, then weekly for up to 8 weeks post-treatment.
We delivered me-IPA in three concentrations: 0.035% (0.7 mg), 0.07% (1.4 mg) and 0.14% (2.8 mg), all incorporated into the bait matrix, with 2 males and 2 females per treatment group. Two males and two females received baits filled with sterile water and no IPA. Bait offering times and mongoose anesthesia are described above. We collected blood samples prior to treatment on day 1, and then weekly up to 4 weeks post-treatment.
We tested serum concentration data for normality and estimated means for serum IPA concentrations of different treatment groups. We used a linear mixed model to compare mean serum et-IPA concentrations pooled across individuals. Bait type (matrix/blister pack) was a fixed effect in addition to experimental day, whereas animal ID was a random effect. All procedures were run using common statistical software (Table of Materials) and significance was evaluated at &#945; = 0.05.

<table><tbody><tr><td>Acetonitrile, Optima grade</td><td>Fisher</td><td>A996</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td>XS204</td><td></td></tr><tr><td>C18 column, 2.1 x 50 mm, 2.5-&amp;micro;m particle size</td><td>Waters Corp.</td><td>186003085</td><td></td></tr><tr><td>ESI Source</td><td>Agilent&amp;nbsp;</td><td>G1958-65138</td><td></td></tr><tr><td>Ethyl-iophenoxic acid, 97 %</td><td>Sigma Aldrich</td><td>N/A</td><td>Lot MKBP5399V</td></tr><tr><td>Formic acid, LC/MS grade</td><td>Fisher</td><td>A117</td><td></td></tr><tr><td>LCMS software</td><td>Agilent</td><td>MassHunter Data Acquisition and Quantitative Analysis</td><td></td></tr><tr><td>Methyl-iophenoxic acid, 97 % (w/w)</td><td>PR EuroChem Ltd.</td><td>N/A</td><td>Lot PR0709514717</td></tr><tr><td>Microanalytical balance</td><td>Mettler Toledo</td><td>XP6U</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5415C</td><td></td></tr><tr><td>MS/MS</td><td>Agilent</td><td>G6470A</td><td></td></tr><tr><td>N-Evap</td><td>Organomation</td><td>115</td><td></td></tr><tr><td>Oral Rabies Vaccine Baits</td><td>IDT Biologika, Dessau Rossleau, Germany</td><td>N/A</td><td></td></tr><tr><td>Propyl-iophenoxic acid, 99 % (w/w)</td><td>PR EuroChem Ltd.</td><td>N/A</td><td>Lot PR100612108RR</td></tr><tr><td>Repeat pipettor</td><td>Eppendorf</td><td>M4</td><td></td></tr><tr><td>Screw-top autosampler vial caps, PTFE-lined</td><td>Agilent</td><td>5190-7024</td><td></td></tr><tr><td>Sodium chloride, Certified ACS grade</td><td>Fisher</td><td>S271</td><td></td></tr><tr><td>Statistical Software Package</td><td>SAS Institute, Cary, North Carolina, USA</td><td>N/A</td><td></td></tr><tr><td>Trifluoroacetic acid, 99 %</td><td>Alfa Aesar</td><td>L06374</td><td></td></tr><tr><td>UPLC</td><td>Agilent</td><td>1290 Series</td><td></td></tr><tr><td>Vortex Mixer</td><td>Glas-Col</td><td>099A PV6</td><td></td></tr><tr><td>0.2-mL pipettor tips</td><td>Eppendorf</td><td>30089.413</td><td></td></tr><tr><td>0.5-mL pipettor tips</td><td>Eppendorf</td><td>30089.421</td><td></td></tr><tr><td>1.5-mL microcentrifuge tubes</td><td>Fisher</td><td>14-666-325</td><td></td></tr><tr><td>1250-&amp;micro;L capacity pipette tips</td><td>GeneMate</td><td>P-1233-1250</td><td></td></tr><tr><td>1-mL pipettor tips</td><td>Eppendorf</td><td>30089.43</td><td></td></tr><tr><td>2-mL amber screw-top autosampler vials</td><td>Agilent</td><td>5182-0716</td><td></td></tr><tr><td>5-mL pipettor tips</td><td>Eppendorf</td><td>30089.456</td><td></td></tr><tr><td>80-position microcentrifuge tube rack</td><td>Fisher</td><td>05-541-2</td><td></td></tr><tr><td>8-mL amber vials with PTFE-lined caps</td><td>Wheaton</td><td>224754</td><td></td></tr><tr><td>70 % (v/v) isopropanol</td><td>Fisher</td><td>A459</td><td></td></tr><tr><td>100-1000 &amp;micro;L air displacement pipette</td><td>Eppendorf</td><td>ES-100</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Analysis of Iophenoxic Acid Analogues in Small Indian Mongoose Herpestes Auropunctatus Sera for Use as an Oral Rabies Vaccination Biological Marker at JoVE.co

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.

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 ...

analysis-iophenoxic-acid-analogues-small-indian-mongoose-herpestes

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

5.8K Views.  National Wildlife Research Center. 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.

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

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

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

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, ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

Quantitation of Rabies Virus in Various Bovine Brain Structures

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major zoonotic risk. Here, our objective was to utilize a laboratory protocol to determine the relative copy number of the rabies virus (RABV) genome in different bovine brain anatomical structures using quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR quantifies the specific number of gene copies present in a sample based on fluorescence emitted after amplification that is directly proportional to the amount of target nucleic acid present in the sample. This method is advantageous owing to its short duration, reduced risk of contamination, and potential to detect viral nucleic acids in different samples more easily compared to other techniques. The brains of six rabid animals were divided into six anatomical structures, namely the Ammon&#8217;s horn, cerebellum, cortex, medulla, pons, and thalamus. All brains were identified as positive for RABV antigens based on a direct immunofluorescence test. The same anatomical structures from the brains of four RABV-negative bovines were also assessed. RNA was extracted from each structure and used for qRT-PCR. An assay was performed to determine the copy numbers of RABV genes using an in vitro transcribed nucleoprotein gene. The standard curve used to quantify viral RNA exhibited an efficiency of 100% and linearity of 0.99. Analysis revealed that the cortex, medulla, and thalamus were the ideal CNS portions for use in RABV detection, based on the observation that these structures possessed the highest levels of RABV. The test specificity was 100%. All samples were positive, no false positives were detected. This method can be used to detect RABV in samples that contain low levels of RABV during diagnosis of BPR.

This protocol presents a qRT-PCR-based approach for determining the rabies virus nucleoprotein (N) gene copy number within various bovine brain anatomical structures using in vitro transcription.

Bovine paralytic rabies (BPR) is a form of viral encephalitis that is of substantial economic importance throughout Latin America, where it poses a major ...

Quick and Early Detection of Rabies Antibodies to Evaluate the Immune Response in a Patient

Detection of Rabies IgG and IgM Antibodies Using the Rabies Indirect Fluorescent Antibody Test

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. This test provides rapid results and can be used to detect rabies antibodies in several different scenarios. The rabies IFA test is especially useful for the quick and early detection of antibodies to evaluate the immune response in a patient who has developed rabies. Although other methods for antemortem rabies diagnosis take precedence, this test may be utilized to demonstrate recent rabies virus exposure through antibody detection. The IFA test does not provide a virus-neutralizing antibody (VNA) titer, but the pre-exposure prophylaxis (PrEP) response can be evaluated through positive or negative antibody presence. This test can be utilized in various situations and can provide results for a number of different targets. In this study, we used several paired serum samples from individuals who received PrEP and demonstrated their rabies antibody presence over time using the IFA test.

Author Spotlight: Rabies-Specific Antibody Isotypes Detection in Sera or Cerebral Spinal Fluid Using an IFA Test 

The aim of this manuscript is to examine the use of the rabies indirect fluorescent antibody test for the detection of rabies-specific IgG and IgM antibodies.

The rabies indirect fluorescent antibody (IFA) test was developed to detect various rabies-specific antibody isotypes in sera or cerebral spinal fluid. ...

An Immunohistochemistry Staining for the Detection of Rabies Virus Antigens

Using a microtome, make a 5-micrometer paraffin section. Float the section on a water bath at 38 degrees Celsius, and collect it onto glass slides. Label the slides with a reagent-resistant pen. Place the slides onto a tray, and melt in an oven at 55 to 60 degrees Celsius for one hour.
Then, remove the slides from the oven, and immediately deparaffinize them using three consecutive xylene rinses of five minutes each. After this, rehydrate the sections on the slide by sequential immersions in decreasing dilutions of ethanol to deionized water, as outlined in the text protocol.
Treat the slides with the protease for 30 minutes for proteolytic antigen retrieval, then, rinse the slides in PBST for 10 minutes, and treat them with 3% hydrogen peroxide for 10 minutes. After this, wash the slides again with PBST for 10 minutes.
To begin, remove one slide from the buffer, making sure to only handle one slide at a time, while the others remain submerged, and use a paper towel to blot off excess buffer from around the tissue section. Place the slides into a humidity chamber, and incubate with normal goat serum at room temperature for 15 minutes.
Next, incubate the slides in the humidity chamber with the optimal predetermined dilution primary anti-rabies antibody and negative control antibodies at room temperature for 60 minutes with no washes in between. After this, wash the slides with PBST for 10 minutes, and incubate in a humidity chamber with the biotinylated antibody at room temperature for 15 minutes.
Wash the slides again with PBST for 10 minutes. Incubate the slides in a humidity chamber with streptavidin-HRP complex at room temperature for 15 minutes, and wash the PBST for 10 minutes. Then, incubate with AEC in a humidity chamber at room temperature for 10 minutes. Wash the slides in deionized water for 10 minutes and counterstain with Gill&#39;s hematoxylin diluted 1 to 2 with deionized water for two minutes.
After this, rinse off the excess hematoxylin by dip-rinsing the slides in deionized water. Rinse the slides in Scott&#39;s tap water for 30 seconds, and wash in deionized water for 10 minutes. Remove the slides one at a time and mount them with water-soluble mounting medium. Then, use a light microscope to read the slides.

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Development of a Negative Selectable Marker for Entamoeba histolytica

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

Enhanced Rabies Surveillance Using a Direct Rapid Immunohistochemical Test

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

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

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

Quantitation of Rabies Virus in Various Bovine Brain Structures

Detection of Rabies IgG and IgM Antibodies Using the Rabies Indirect Fluorescent Antibody Test

Barwienie immunohistochemiczne do wykrywania antygenów wirusa wścieklizny