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 ...

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 border="0" cellpadding="0" cellspacing="0" style="width:1036px;" width="1036">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">Acetonitrile, Optima grade</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">A996</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">Analytical balance</td>
			<td style="width:193px;">Mettler Toledo</td>
			<td style="width:304px;">XS204</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:380px;">C18 column, 2.1 x 50 mm, 2.5-&#181;m particle size</td>
			<td style="width:193px;">Waters Corp.</td>
			<td style="width:304px;">186003085</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">ESI Source</td>
			<td style="width:193px;">Agilent&#160;</td>
			<td style="width:304px;">G1958-65138</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:380px;">Ethyl-iophenoxic acid, 97 %</td>
			<td style="width:193px;">Sigma Aldrich</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;">Lot MKBP5399V</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:380px;">Formic acid, LC/MS grade</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">A117</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:380px;">LCMS software</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">MassHunter Data Acquisition and Quantitative Analysis</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:380px;">Methyl-iophenoxic acid, 97 % (w/w)</td>
			<td style="width:193px;">PR EuroChem Ltd.</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;">Lot PR0709514717</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">Microanalytical balance</td>
			<td style="width:193px;">Mettler Toledo</td>
			<td style="width:304px;">XP6U</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">Microcentrifuge</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">5415C</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">MS/MS</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">G6470A</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:380px;">N-Evap</td>
			<td style="width:193px;">Organomation</td>
			<td style="width:304px;">115</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:380px;">Oral Rabies Vaccine Baits</td>
			<td style="width:193px;">IDT Biologika, Dessau Rossleau, Germany</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">Propyl-iophenoxic acid, 99 % (w/w)</td>
			<td style="width:193px;">PR EuroChem Ltd.</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;">Lot PR100612108RR</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:380px;">Repeat pipettor</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">M4</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:380px;">Screw-top autosampler vial caps, PTFE-lined</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">5190-7024</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:380px;">Sodium chloride, Certified ACS grade</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">S271</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:380px;">Statistical Software Package</td>
			<td style="width:193px;">SAS Institute, Cary, North Carolina, USA</td>
			<td style="width:304px;">N/A</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:380px;">Trifluoroacetic acid, 99 %</td>
			<td style="width:193px;">Alfa Aesar</td>
			<td style="width:304px;">L06374</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;width:380px;">UPLC</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">1290 Series</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:380px;">Vortex Mixer</td>
			<td style="width:193px;">Glas-Col</td>
			<td style="width:304px;">099A PV6</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">0.2-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.413</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">0.5-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.421</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">1.5-mL microcentrifuge tubes</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">14-666-325</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:380px;">1250-&#181;L capacity pipette tips</td>
			<td style="width:193px;">GeneMate</td>
			<td style="width:304px;">P-1233-1250</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:380px;">1-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.43</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:380px;">2-mL amber screw-top autosampler vials</td>
			<td style="width:193px;">Agilent</td>
			<td style="width:304px;">5182-0716</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:380px;">5-mL pipettor tips</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">30089.456</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">80-position microcentrifuge tube rack</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">05-541-2</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:380px;">8-mL amber vials with PTFE-lined caps</td>
			<td style="width:193px;">Wheaton</td>
			<td style="width:304px;">224754</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:380px;">70 % (v/v) isopropanol</td>
			<td style="width:193px;">Fisher</td>
			<td style="width:304px;">A459</td>
			<td style="width:159px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:380px;">100-1000 &#181;L air displacement pipette</td>
			<td style="width:193px;">Eppendorf</td>
			<td style="width:304px;">ES-100</td>
			<td style="width:159px;"></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.

Representative ion chromatograms from a me-IPA analysis are presented in Figure 1. The control mongoose serum (Figure 1A) illustrates the retention time of et-IPA (surrogate analyte) and the absence of me-IPA at the indicated retention time. The quality control sample (Figure 1B) illustrates the baseline separation of me-IPA from et-IPA as well as the quantifier and qualifier transitions for me-IPA. ...

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 ...

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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

An Analytical Tool-box for Comprehensive Biochemical, Structural and Transcriptome Evaluation of Oral Biofilms Mediated by Mutans Streptococci

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and composition 1, 2. In general, biofilms develop from initial microbial attachment on a surface followed by formation of cell clusters (or microcolonies) and further development and stabilization of the microcolonies, which occur in a complex extracellular matrix. The majority of biofilm matrices harbor exopolysaccharides (EPS), and dental biofilms are no exception; especially those associated with caries disease, which are mostly mediated by mutans streptococci 3. The EPS are synthesized by microorganisms (S. mutans, a key contributor) by means of extracellular enzymes, such as glucosyltransferases using sucrose primarily as substrate 3.
Studies of biofilms formed on tooth surfaces are particularly challenging owing to their constant exposure to environmental challenges associated with complex diet-host-microbial interactions occurring in the oral cavity. Better understanding of the dynamic changes of the structural organization and composition of the matrix, physiology and transcriptome/proteome profile of biofilm-cells in response to these complex interactions would further advance the current knowledge of how oral biofilms modulate pathogenicity. Therefore, we have developed an analytical tool-box to facilitate biofilm analysis at structural, biochemical and molecular levels by combining commonly available and novel techniques with custom-made software for data analysis. Standard analytical (colorimetric assays, RT-qPCR and microarrays) and novel fluorescence techniques (for simultaneous labeling of bacteria and EPS) were integrated with specific software for data analysis to address the complex nature of oral biofilm research.
The tool-box is comprised of 4 distinct but interconnected steps (Figure 1): 1) Bioassays, 2) Raw Data Input, 3) Data Processing, and 4) Data Analysis. We used our in vitro biofilm model and specific experimental conditions to demonstrate the usefulness and flexibility of the tool-box. The biofilm model is simple, reproducible and multiple replicates of a single experiment can be done simultaneously 4, 5. Moreover, it allows temporal evaluation, inclusion of various microbial species 5 and assessment of the effects of distinct experimental conditions (e.g. treatments 6; comparison of knockout mutants vs. parental strain 5; carbohydrates availability 7). Here, we describe two specific components of the tool-box, including (i) new software for microarray data mining/organization (MDV) and fluorescence imaging analysis (DUOSTAT), and (ii) in situ EPS-labeling. We also provide an experimental case showing how the tool-box can assist with biofilms analysis, data organization, integration and interpretation.

Biofilms formed on tooth surfaces are highly complex and exposed to constant innate and exogenous environmental challenges, which modulate their architecture, physiology and transcriptome. We developed a toolbox to examine the composition, structural organization and gene expression of oral biofilms, which can be adapted to other areas of biofilm research.

Biofilms are highly dynamic, organized and structured communities of microbial cells enmeshed in an extracellular matrix of variable density and ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and frequently of indigenous origin. The ability of some anaerobic pathogens to invade human cells gives them adaptive measures to escape innate immunity as well as to modulate host cell behavior. However, ensuring that the anaerobic bacteria are live during experimental investigation of the events may pose challenges. Porphyromonas gingivalis, a Gram-negative anaerobe, is capable of invading a variety of eukaryotic non-phagocytic cells. This article outlines how to successfully culture and assess the ability of P. gingivalis to invade human umbilical vein endothelial cells (HUVECs). Two protocols were developed: one to measure bacteria that can successfully invade and survive within the host, and the other to visualize bacteria interacting with host cells. These techniques necessitate the use of an anaerobic chamber to supply P. gingivalis with an anaerobic environment for optimal growth.
The first protocol is based on the antibiotic protection assay, which is largely used to study the invasion of host cells by bacteria. However, the antibiotic protection assay is limited; only intracellular bacteria that are culturable following antibiotic treatment and host cell lysis are measured. To assess all bacteria interacting with host cells, both live and dead, we developed a protocol that uses fluorescent microscopy to examine host-pathogen interaction. Bacteria are fluorescently labeled with 2&#39;,7&#39;-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and used to infect eukaryotic cells under anaerobic conditions. Following fixing with paraformaldehyde and permeabilization with 0.2% Triton X-100, host cells are labeled with TRITC phalloidin and DAPI to label the cell cytoskeleton and nucleus, respectively. Multiple images taken at different focal points (Z-stack) are obtained for temporal-spatial visualization of bacteria. Methods used in this study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

This article presents two protocols: one to measure anaerobic bacteria that can successfully invade and survive within the host, and the other to visualize anaerobic bacteria interacting with host cells. This study can be applied to any cultivable anaerobe and any eukaryotic cell type.

Anaerobic bacteria far outnumber aerobes in many human niches such as the gut, mouth, and vagina. Furthermore, anaerobic infections are common and ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected with Mycobacterium tuberculosis. Despite decades of research, many of the mechanisms behind the success of M. tuberculosis as a pathogenic organism remain to be investigated, and the development of safer, more effective antimycobacterial drugs are urgently needed to tackle the rise and spread of drug resistant tuberculosis. However, the progression of tuberculosis research is bottlenecked by traditional mammalian infection models that are expensive, time consuming, and ethically challenging. Previously we established the larvae of the insect Galleria mellonella (greater wax moth) as a novel, reproducible, low cost, high-throughput and ethically acceptable infection model for members of the M. tuberculosis complex. Here we describe the maintenance, preparation, and infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux. Using this infection model, mycobacterial dose dependent virulence can be observed, and a rapid readout of in vivo mycobacterial burden using bioluminescence measurements is easily achievable and reproducible. Although limitations exist, such as the lack of a fully annotated genome for transcriptomic analysis, ontological analysis against genetically similar insects can be carried out. As a low cost, rapid, and ethically acceptable model for tuberculosis, G. mellonella can be used as a pre-screen to determine drug efficacy and toxicity, and to determine comparative mycobacterial virulence prior to the use of conventional mammalian models. The use of the G. mellonella-mycobacteria model will lead to a reduction in the substantial number of animals currently used in tuberculosis research.

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Galleria mellonella was recently established as a reproducible, cheap, and ethically acceptable infection model for the Mycobacterium tuberculosis complex. Here we describe and demonstrate the steps taken to establish successful infection of G. mellonella with bioluminescent Mycobacterium bovis BCG lux.

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

An Automated Culture System for Use in Preclinical Testing of Host-Directed Therapies for Tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent of COVID-19. Mtb has evolved to persist in its intracellular environment, evade host defenses, and has developed resistance to many anti-tubercular drugs. One approach to solving resistance is identifying existing&#160;approved drugs that will boost the host immune response to Mtb. These drugs could then be repurposed as adjunctive host-directed therapies (HDT) to shorten treatment time and help overcome antibiotic resistance.
Quantification of intracellular Mtb growth in macrophages is a crucial aspect of assessing potential HDT. The gold standard for measuring Mtb growth is counting colony-forming units (CFU) on agar plates. This is a slow, labor-intensive assay that does not lend itself to rapid screening of drugs. In this protocol, an automated, broth-based culture system, which is more commonly used to detect Mtb in clinical specimens, has been adapted for preclinical screening of host-directed therapies. The capacity of the liquid culture assay system to investigate intracellular Mtb growth in macrophages treated with HDT was evaluated. The HDTs tested for their ability to inhibit Mtb growth were all-trans Retinoic acid (AtRA), both in solution and encapsulated in poly(lactic-co-glycolic acid) (PLGA) microparticles and the combination of interferon-gamma and linezolid. The advantages of this automated liquid culture-based technique over the CFU method include simplicity of setup, less labor-intensive preparation, and faster time to results (5-12 days compared to 21 days or more for agar plates).

Rapid and efficient quantification of intracellular M. tuberculosis growth is crucial for pursuing improved therapies against tuberculosis (TB). This protocol describes a broth-based colorimetric detection assay using an automated liquid culture system to quantify Mtb growth in macrophages treated with candidate host-directed therapies.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), was the most significant infectious disease killer globally until the advent ...