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

Summary

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.

Abstract

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.

Introduction

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 Hemisphere^1,2,3. 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 coordinate....

Protocol

All procedures were approved by the USDA National Wildlife Research Center'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.

Results

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. .......

Discussion

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 .......

Materials

List of materials used in this article

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