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

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 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 possible^5,6,7,8.

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 uptake^18,19. 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 whiskers^10,21.

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, raccoons^9,25, 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 marsupials^28,29, 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 "bait matrix") 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/kg^27,36 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 >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 °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 α = 0.05.

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.

1. Preparation of solutions and standards

1. Purchase me-IPA and et-IPA.
2. 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 (≥ 18 MΩ). 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.
3. 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.
4. Prepare concentrated IPA stock solutions of me-IPA and et-IPA in ACN at concentrations of approximately 1,000 µg/mL.
   1. Weigh approximately 10 mg of me-IPA on a microbalance and record the mass to ± 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.
   2. 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.
5. For the 25x-7 me-IPA stock (Table 1), prepare a stock of me-IPA in ACN at approximately 200 µg/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 µL glass syringe. Dilute to volume with ACN. Transfer the stock to an amber 8 mL glass vial with PTFE-lined cap. Store at RT.
6. 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.
7. For the 25x surrogate stock, prepare a surrogate stock of me-IPA in ACN at approximately 10 µg/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 µL glass syringe, and then dilute to volume with ACN.
   1. Transfer ~8 mL to an amber 8-mL glass vial with PTFE-lined cap. Store at RT. Transfer the remaining stock to hazardous waste.
8. Prepare 4x stocks containing both analytes in 2 mL screw-top glass autosampler vials as described in Table 2.
   1. 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.
   2. Add 0.85 mL of ACN using a repeat pipettor with 1 mL capacity tip. Cap the vial securely and invert 5x to mix.
9. Prepare the standard curve in 2 mL screw-top autosampler vials as described in Table 3.
   1. 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.

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 2.3−2.6 in a class II biosafety cabinet.

1. For each sample, prepare a 1.5 mL microcentrifuge tube containing 200−300 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.
2. For each sample, label two 1.5 mL microcentrifuge tubes: one as "A" and the other as "B". Arrange the tubes in an 80-position plastic rack.
3. 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, 100−1,000 µL air displacement pipette with 1,000 µL tips, containers with approximately 100 mL each of diluent and ultrapure DI water, and a biohazard waste container.
4. Remove serum samples from frozen storage and warm to RT in the biosafety cabinet. Vortex mix each serum sample prior to sampling.
5. Using a repeat pipettor with 0.5 mL capacity tip, dispense 0.050 mL of mongoose serum into tube "A" and add 0.050 mL of 25x surrogate stock. Then add 0.950 mL of diluent to tube "A" using a repeat pipettor with 5 mL capacity tip. Cap securely and vortex mix for 10−15 s.
6. Dispense the pre-weighed NaCl from step 2.1 into tube "A" and vortex mix 3x for 8−12 s. Wipe down the outside surfaces of the vial rack containing tube "A" using 70% (v/v) isopropanol.
   NOTE: The rack of samples may now be removed from the class II biosafety cabinet.
7. Centrifuge tube "A" 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 "B" using a 100−1,000 µL air displacement pipette. Transfer the remaining solution in tube "A" to hazardous waste and discard the empty tube in a biohazardous waste container.
8. Remove ACN and TFA from tube "B" with a gentle flow of N₂ gas in a 45 °C water bath.
9. Add 0.250 mL of ACN to tube "B" using a repeat pipettor, vortex mix for 4−5 s, and then centrifuge briefly (2−4 s) at 12,000 x g to collect the liquid in the bottom of the tube.
10. Add 0.750 mL of ultrapure DI water to tube "B" using a repeat pipettor with 5 mL capacity tip, vortex mix for 4−5 s, and then centrifuge for 1 min at 12,000 x g to clarify the sample.
11. Transfer 0.75 mL of the supernatant to an autosampler vial using a 1,000 µL air displacement pipette. Discard pipette tips in biohazard waste container.
12. Cap autosampler vials securely and analyze by LC-MS/MS (section 4). Transfer the remaining solution in tube "B" to hazardous waste and discard the empty tube to a biohazardous waste container. Dispose of all biohazardous waste by autoclaving or incineration.

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.

1. Prepare four 1.5 mL microcentrifuge tubes containing 200−300 mg of NaCl. Arrange the tubes in an 80-position plastic rack.
2. For each QC sample, label two 1.5 mL microcentrifuge tubes: one as "A" and the other as "B". Arrange the tubes in an 80-position plastic rack.
3. Repeat step 2.3.
4. Remove control mongoose serum from frozen storage and warm to RT in the biosafety cabinet. Vortex mix the control serum prior to sampling.
5. Dispense 0.050 mL of control mongoose serum into the four 1.5-mL "A" tubes using a repeat pipettor with 0.5 mL capacity tip.
6. 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 10−15 s.
7. Perform the extraction procedure as described in steps 2.6−2.12.

4. LC-MS/MS analysis

1. 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 °C before setting the flow rate to 0.800 mL/min.
2. 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.
3. Inject all standards and samples and acquire MRM ion chromatograms using parameters listed in Table 5.
4. After sequence completion, turn off the LC-MS/MS and dispose of all autosampler vials as hazardous waste.

5. Quantification

1. 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 → 428.7) divided by the MRM transition for et-IPA (570.7 → 442.7). Construct a 7-level calibration curve using a second order quadratic function that is weighted 1/x and ignores the origin.
2. Calculate the serum concentration (C_serum) of me-IPA using the following equation:
   
   where c_instrument is the concentration determined by the instrument from the calibration curve in units of µg/mL, 1.25 is the dilution factor , V_final is the final sample volume (1.0 mL), and V_serum is the serum volume in mL (0.050 mL nominal).

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. Figure 1C shows a representative sample from the study with an observed serum concentration of 33.5 µg/mL me-IPA. 

A representative calibration curve from a me-IPA analysis is presented in Figure 2. The 7-level, 14-point standard curve ranges from 0.00202 to 8.27 µg/mL me-IPA with a correlation coefficient (r²) of 0.9998. Correlation coefficients ranged from 0.9998 to 0.99997 for the five me-IPA analyses. The surrogate analyte et-IPA concentration was 0.502 µg/mL in all standards.

Table 6 presents the accuracy and precision results for control mongoose serum fortified with 0, 1.3, 31, and 82 µg/mL me-IPA (n = 10 at each level). The results were collected from five separate analyses. Percent recoveries ranged from 96.9% to 109%. The percent relative standard deviation (% RSD) at the three fortification levels was 3.4%, 1.7%, and 2.3%, respectively.

The signal-to-noise ratio (S/N) observed in quality control samples (n = 10 negative controls; n = 10 at 1.3 µg/mL me-IPA) was used to determine the detection limit (DL; 3 x S/N) and quantitation limit (QL; 10 x S/N). The DL and QL for me-IPA in mongoose serum were 0.012 µg/mL and 0.042 µg/mL, respectively.

The peak area responses of the qualifier MRM transition divided by the quantifier MRM transition ( was calculated for all standards and samples. This ratio for each sample was then divided by the average ratio observed in the calibration standards to determine the qualifier percent match. The qualifier ratio for the sample shown in Figure 1C was 0.439, with a 96.3% match.

The recovery of et-IPA surrogate analyte was calculated for all QC samples and unknown samples by dividing the et-IPA peak area response by the average et-IPA peak area response observed in the calibration standards. Average surrogate analyte recoveries were 91.0% (negative controls), 91.4% (1.3 µg/mL), 92.8% (31 µg/mL), and 95.4% (82 µg/mL).

No interference peaks for either the quantifier or qualifier transitions of me-IPA were observed in control mongoose serum (Figure 1A).

The extraction procedure and instrumental conditions used to determine et-IPA in mongoose serum (Figure 3 and Table 7) was identical to the me-IPA method, but with the following changes. Propyl-iophenoxic acid (pr-IPA) was used as the surrogate analyte and an older, less sensitive LC-MS/MS was used. The source drying gas temperature was 350 °C with a flow of 12 L/min and a nebulizer pressure of 35 psi. The capillary voltage was -2,500 V. The source had no sheath gas or means to adjust nozzle voltage. The quantifier MRM transition for et-IPA was 570.7 → 442.8 (584.7 → 456.8 for pr-IPA). The fragmentor was 80 V and the collision energy was 10 V for both analytes. The qualifier MRM for et-IPA was 570.7 → 126.8 with a collision energy of 40 V.

The testing of all mongoose serum samples for et-IPA was performed over eight analyses. The 7-level calibration curve ranged from 0.00207 to 8.48 µg/mL with correlation coefficients (r²) ranging from 0.9990 to 0.9999. The surrogate analyte pr-IPA concentration was 0.512 µg/mL. Table 7 presents the accuracy and precision results for control mongoose serum fortified with 0, 1.3, 13, 32, 85, and 170 µg/mL me-IPA. The results were collected from eight separate analyses. Percent recoveries ranged from 89.5% to 115%. The % RSD at the five fortification levels was 4.3%, 1.5%, 2.3%, 5.6%, and 1.1%, respectively. The S/N observed in quality control samples (n = 21 negative controls; n = 21 at 1.3 µg/mL me-IPA) was used to determine the DL and QL. The DL and QL for me-IPA in mongoose serum were 0.12 µg/mL and 0.42 µg/mL, respectively. The average surrogate analyte recovery from quality control samples was 86.8% (n = 75). No interference peaks for either the quantifier or qualifier transitions of et-IPA were observed in control mongoose serum.

All mongooses offered et-IPA baits consumed ≥25% of the bait within the 24 hour time constraint and had quantifiable levels of et-IPA in their sera (Table 8). From the mixed model analysis, overall mean serum IPA concentrations were marginally higher from baits with 2.8 mg of biomarker in the bait matrix (17.5 µg/mL, 95% CI 11.7−23.3 µg/mL) in comparison to the blister pack (9.8 µg/mL, 95% CI 4.0−15.6 µg/mL) (F = 3.6, P = 0.07). Concentrations for both bait types decayed with experimental day (β = -0.15 ± 0.04, F = 14.4, P = 0.0005). Individual level variability in serum IPA concentrations was observed (animal ID covariance parameter estimate = 46.6 ± 20.7). All mongooses consumed 100% of control baits with only the empty foil blister pack remaining. Mean concentration of et-IPA residue in serum was variable by time period and did not appear to decrease consistently over time (Figure 3).

All mongooses offered me-IPA baits consumed ≥ 25% of the bait within the 24 hour time limit and had quantifiable levels of me-IPA in their sera (Table 9). Mean serologic concentration of et- and me-IPA in mongoose sera appeared dependent upon the concentration of IPA in the bait. Higher concentrations of IPA in the bait resulted in higher serum residues even in cases where the overall dose (2.8 mg in the case of et-IPA) remained the same. Interestingly, me-IPA appeared to produce a more even degradation pattern over time with an initial spike at day 1, followed by a steady decline until day 14 where concentrations appeared to plateau (Figure 4).

Figure 1: Representative ion chromatograms. (A) Representative MRM ion chromatogram of control mongoose serum fortified with surrogate analyte ethyl-iophenoxic acid (et-IPA). The arrow indicates the retention time for methyl-iophenoxic acid (me-IPA). (B) Representative MRM ion chromatogram of control mongoose serum fortified with 31 µg/mL me-IPA. The relative intensities of the quantifier (Quant) and qualifier (Qual) transitions for me-IPA are shown. (C) Representative MRM ion chromatogram of a mongoose serum sample with an observed me-IPA concentration of 33.5 µg/mL. Please click here to view a larger version of this figure.

Figure 2: Representative calibration curve. An original calibration curve generated by the data analysis software for methyl-iophenoxic acid (me-IPA). Please click here to view a larger version of this figure.

Figure 3: Mean serum ethyl IPA (et-IPA) concentration by bait type and concentration over time. Please click here to view a larger version of this figure.

Figure 4: Mean serum methyl IPA (me-IPA) concentration by bait concentration over time. All me-IPA concentrations were incorporated into the external bait matrix. Please click here to view a larger version of this figure.

Table 1: Preparation of 25x me-IPA stocks in ACN (in 8-mL amber glass vials).

Table 2: Preparation of 4x IPA stocks in ACN (in 2-mL autosampler vials).

Table 3: Preparation of me-IPA standards in 75/25 water/ACN (in 2-mL autosampler vials).

Table 4: Quality control sample fortification (prepare in 1.5-mL microcentrifuge tubes).

Table 5: LC-MS/MS parameters. Quantifier product ions are bolded.

Table 6: QC results for me-IPA in mongoose serum (µg/mL).

Table 7: QC results for et-IPA in mongoose serum (µg/mL).

Table 8: Mean (stand deviation [SD]) ethyl-IPA serum concentration by bait type. ND = not detected, NA = Not applicable.

Table 9: Mean (SD) methyl-IPA serum concentration by dose. ND = Not detected, NA = Not applicable. All methyl IPA concentrations were incorporated into the external bait matrix.

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 <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 µg/mL, respectively. Similar research performed at Instituto de Investigación en Recursos Cinegéticos, Ciudad Real, Spain, using butyl and pentyl-IPA found day 14 concentrations of approximately 45 and 10 µg/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 (4−8 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 4−6 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.