DOI: 10.5281/zenodo.15132004
title: Long-term carriage of Mycoplasma  ovipneumoniae in bighorn sheep and implications for test and remove protocols

description: Data information:

Each data fils is associated with a model in the manuscript "Long-term carriage of Mycoplasma ovipneumoniae in bighorn sheep and implications for test and remove protocols" and is explained in detail below.

Movi.csv contains the data used in the model evaulating the seasonal effects on testing positive for Mycoplasma ovipneumoniae (M. ovi). 

Movi_PrevMovi.csv contains the data used in evauating the effects of previous M. ovi status, season, and age on the probability of testing positive for M. ovi.

Scenarios.csv contains the data used in hypothetica scenarios of test and remove. "Repeated" indicates that the animal would have been removed at that capture event under a repeated positive removal criteria and "Single" indicates that the animal would have been removed under a single positive removal criteria.

For each dataset "Movi" indicates if an animal tested positive for M. ovi, "AID" indicates the unique animal identifier, "Herd" indicats the population the animal belongs to, "Season" indicates when the test was taken (Spring tests were taken in March and Winter tests were taken in December), "age" indicates the age of the animal when the test was taken, and "CC" indicates if an animal was classifed as a chronic carrier. Vairables that start with "prev" indicate the value of that variable at the previous capture event.

Methods:

Animal Capture and Handling

We captured female bighorn sheep in the Jackson, Upper Shoshone, and Whiskey Mountain populations beginning in 2015 and beginning in 2021 for the West Side population using helicopter net gunning (Jackson, Whiskey Mountain, and West Side; Wagler et al. 2022) or ground darting with chemical immobilization (Upper Shoshone; Kock et al. 1987). Most captures were adults (>2 years), but in the Jackson, Whiskey Mountain, and West Side populations we also captured juveniles (≤ 2 years) that had previously been captured as neonates (2019–2023). Each March and December following the initial capture event, we recaptured as many collared individuals as possible and captured new animals to maintain target sample sizes in each population (N = 25 in Jackson, 15 in Upper Shoshone, 15 in West Side, and 30 in Whiskey Mountain) until spring 2023. Recapture rates were variable and ranged from 0.09 to 0.88. Some recaptures were not possible because of weather conditions or animals being in dangerous terrain or Wilderness areas. We had permission to capture animals in the Bridger Wilderness (West Side) and the Glacier Addition of the Fitzpatrick Wilderness (Whiskey Mountain); however, we missed recapture of some animals in the Whiskey Mountain and Jackson populations because of restrictions in those Wilderness areas. December captures occurred during the end of the mating season and when most of the migrants were on low-elevation winter ranges. March captures occurred in late winter when animals were in poorer nutritional condition (Smiley et al. 2022) and before spring migration and parturition (Argov et al. 2024). Upon capture, we assigned a unique identifier (Animal ID) to each animal fitted animals with a GPS collar (Advanced Telemetry Systems, Isanti, MN, USA; VECTRONIC Aerospace Gmbh, Berlin, Germany). We assessed nutritional condition using body palpation to estimate a body condition score and measured rump fat with ultrasonography (5-MHz transducer; Ibex Pro, E.I. Medical Imaging, Loveland, CO, USA; (Stephenson et al. 2020). We calculated percent ingesta-free body fat (hereafter body fat) for all animals using an equation developed for bighorn sheep (Stephenson et al. 2020). We estimated the age of animals based on incisor replacement, tooth wear, and horn annuli (Valdez and Krausman 1999). We cleaned capture equipment between animals with a solution of Virkon S Disinfectant (Lanxess, Cologne, Germany) or Re-Juv-Nal (Hillyard, Denver, Colorado, USA) to prevent pathogen transmission. All animal and handling was approved by Institutional Animal Care and Use Committee (20150316KM00148, 20180305KM00296, 20200305KM00412-03), the Wyoming Game and Fish Department (Chapter 33-1278), and were in accordance with the guidelines of the American Society of Mammalogists (Sikes 2016).

Pathogen detectionWe collected one nasal swab at each capture event to test for the presence of M. ovi by culture followed by PCR (detection probability of 0.85; Butler et al. 2017; Figure 1). We inserted nasal swabs deep into each of the nares and rotated them against the mucosa before removal. We stored polyester swabs (Puritan Medical Products, Pittsfield, Maine, USA) in tryptic soy broth (TSB; Hardy Diagnostics, Santa Maria, California, USA) with 15% glycerol (Butler et al. 2017); Alfa Aesar - Thermo Fisher Scientific, Ward Hill, Massachusetts, USA) and frozen on dry ice for transport. We placed swabs in enrichment broth (modified TSB-1 Jennings-Gaines et al. 2016), and culture (Wood et al. 2017) and PCR (Manlove et al. 2019) were performed at the Wyoming Game and Fish Wildlife Health Laboratory (Laramie, Wyoming, USA). For PCR, DNA was extracted from enrichment broth using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, Maryland, USA).

Statistical analysisWe first evaluated the effects of the seasonal timing of testing (i.e. March or December) and age on the probability of testing positive for M. ovi. We assessed the probability of testing positive for M. ovi regardless of previous infection status and included M. ovi status as the response and age and timing of test as explanatory variables. We included a random intercept for animal ID nested within population.Removal criteria (i.e., how many positive tests constitute removal) have varied across test and remove protocols where some protocols require removal of all animals that test positive and some protocols require repeated positives for removal. To assess the likelihood of remaining positive for M. ovi after testing positive once, we used two additional models. These models included M. ovi status during the previous season (i.e., previous March or previous December) or previous year (i.e., 24 months prior) as an explanatory variable. We retained age and the timing of test as predictor variables. We used these additional models to inform the likelihood of remaining positive at two different time scales (season or year); consequently, we subset data to animals where their previous M. ovi status was known. We included a random intercept for population but not for Animal ID because consistently testing positive for M. ovi was a repeatable characteristic of the animal (i.e., Animal ID and consistently testing positive for M. ovi).We fit all three mixed-effects binomial regression models using the glmmTMB package (Brooks et al. 2017) in Program R version 4.3.2 (R Core Team 2023). Younger and older animals were more likely to test positive for M. ovi than middle aged animals in a different study area (Plowright et al. 2017). For each model we first evaluated which shape for the age term fit the data best by comparing global models with a linear, quadratic, and logarithmic age relationship and used the shape for age with the lowest AICc in the global model moving forward. We built a global model consisting of all variables of interest and used Akaike information criterion adjusted for small sample size (AICc) to compare every possible combination of variables from the global model (Burnham and Anderson 2002). We evaluated all models within 2 AICc of the top model and retained the model with the most variables because we were interested in the effects of all predictors, even if they were marginally informative. To understand how different removal criteria influence the number of animal removals, we compared the proportion of animals that qualified for removal from four hypothetical scenarios of test and remove occurring over two consecutive capture events that were one year apart. We subset this analysis to animals that were ≥2 years old because most test and remove efforts are focused on adults. The four scenarios included two different removal criteria (single positive test and repeated positive test) implemented with captures (i.e., testing for M. ovi) occurring in two different seasons (December and March). For each scenario, we subset our data to two consecutive capture events within the respective months (i.e., one year apart) and only included animals that were captured at each capture event. Under the single positive removal criteria, we quantified the proportion of animals that tested positive once out of the two consecutive M. ovi tests. Under the repeated positive removal criteria, we quantified the proportion of animals that had two positive M. ovi tests. We quantified the proportion of animals that qualified for removal under all four hypothetical scenarios for each consecutive two capture window (n = 8 capture windows for March, n = 7 for December).