INTRODUCTION

Pronghorn (Antilocapra americana) is considered a keystone species of North American grasslands, and its population viability is an indication of a healthy grassland ecosystem. Historically, pronghorn populations were estimated at approx. 30 million in the early and mid-1900s,^35,59 but they declined to approx. 25,000 in the 1920s due to the movement of settlers resulting in habitat loss and fragmentation, land cultivation, and unrestricted harvest.⁶⁰ Intensive management with large-scale translocations allowed these populations to flourish to an estimated 1 million in 1984.^38,60 In Texas, the Texas Parks and Wildlife Department initiated a statewide restoration effort in 1939,²³ primarily sourcing individuals from other populations within the state.

Previously, pronghorn numbers had been depleted in the Trans-Pecos region of Texas, initiating a restocking and recovery project as well as research projects centered around documentation of causative factors of decline. In addition, they serve as a financially important source of income for many private landowners in Texas. In this region, pronghorn may experience significant population growth during years of ample precipitation⁵⁰ and the lack of precipitation predicted herd declines.²³ Some populations in the Texas Panhandle, regardless of drought, have flourished and are currently stable due to adequate fawn recruitment and low adult mortality. In fact, these populations are a source for translocations,²⁰ a key component to pronghorn restoration in North America.²¹ Although posttranslocation aerial surveys documented declines in restoration areas in the Trans-Pecos, population numbers remain above the historic lows seen in the early 2010s.

The overall objectives of any pronghorn restoration project are to evaluate the success of restoration efforts; monitor mortality and factors affecting survival; and document movements in relation to release sites, habitat components, and fencing.²⁰ The health status of a population can be examined by performing complete blood count and biochemical assessments of a healthy population to set normative values for future use. These tests can be useful to indicate infection, inflammation, disease processes, and organ system function, providing a comprehensive view of individual and population health. Normative values can vary among pronghorn populations, making it vital to develop normal blood parameters from healthy individuals and cautiously interpreting data from other geographic regions. In addition, presence or absence survey of disease pathogens throughout the population can identify areas of concern for naïve or translocated individuals and characterize potential disease threats to pronghorn populations.

Reference intervals (RI) are determined by 95% of a “healthy” population comprised of a recommended minimum of 120 individuals.¹⁷ Inclusion and exclusion criteria allow the healthy population to be established and sampling protocols set in place to promote consistency and eliminate bias. The objectives of this study were to provide baseline hematology and biochemical reference values and the presence of antibodies to the hemorrhagic disease viruses Epizootic hemorrhagic disease virus (EHDV) and Bluetongue virus (BTV) to assess the health status of newly captured pronghorn. This information provides baseline monitoring values to examine the impact of habitat and population health in current and future pronghorn populations as well as the presence of viral pathogens to guide future restoration efforts.

MATERIALS AND METHODS

Study site

Captures occurred on private ranches during 27–29 January 2014, 25–26 January 2016, 29 January–01 February 2017, 29–31 January 2018, and 27–29 January 2020 near Dalhart and Pampa, TX. Pronghorn were then translocated to the Marfa Northwest, Northeast, and Southwest Restoration areas and the Marathon Restoration Area as part of a larger pronghorn restoration effort in the Trans-Pecos region of Texas (Fig. 1). The Marfa Plateau is a grassland basin located in Brewster, Presidio, and Jeff Davis counties. It is bounded by three mountain ranges: the Chinati, Sierra Vieja, and Davis mountains. The elevation ranges from 1,067 to 1,372 m (3,500–4,500 ft), with topography similar to rolling terrain in the foothills bordering the mountain ranges. Average rainfall is 35.6–40.6 cm (14–16 in.), but can vary annually, with precipitation and severe thunderstorms occurring between June and October. A variety of plants, including brush and forb species, and excellent water distribution with ample permanent water sources make this region suitable for pronghorn.^7,10,24,37

Figure 1.

Capture-and-release sites for free-ranging pronghorn in the Panhandle and Trans-Pecos regions of Texas from 2014 to 2020. Figure is not to scale.

RESULTS

Baseline hematology and biochemical reference values for pronghorns of normal health status, both sexes, and adult age group are shown in Table 1. Differences based on sex and age were present for many hematology and biochemical values; however, narrow differences in mean values did not warrant the production of blood normals by class (Table 2). When testing for differences in hematology and biochemical values due to the presence of a rectal temperature >39.7°C, mean values and statistical results indicated elevations in sodium and chloride (Table 3). There were statistically significant effects of anisocytosis, poikilocytosis, reactive lymphocytes, and hemolysis on various blood cell parameters (  Supplemental Table 1 (04-Reeves Supplemental Table 1.docx)) compared with normal values established in this study (Table 3), with clinical relevance remaining unknown. In the overall population, antibodies to BTV were identified in 46.8% (195/417) of pronghorns and EHD was identified in 89.4% (194/217) of pronghorns; 84.8% (184/217) tested positive for both viruses. Based on year, BTV antibodies were identified in 90.0% (18/20) of pronghorns in 2014, 91.8% (89/97) in 2016, 88.0% (88/100) in 2017, 91.3% (95/104) in 2018, and 89.7% (87/97) in 2020. Based on year, EHDV antibodies were identified in 100% (20/20) of pronghorns in 2014, 85.6% (83/97) in 2016, and 91% (91/100) in 2017, and it was not tested for in 2018 and 2020. In the normal population used to develop RI, BTV antibodies were identified in 94.6% (229/242) and EHDV antibodies were identified in 89.2% (124/139) of pronghorns; 86.3% (120/139) tested positive for both viruses. The significant effects of disease on blood parameters by year and with the presence of exposure to BTV and EHDV are shown in Table 4.

Table 1.

Hematology and serum biochemistry RI for pronghorn based on data from clinically healthy free-ranging individuals of both sexes and adult age group in West Texas, 2014–2020.

Table 2.

Differences in hematology and biochemical parameters based on sex and age for pronghorn in the Panhandle of Texas, 2014–2020. Mean ± SEM values are presented.^a

Table 3.

Differences in hematology and biochemical parameters based on elevation of internal body temperature and laboratory abnormalities for pronghorn in the Panhandle of Texas, 2014–2020. Mean ± SEM values are presented.

Table 4.

Differences in hematological and biochemical parameters when positive for exposure to BTV and EHDV and by year for pronghorn in the Panhandle of Texas, 2014–2020.^a,b

DISCUSSION

To date, only a few studies have published RI for pronghorn populations during the 1970s–1990s residing in Oregon and Canada (  Supplemental Table 1 (04-Reeves Supplemental Table 1.docx)).^5,6,15 This study presents comprehensive RI from a large population of pronghorn sampled from 2014 to 2020 and is beneficial for continued monitoring of pronghorn populations in Texas. Baseline RI were compared with those previously published for pronghorn,^5,6,15 and when values were unavailable, to published intervals of related species of ruminants (mule deer [Odocoileus hemionus], white-tailed deer [Odocoileus virginianus], and bovine, caprine, ovine species) as provided by TVMDL and in the literature.^3,51 Biochemical testing was comprised of 16 analytes in which many (sodium, magnesium, calcium, phosphorus, blood urea nitrogen, creatinine, glucose, albumin, globulins, and total protein) were similar to those reported previously for pronghorn.^5,6,15 For values that did not fall within reported pronghorn ranges, two (chloride and total bilirubin) fell within or close to published reference ranges for ovine species as referenced by TVMDL. Multiple analytes (creatinine kinase [CK], gamma glutamyl transferase [GGT], aspartate transferase [AST], and potassium) either varied in upper and lower reference limits or were not similar to any of the reference species mentioned above.^3,51

Hematology testing consisted of 15 analytes of which most (hematocrit, hemoglobin, red blood cell concentration, mean cell hemoglobin concentration, mean cell hemoglobin, white blood cell concentration, lymphocytes, monocytes, and neutrophils) were similar to those in previous reports in pronghorn.^3,6,51 Others (mean cell volume, basophils, plasma protein, platelet concentration, and fibrinogen) were similar to referenced species of ruminants as referenced by TVMDL and cervids;^3,51 however, none were similar to one species alone. Multiple analytes (eosinophils and platelets) either fell outside or resulted in tighter ranges than reference ranges for other ruminants or cervids.^3,51 Although a few reference values exist for historic pronghorn populations, the establishment of values including current populations and the large sample size present in this study indicate the documented values to be a reliable source for current and future pronghorn populations in Texas.

Prolonged capture in hoof stock species can result in a life-threatening complication termed capture myopathy, or exertional rhabdomyolysis.^4,36 One of the most notable clinical findings is pronounced elevation in potassium that can sensitize the heart to catecholamines and thus acute death can occur.^4,36 Cattle have a high level of potassium intake in their diet; as such, they have higher renal excretory rates.^47,48 Catecholamines (specifically in skeletal muscle) stimulate the uptake of potassium by cells and the shift of sodium out of the cells, decreasing serum potassium and increasing sodium. Tissue necrosis and exercise can cause excretion of potassium from skeletal muscle and potassium release occurs from platelets during clotting. In addition, several days after the inciting event, elevations in myoglobin due to muscular damage can cause renal failure and death.^4,36 Two enzymes associated with capture myopathy and typically increased in the case of muscular damage are CK and AST.¹³ Although typically evaluated for muscular disease, these values can be elevated after exercise in normal individuals.¹³ CK, AST, and potassium values were elevated in pronghorn in this study compared with other analytes, suggesting the cause of increase could be due to elevated movements across landscapes naturally, high levels of exercise during capture, or a higher use of muscle in free-ranging populations, and/or these differences might represent differences in laboratories and methods used to determine these analytes. In addition, elevations in CK levels were documented in males compared with females and in fawns compared with adults (Table 2). These findings likely indicate greater levels of movement; longer chase times during capture; and/or longer exercise times before actual capture, but they would need to be evaluated further.

GGT is an enzyme that is common in the kidneys, the gastrointestinal system, and hepatobiliary system in many species and can serve as an indicator of cell damage during acute and excessive training or exercise.^14,42,45 In some donkeys and burros, their GGT values are 2–3 times higher than those of related equine species,⁸ suggesting differences at the species level, with fewer similarities to related species. Although our GGT values were not specifically elevated compared with those of other pronghorn populations, they were different, suggesting that different pronghorn populations may vary in GGT activity and could be related to differences in locations and/or diet sources among landscapes.

When individuals presented with elevated temperature (rectal temperature ≥39.7°C), greater levels of sodium and chloride electrolytes were observed. Although elevated, these blood chemistry values were not beyond “normal,” suggesting this may not be clinically relevant in free-ranging pronghorn. Elevations in sodium and chloride can be caused by elevations in catecholamines (epinephrine),¹ fluid loss in the respiratory tract from panting and fever, renal disease, and increased intake.^54,55 Elevations in both values and stress upon capture suggest these likely resulted from panting, fever, and potential elevations in cortisol, although cortisol levels would need to be directly measured and examined further.

When sex (male, female) and age (adult, fawn) were considered, there were differences noted in hematology and biochemical values; however, the differences were not of enough magnitude to warrant building RI by sex or age. This is likely due to the reduced number of males and fawns included in this study, because neither were the primary target demographic for translocation efforts. Elevations in potassium and AST were noted for males compared with females. Although not outside of the normal reference ranges, this could relate to tissue damage induced by capture and handling, because males may demonstrate more exercise and muscle use during capture.^19,31 For biochemical values, although many were slightly different, calcium and phosphorus were elevated in fawns compared with adults. This information is likely due to fawns being in a stage of growth due to enhanced intestinal absorption and decreased renal excretion, thought to be needed to facilitate bone mineralization.^22,34 These values, although higher, were not largely different from adults. We suspect with neonates, not included in this study, these values would be much more elevated and with more sample numbers, we might see a larger difference between fawns and adults. In addition, glucose and AST were reported to be higher in fawns compared with adults. Elevations in glucose and AST have been associated with stressful events and exercise, respectively, increasing suspicion that the elevations in these values could be related to a higher stress component and longer chase times during capture in fawns than adults,¹⁶ but comparison of “chase times” to these findings would need to be further explored.

Furthermore, we evaluated the effect of abnormalities reported by the testing laboratory such as hemolysis (lysis of red blood cells), poikilocytosis (change in red blood cell shape), and anisocytosis (change in cell size and diameter) on blood analytes. These abnormalities can be caused by mishandling of samples during collection and/or storage, during disease processes, or at low levels in clinically normal individuals. Although values varied with the presence or absence of testing abnormalities, none of the values fell outside or near reference limits for the reported values (Table 3). Caution should be taken with interpretation of blood values if abnormalities are reported, and careful handling of samples should always be practiced preventing iatrogenic causes such as underfilled blood tubes, using appropriately sized needles, and not forcing blood through needles by using high pressures. In addition, these abnormalities could falsely report values from ill individuals as normal, making it vital to handle samples with care at time of collection and during storage until analysis.

Finally, we examined the effect of testing positive for antibodies to agents of hemorrhagic disease, including EHDV and BTV, or both pathogens on blood analytes tested in this study. Both pathogens were present in the population with EHDV at a higher prevalence; however, a similar prevalence of exposure to both pathogens was found than in BTV alone. In 2010 and 2011, studies found 96–100% exposure prevalence to BTV and 92–93% exposure prevalence to EHDV in the Trans-Pecos region, respectively.⁵⁶ In 2011, exposure prevalence to BTV and EHDV in the Panhandle was 87 and 50.5%, respectively.⁵⁶ In other regions of the United States, BT and EHD have been shown to cause significant mortality events and large impacts on pronghorn populations;⁵³ however, in southern states, whereas exposure rates reaching near 80% were documented, the same mortality rates were not evident.^26,27,38 When assessed by year, the prevalence of both pathogens was similar from 2014 to 2017, but were unable to be compared in 2018 and 2020 because EHDV was not included in the testing protocol for captures taking place during these years. Few blood parameters differed by year (Table 4), including declines in red blood cell parameters in 2014 specifically. During this year, evidence of hemorrhagic disease did not exceed or differ greatly from that of other years, suggesting this to be an incidental finding and/or a result of small sample sizes.

Hemorrhagic disease pathogens primarily affects white-tailed deer, but can affect mule deer and pronghorn, among other ruminant species.⁴⁴ The viruses are transmitted to deer by Culicoides midges (also known as “no-see-ums”).²⁹ Clinical signs of disease can begin within 10 d of infection and include fever and swollen head, neck, tongue, and eyelids.⁵⁸ This may result in a decrease in appetite and weakness, resulting in death within 36 h. There is no treatment for hemorrhagic diseases, or prevention currently in place, in wildlife populations. These clinical signs and symptoms can mimic foot and mouth disease, an exotic disease not currently present in the United States, but more severe in clinical signs and mortality. Some individuals develop an immunity to the virus and are able to survive,^9,11,38 although it is suggested that areas with high white-tailed deer presence or smaller, isolated populations may be at greater risk of impact. In Table 4, many analytes differed based on the presence of disease with suspected increases in inflammatory cells, decreases in red blood cells, and elevations in values suggesting dehydration. Although differences were apparent, these values did not fall outside of normal parameters, questioning their clinical significance. It is suspected that these individuals have evolved with the presence of these pathogens in their population and likely formed an immunity that allowed them to survive in their presence.

This study developed a baseline reference profile for free-ranging pronghorn populations in northwestern Texas. In addition, it identifies high presence of hemorrhagic disease with a likely result of immunity within this population. Continued monitoring of these pathogens is recommended in the event of a mortality event or the potential spread to small, isolated, naive populations. Although significant differences were noted when pathogens were present, the removal of positive individuals to develop the normal parameters were not indicated. These values can be used to monitor current population health, translocation success, disease spread, and the differences in blood analytes across population and environments.