Timothy N. Holt

Increased prevalence of EPAS1 variant in cattle with high-altitude pulmonary hypertension

High-altitude pulmonary hypertension (HAPH) has heritable features and is a major cause of death in cattle in the Rocky Mountains, USA. Although multiple genes are likely involved in the genesis of HAPH, to date no major gene variant has been identified. Using whole-exome sequencing, we report the high association of an EPAS1 (HIF2α) double variant in the oxygen degradation domain of EPAS1 in Angus cattle with HAPH, mean pulmonary artery pressure >50 mm Hg in two independent herds. Expression analysis shows upregulation of 26 of 27 HIF2α target genes in EPAS1 carriers with HAPH. Of interest, this variant appears to be prevalent in lowland cattle, in which 41% of a herd of 32 are carriers, but the variant may only have a phenotype when the animal is hypoxemic at altitude. The EPAS1 variant will be a tool to determine the cells and signalling pathways leading to HAPH.

High-altitude pulmonary hypertension in cattle (brisket disease): Candidate genes and gene expression profiling of peripheral blood mononuclear cells

High-altitude pulmonary hypertension (HAPH) is a consequence of chronic alveolar hypoxia, leading to hypoxic vasoconstriction and remodeling of the pulmonary circulation. Brisket disease in cattle is a naturally occurring animal model of hypoxic pulmonary hypertension. Genetically susceptible cattle develop severe pulmonary hypertension and right heart failure at altitudes >7,000 ft. No information currently exists regarding the identity of the pathways and gene(s) responsible for HAPH or influencing severity. We hypothesized that initial insights into the pathogenesis of the disease could be discovered by a strategy of (1) sequencing of functional candidates revealed by single nucleotide polymorphism (SNP) analysis and (2) gene expression profiling of affected cattle compared with altitude-matched normal controls, with gene set enrichment analysis (GSEA) and Ingenuity pathway analysis (IPA). We isolated blood from a single herd of Black Angus cattle of both genders, aged 12–18 months, by jugular vein puncture. Mean pulmonary arterial pressures were 85.6±13 mmHg STD in the 10 affected and 35.3±1.2 mmHg STD in the 10 resistant cattle, P<0.001. From peripheral blood mononuclear cells, DNA was hybridized to an Affymetrix 10K Gene Chip SNP, and RNA was used to probe an Affymetrix Bovine genome array. SNP loci were remapped using the Btau 4.0 bovine genome assembly. mRNA data was analyzed by the Partek software package to identify sets of genes with an expression that was statistically different between the two groups. GSEA and IPA were conducted on the refined expression data to identify key cellular pathways and to generate networks and conduct functional analyses of the pathways and networks. Ten SNPs were identified by allelelic association and four candidate genes were sequenced in the cohort. Neither endothelial nitric oxide synthetase, NADH dehydrogenase, TG-interacting factor-2 nor BMPR2 were different among affected and resistant cattle. A 60-gene mRNA signature was identified that differentiated affected from unaffected cattle. Forty-six genes were overexpressed in the affected and 14 genes were downregulated in the affected cattle by at least 20%. GSEA and Ingenuity analysis identified respiratory diseases, inflammatory diseases and pathways as the top diseases and disorders (P<5.14×10−14), cell development and cell signaling as the top cellular functions (P<1.20×10−08), and IL6, TREM, PPAR, NFkB cell signaling (P<8.69×10−09) as the top canonical pathways associated with this gene signature. This study provides insights into differences in RNA expression in HAPH at a molecular level, and eliminates four functional gene candidates. Further studies are needed to validate and refine these preliminary findings and to determine the role of transcribed genes in the development of HAPH.

An investigation into beef calf mortality on five high-altitude ranches that selected sires with low pulmonary arterial pressures for over 20 years

Producer reports from ranches over 2,438 meters in southwest Colorado suggest that the mortality of preweaned beef calves may be substantially higher than the national average despite the selection of low pulmonary pressure herd sires for over 20 years. Diagnostic investigations of this death loss problem have been limited due to the extensive mountainous terrain over which these calves are grazed with their dams. The objective of the current study was to determine the causes of calf mortality on 5 high-altitude ranches in Colorado that have been selectively breeding sires with low pulmonary pressure (<45 mmHg) for over 20 years. Calves were followed from branding (6 weeks of age) in the spring to weaning in the fall (7 months of age). Clinical signs were recorded, and blood samples were taken from sick calves. Postmortem examinations were performed, and select tissue samples were submitted for aerobic culture and/or histopathology. On the principal study ranch, 9.6% (59/612) of the calves that were branded in the spring either died or were presumed dead by weaning in the fall. In total, 28 necropsies were performed: 14 calves (50%) had lesions consistent with pulmonary hypertension and right-sided heart failure, and 14 calves (50%) died from bronchopneumonia. Remodeling of the pulmonary arterial system, indicative of pulmonary hypertension, was evident in the former and to varying degrees in the latter. There is a need to better characterize the additional risk factors that complicate pulmonary arterial pressure testing of herd sires as a strategy to control pulmonary hypertension.

Mean pulmonary arterial pressures in Angus steers increase from cow–calf to feedlot–finishing phases1

Pulmonary arterial hypertension due to hypoxia-induced pulmonary vascular remodeling is the predominant cause of right-sided congestive heart failure (CHF) in cattle. Historically, heart failure was problematic only at altitudes over 2,134 m. However, anecdotal reports suggest that the incidence of heart failure is increasing in feedlot cattle at moderate altitude (800 to 1,600 m), with late-fed, or fat, cattle at greatest risk. The goal of this study was to evaluate pulmonary arterial pressures (PAP) in a cohort of male Angus calves from suckling to finishing to better understand why heart failure is particularly problematic in fat cattle. It was hypothesized that mean PAP would increase through the feeding period and that the calves with the greatest pressures at high altitude would have the greatest pressures as fat cattle. A total of 362 PAP measurements were obtained from 153 calves. Calves were tested at altitudes of 2,170 (4 and 6 mo old), 1,560 (13 mo old), and 1,300 m (13 and 18 mo old). Mean PAP were greater in 18-mo-old steers than any other age group (mean = 50.3 mm Hg; 95% confidence interval 48.2 to 52.4; < 0.05). Calves that had the greatest mean pressure at 6 mo of age tended to have the greatest mean pressures at 18 mo of age ( = 0.45, < 0.001). The increase in mean PAP with increasing age and adiposity likely predisposed the steers to an increased risk of CHF during the finishing phase.

Pulmonary arterial pressures, arterial blood-gas tensions, and serum biochemistry of beef calves born and raised at high altitude

High-altitude exposure is physiologically challenging. This is particularly true for animals native to low-altitude environments, such as British breeds of cattle. The objective of this study was to document the effect of high altitude on select physiological parameters of healthy beef calves (Bos taurus) born and raised on a high-altitude ranch typical of the Rocky Mountain region. Pulmonary arterial pressures, arterial blood-gas tensions, serum biochemistry, and hematocrit were evaluated. The calves studied were a composite of British (50%-75%) and Continental (25%-50%) breeds born on one ranch at an altitude of 2410 m. Calves were sampled at an altitude of 2410 m when 1 month old and again at an altitude of 2730 m when 3 and 6 months old. Between 3 and 6 months of age, calves had access to grazing from 2730 m to approximately 3500 m above sea level. On each occasion, 16 to 50 calves were sampled. Only calves that remained healthy throughout all three testing periods were included in the dataset. Calves with the highest pulmonary arterial pressures at 1 month of age tended to have the highest pressures at 6 months of age (r = 0.43, P = 0.16, n = 12). Respiratory alkalosis was greatest at 6 months of age (pH 7.48 ± 0.06). Mean alveolar-arterial oxygen pressure gradients were 11.7 and 11.6 mmHg at 3 and 6 months of age, indicating poor transfer of oxygen from the alveoli into the pulmonary blood. Median values for blood lactate ranged from 1.4 to 3.4 mmol/L indicat- ing substantial anaerobic respiration at all ages. Mean hematocrits were # 35.7%, only slightly higher than values obtained from age-matched calves at sea level. These results suggest that the provision of oxygen to the peripheral tissues of beef calves may be compromised at altitudes over 2410 m. This may have implications for diseases of the cardiopulmonary system.

Evaluation of a point-of-care glucose and β-hydroxybutyrate meter operated in various environmental conditions in prepartum and postpartum sheep.

OBJECTIVE To compare β-hydroxybutyrate (BHB) and glucose concentrations measured with a dual-purpose point-of-care (POC) meter designed for use in humans and a laboratory biochemical analyzer (LBA) to determine whether the POC meter would be reliable for on-farm measurement of blood glucose and BHB concentrations in sheep in various environmental conditions and nutritional states. ANIMALS 36 pregnant mixed-breed ewes involved in a maternal feed restriction study. PROCEDURES Blood samples were collected from each sheep at multiple points throughout gestation and lactation to allow for tracking of gradually increasing metabolic hardship. Whole blood glucose and BHB concentrations were measured with the POC meter and compared with serum results obtained with an LBA. RESULTS 464 samples were collected. Whole blood BHB concentrations measured with the POC meter compared well with LBA results, and error grid analysis showed the POC values were acceptable. Whole blood glucose concentrations measured with the POC meter had more variation, compared with LBA values, over the glucose ranges evaluated. Results of error grid analysis of POC-measured glucose concentrations were not acceptable, indicating errors likely to result in needless treatment with glucose or other supplemental energy sources in normoglycemic sheep. CONCLUSIONS AND CLINICAL RELEVANCE The POC meter was user-friendly and performed well across a wide range of conditions. The meter was adequate for detection of pregnancy toxemia in sheep via whole blood BHB concentration. Results should be interpreted with caution when the POC meter is used to measure blood glucose concentrations.

The altitude at which a calf is born and raised influences the rate at which mean pulmonary arterial pressure increases with age.

Right heart failure secondary to pulmonary hypertension is a leading cause of mortality among suckling beef calves in the Rocky Mountain region. The objective of this study was to track changes in pulmonary arterial pressures (PAP) in healthy calves born and raised at altitudes ranging from 1,470 to 2,730 m. It was hypothesized that calves located at higher altitudes would show a greater increase in mean PAP (mPAP) with age than would be experienced by calves located at lower altitudes. The rationale is that high altitude hypobaric hypoxia causes a greater rate of vascular remodeling and, consequently, greater resistance to blood flow than calves located at lower altitudes. A prospective study was conducted on 5 cohorts of suckling calves from 4 herds located at altitudes of 1,470, 2,010, 2,170, and 2,730 m. In total, 470 PAP measurements were obtained from 258 calves. As hypothesized, calves located at altitudes ≥2,170 m showed a significant increase in mPAP with age ( ≤ 0.002) whereas calves at 1,470 m did not ( = 0.16). Except for calves at 2,170 m ( < 0.001), systolic PAP did not increase with age ( ≥ 0.16). Diastolic PAP increased with age at altitudes ≥ 2,170 m ( ≤ 0.09) but did not change in calves at 1,470 m ( = 0.20). In summary, mPAP and the rate at which mPAP increases with age are positively associated with the altitude at which calves are born and raised.

Technical note: A comparison of DNA collection methods in cattle and yaks

A variety of biological materials are suitable for the analysis of bovine DNA. The objective of this study was to evaluate the ease of collection, storage, and cost as well as quality and quantity of DNA samples obtained from Bos taurus (European cattle) and Bos grunniens (yak) using 2 different sample types: whole blood sampling and nasal swabs. Hair follicle DNA samples from yaks were also analyzed. Deoxyribonucleic acid samples were collected from 1 herd of Black Angus yearling bulls (n = 166) and 1 herd of yaks (n = 24). A NanoDrop Bioanalyzer ND1000 was used to quantify DNA. To assess DNA purity, absorbance ratios were determined at wavelengths of 260 nm relative to 280 nm and 260 nm relative to 230 nm. Single nucleotide polymorphism genotyping was performed using a competitive allele-specific PCR (KASP) genotyping system and the call rates to 3 specific SNP were compared. Using a commercially available nonautomated ethanol DNA extraction technique, nasal swabs yielded a greater quantity of DNA than blood (P < 0.0001) and a greater quality DNA sample than blood (P < 0.0001). Blood and nasal swab performance in SNP genotyping assays were similar (P = 0.5). The greater expense of nasal swabs was offset by their ease of use: less time, skill, and equipment was needed to obtain a sample and the storage of samples was more convenient (room temperature). In yaks, accessing the coccygeal vein, which is relatively straightforward in cattle, was difficult. Nasal swabbing and hair follicle sampling in yaks was performed relatively easily. Yak hair follicles were a poor source of DNA. In conclusion, DNA collection using nasal swabs was more convenient and provided a greater quantity of DNA and better quality sample than blood collection in both Angus and yak. Notably, yak hair was a poor source of DNA, and yak blood was difficult to obtain.

Prairie Rattlesnake Envenomation in 27 New World Camelids

BACKGROUND Morbidity and case fatality from rattlesnake envenomation is regionally specific because of variability in relative toxicity of the species of snake encountered. A previous report of rattlesnake envenomation in New World camelids (NWC) from the western coastal United States documented high case fatality rates and guarded prognosis for survival. HYPOTHESIS/OBJECTIVES To describe clinical findings, treatments, and outcome of NWC with prairie rattlesnake (Crotalus viridis viridis) envenomation in the Rocky Mountain region of the United States. ANIMALS Twenty-seven NWC admitted to the Colorado State University Veterinary Teaching Hospital for evaluation of acute rattlesnake envenomation between 1992 and 2012. METHODS Medical records of NWC evaluated for rattlesnake envenomation as coded by the attending clinician and identified by a database search were reviewed retrospectively. Month of admission, signalment, area of bite, clinical and clinicopathologic data, treatments, and outcome were recorded. RESULTS Twenty-five llamas and 2 alpacas were admitted for envenomation. Llamas were overrepresented compared to hospital caseload. The face was the most common site of envenomation, observed in 96% of recorded cases. Presenting clinical signs included fever, tachypnea, tachycardia, and respiratory distress. Nine animals required a tracheotomy. Median hospitalization time was 3 days and overall survival rate was 69%. CONCLUSIONS AND CLINICAL IMPORTANCE Case fatality rate for prairie rattlesnake envenomation in NWC was lower than that reported in the Western coastal region of the United States and similar to that reported for prairie rattlesnake envenomation in horses.

Inadvertent transvaginal administration of sodium phosphate enemas in 2 alpaca crias.

Objective – To describe the clinical presentation and resultant metabolic disturbances following retroperitoneal administration of hyperphosphate enemas in alpaca crias. Case or Series Summary – Two crias presented to the Colorado State University Veterinary Teaching Hospital after inadvertent transvaginal retroperitoneal hyperphosphate enema administration. The first cria developed an acute onset of neurologic signs, severe hypernatremia, and died soon after presentation. The second cria developed severe hyperphosphatemia, hypocalcemia, and acidemia. The metabolic derangements normalized within 24 hours of intensive treatment with calcium supplementation and IV crystalloid fluid diuresis. The cria was discharged after 1 week in the hospital. New or Unique Information Provided – This report provides a description of electrolyte disturbances secondary to inadvertent transvaginal retroperitoneal administration of hyperphosphate enemas in 2 crias and attendant clinical signs of these disturbances. Management of hyperphosphatemia and hypocalcemia in 1 cria via aggressive fluid therapy with calcium supplementation led to a rapid and sustained normalization of phosphorus, calcium, and acid-base balance.OBJECTIVE To describe the clinical presentation and resultant metabolic disturbances following retroperitoneal administration of hyperphosphate enemas in alpaca crias. CASE OR SERIES SUMMARY Two crias presented to the Colorado State University Veterinary Teaching Hospital after inadvertent transvaginal retroperitoneal hyperphosphate enema administration. The first cria developed an acute onset of neurologic signs, severe hypernatremia, and died soon after presentation. The second cria developed severe hyperphosphatemia, hypocalcemia, and acidemia. The metabolic derangements normalized within 24 hours of intensive treatment with calcium supplementation and IV crystalloid fluid diuresis. The cria was discharged after 1 week in the hospital. NEW OR UNIQUE INFORMATION PROVIDED This report provides a description of electrolyte disturbances secondary to inadvertent transvaginal retroperitoneal administration of hyperphosphate enemas in 2 crias and attendant clinical signs of these disturbances. Management of hyperphosphatemia and hypocalcemia in 1 cria via aggressive fluid therapy with calcium supplementation led to a rapid and sustained normalization of phosphorus, calcium, and acid-base balance.