Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331
Performing Department
Animal and Rangeland Sciences
Non Technical Summary
Hypothyroidism is a common endocrine disease in which the thyroid gland does not produce enough thyroid hormone (triiodothyronine and/or thyroxine).Hypothyroidism has been reported in every domestic animal.Primary hypothyroidism is the most common form of hypothyroidism, but other causes include deficiency of thyrotropin-releasing hormone or deficiency of thyroid-stimulating hormone, or consumptive hypothyroidism from excessive inactivation of thyroid hormone. Subclinical hypothyroidism can occur when there is elevated thyroid-stimulating hormone but a normal thyroxine concentration.Hypothyroidism is a serious clinical entity in llamas and alpacas, associated with a variety of symptoms. Smith and colleagues reported on a syndrome in hypothyroid llamas that presented with depression, low body weight, fleece abnormalities, anemia, and erythrocyte dyscrasia.Hamir and coworkers commented that thyroid nodular hyperplasia and cysts may be partially responsible for infertility in female llamas.McLaughlin and colleagues described symptoms of hypothyroidism in newborn llamas including reluctance to suckle, lack of vigor, unable to thermoregulate, and poor growth.Unfortunately, hypothyroidism in llamas and alpacas is rarely diagnosed by veterinarians because of confusion that exists around interpreting the test results.A number of studies have investigated thyroid function in healthy and unhealthy llamas using radioimmunoassay.Radioimmunoassay is considered to be the "gold standard" for which all other methods for endocrine concentration determination must be validated against.However, radioimmunoassay's have several disadvantages over other assay techniques, including short shelf-lives of the radioactive reagents, radiation safety hazards for personnel, and stringent requirements for waste disposal. As a result, measuring hormone concentrations by radioimmunoassay is more expensive and time-consuming compared to other methods.The majority of commercial automated immunoassay analyzers in use today utilize chemiluminescencebecause chemiluminescence is equal to or exceeds that of radiolabels in analytical sensitivity.There have been no studies published to date in which chemiluminescence has been utilized for determining any endocrine concentrations in llamas or alpacas. Research is needed to validate these chemiluminescence assays, so that clinical diagnoses (e.g. hypothyroidism) made following results obtained from these assays are reliable.Funding a project in a minor agricultural species area (e.g. llamas, alpacas) may appear on the surface to be outside of the Animal Health and Disease (AHD) program objectives. On the contrary, this funding request is particularly relevant as the economic and cultural importance of camelids to the lives of the people living in the areas where they are domesticated (e.g. Argentina, Bolivia, Chile, Peru) where they are the main livestock animal used as a source of food, wool, and transportation.Funding a project in this area will demonstrate AHD's commitment to public welfare in addition to improving animal health and advancing scientific knowledge. By initially limiting the scope of the proposed research to camelids, the project can be completed in a timely manner. However, with validation of this new technology, similar studies can then be performed in other agricultural animals.
Animal Health Component
90%
Research Effort Categories
Basic
5%
Applied
90%
Developmental
5%
Goals / Objectives
In a previous investigation,blood samples were collected into uncoated serum collection tubes without a clot activator from the internal jugular veins of 67 healthy alpacas (males=18, geldings=18, females=31) and 63 healthy llamas (females=22, males=23, geldings=18). Health status was confirmed by a complete physical examination performed prior to blood sample collection. Ages ranged from 1-21 years. Blood samples were allowed to clot and were then centrifuged. Total thyroxine (Immulite, DPC #LKT45) and triiodothyronine (Immulite, DPC #LKT31) concentrations were determined on an automated chemiluminescence analyzer (Immulite 1000, Siemens Health Care). This system utilizes a homologous solid-phase competitive two-site chemiluminescent enzyme immunometric assay (Immulite, Diagnostic Products Corporation, Los Angeles, CA, USA). All samples were run on a single assay for total thyroxine and T3, respectively. The remaining sera were stored at -20°C for additional analysis. The triiodothyronine and total thyroxine concentrations as determined by chemiluminescence are summarized in Table 1. Using linear regression, there was no significant relationship between age and the concentration of total thyroxine (R2=0.0009) or triiodothyronine (R2=0.0001).The major goal of the proposed researchis to validate the chemiluminescence assays for thyroid hormones (thyroxine and triiodothyronine) in llamas and alpacas. This will be accomplished by measuring the thyroid hormone concentrations using radioimmunoassay and then comparing the reliability between the assay methods. Thyroid-stimulating hormone concentrations will also be determined using both assay methods to compare the reliability for this measurement as well. Samples from animals previously collected will be used. Although some hormones degrade over time even when kept frozen, the proposed hormones to be tested (thyroxine, triiodothyronine, and thyroid-stimulatinghormone) have been shown to be stable (no sign of degradation) after 29 years in storage.SpeciesGendernTotal thyroxine (µg/dL)Triiodothyronine (µg/dL)LlamaFemale224.38-13.062.12-362.1Male233.88-14.1653.89-223.2Gelding183.65-14.8338.23-252.8All634.03-13.9320.78-290.7AlpacaFemale313.74-14.6460.94-276.40Male185.49-18.1966.86-333.00Gelding184.18-15.1859.08-214.20All673.98-16.1051.97-285.00Table 1. Predictive ranges of total thyroxine and triiodothyronine concentrations for llamas and alpacas using a chemiluminescence assay.
Project Methods
AnimalsThe animal work for the proposed research has already been completed. The health of the animals was confirmed by thorough physical examinations prior to blood sample collection. In addition, the hormones to be tested (thyroxine, triiodothyronine, and thyroid-stimulating hormone) have been shown by Gislefoss and colleagues (2015) to remain stable (no sign of degradation) after 29 years in storage.Therefore, the results from the stored samples in the proposed research should be reliable.Chemiluminescence AssayThe total thyroxine and triiodothyronine concentrations have already been completed by chemiluminescence. For determining the thyroid-stimulating hormone concentrations by chemiluminescence, the assay will be adjusted to a standard curve and validated by testing the linearity and precision. This was previously done for total thyroxine and triiodothyronine. For thyroxine, the intra-assay precision had a coefficient of variation (CV) of <10%, with a lower limit of sensitivity of 0.4 µg/dL. The L-thyroxine assay had 70% cross-reactivity between D-thyroxine and tetraiodothyroacetic acid. For triiodothyronine, the intra-assay precision had a CV of ≤ 15.6%, with a lower limit of sensitivity of 35 ng/dL. The triiodo-L-thyronine assay had 100% cross reactivity with triiodo-D-thyronine and ≤ 2.0% cross reactivity with triiodothyroacetic acid, tetraiodothyroacetic acid, L-thyroxine, and D-thyroxine.RadioimmunoassaysDirect-reading one-step solid-phase commercially available radioimmunoassays (IBL America,Minneapolis, MN) will be used as directed per the manufacturer's instructions. These assays have been previously validated for use in the llama by Smith and others. Briefly, serum samples will be incubated in anti-total thyroxine, triiodothyronine or thyroid stimulating hormone antibody-coated tubes bound to 125I. The tubes will be incubated for 60 minutes at 37°C, the supernatants decanted and the remaining radioactivity in each tube will be counted for 1 minute on the gamma counter. As with the chemiluminescence, all of the samples will be run in a single assay. Intra-assay precision and linearity will be determined as previously done for the chemiluminescence. Radioimmunoassay sensitivity will be estimated as the lowest hormone concentration required to displace 10% of bound tracer.Data AnalysisThe Shapiro-Wilk normality test will be performed to ensure the hormone data is of Normal (Gaussian) distribution. Descriptive statistics (mean±standard deviation) will be reported for each assay. The predictive (reference) ranges for each assay will be determined using the middle 95th percentile. Thyroid-stimulating hormone concentrations will be compared between genders using an ANOVA. The relationship of age will also be evaluated by linear regression for thyroid-stimulating hormone. In addition, the relationships between total thyroxine and triiodothyronine concentrations will be compared to thyroid-stimulating hormone by linear regression. For each thyroid hormone (total thyroxine, triiodothyronine and thyroid-stimulating hormone), chemiluminescence and radioimmunoassay will be compared by Pearson's correlation tests, by modified linear regression tests (unweighted Deming regression) and by absolute difference bias plots (Bland-Altman plot). The Pearson coefficient of correlation is used to describe the linear component of correlation. The Bland-Altman plot shows the difference (given as percent of the mean) against the mean of the radioimmunoassay method compared to the chemiluminescence method. The Deming regression analysis also compares the chemiluminescence with the radioimmunoassay method. Data analysis will be performed using the Statistical Applications System (SAS) program (version 6.0). Significance will be defined as p<0.05.