Diagnostic cut-off values for the urinary corticoid:creatinine ratio for the diagnosis of canine Cushing's syndrome using an automated chemiluminescent assay.

BACKGROUND: Cushing's syndrome is one of the most common endocrinopathies in dogs. The preferred screening test for spontaneous Cushing's syndrome is the low-dose dexamethasone suppression test (LDDST). The diagnostic value of urinary cortisol:creatinine ratios (UCCR) is questionable. OBJECTIVES: The aim of this study was to determine diagnostic cut-off values for UCCR testing in comparison with LDDST as a clinical reference standard and to calculate the sensitivity and specificity. METHODS: Data from 2018 to 2020 were obtained retrospectively from a commercial laboratory. Both LDDST and UCCR were measured by automated chemiluminescent immunoassay (CLIA). The maximum interval between both tests was 14 days. The optimal cut-off value for UCCR testing was calculated by the Youden index. The sensitivity and specificity of these cut-off values for the UCCR test and LDDST were assessed by Bayesian latent class models (BLCMs). RESULTS: This study included 324 dogs with both UCCR test and LDDST results. The optimal UCCR cut-off value, calculated by the Youden index, was 47.4 × 10-6 . Any UCCR <40 × 10-6 was interpreted as a negative result, 40-60 × 10-6 as values in a gray zone, and >60 × 10-6 as positive. Using the cut-off of 60 × 10-6 , BLCM showed 91% (LDDST) and 86% (UCCR test) sensitivity and a specificity of 54% (LDDST) and 63% (UCCR test). CONCLUSIONS: Considering an 86% sensitivity and a 63% specificity, UCCR testing may be considered a first-line investigation to rule out Cushing's syndrome using CLIA analysis. Urine samples can be collected noninvasively at home by the owner, reducing the potential impact of stress.

Validation of a dry-slide immunoassay for progesterone analysis in canine plasma in a clinical setting.

BACKGROUND: The identification of canine ovulation is critical for successful breeding. Progesterone measurements are useful for identifying ovulation. Progesterone assays are also quantitative and easily accessed, making them valuable in veterinary practice. OBJECTIVES: We aimed to validate a dry-slide immunoassay (DSI) for use in dogs, including a method comparison with the chemiluminescence assay (CLIA) and mass spectrometry. METHODS: Twenty-nine bitches were prospectively recruited. Accuracy, precision, interference, and stability were evaluated. Method comparison between DSI and CLIA and mass spectrometry was conducted, and bias was calculated. RESULTS: Repeatability was 8.0%-10.8%, and within-laboratory imprecision was 8.8%-11.1% for four concentration levels. Recovery under dilution was 61%-100%, and the method was linear to a concentration of ~50 nmol/L. Recovery after the addition of a high progesterone sample was 76%-83%. Minor changes were seen in one hemolytic and two lipemic samples. Storage at room temperature for 12-24 hours resulted in concentrations that were 57%-96% of the initial concentrations. For samples frozen at -80°C, the concentrations were reduced 17%-27%. There was a significant difference between results from the DSI and CLIA, and a proportional bias was seen when DSI was compared with mass spectrometry, where CLIA correlated better than DSI. CONCLUSIONS: Precision and accuracy were acceptable. A proportional bias was seen between DSI and CLIA. A small amount of interference was seen with hemolysis and lipemia. Progesterone concentrations were decreased in samples stored at room temperature and -80°C. The results support the use of the DSI for ovulation timing but not for artificial insemination with frozen semen since progesterone concentrations might exceed the assay's linearity and precision limits.

The ATP bioluminescence assay: a new application and optimization for viability testing in the parasitic nematode Haemonchus contortus.

The parasitic gastrointestinal nematode Haemonchus contortus causes serious economic losses to agriculture due to infection and disease in small ruminant livestock. The development of new therapies requires appropriate viability testing, with methods nowadays relying on larval motility or development using procedures that involve microscopy. None of the existing biochemical methods, however, are performed in adults, the target stage of the anthelmintic compounds. Here we present a new test for the viability of H. contortus adults and exsheathed third-stage larvae which is based on a bioluminescent assay of ATP content normalized to total protein concentration measured using bicinchoninic acid. All the procedure steps were optimized to achieve maximal sensitivity and robustness. This novel method can be used as a complementary assay for the phenotypic screening of new compounds with potential antinematode activity in exsheathed third-stage larvae and in adult males. Additionally, it might be used for the detection of drug-resistant isolates.

VALIDATION OF CHEMILUMINESCENT ASSAY FOR CANINE THYROID STIMULATING HORMONE IN RED PANDAS (AILURUS FULGENS FULGENS).

Thyroid abnormalities have been anecdotally reported in red pandas (Ailurus fulgens fulgens); however, definitive diagnosis is hampered by a lack of established reference ranges and validated diagnostic tests. The chemiluminescent assay for canine thyroid stimulating hormone (cTSH) has been validated for use in domestic canids and felids. This study aims to validate the cTSH assay for use in red pandas. Validation was performed via serial dilutions of banked serum samples (n = 15) and both inter- and intra-assay testing. High estimated recoveries and low coefficients of variability indicate that the cTSH assay accurately and consistently measures TSH concentrations in red panda serum. Further studies to generate red panda age and sex TSH reference ranges are indicated.

Feline plasma adrenocorticotropic hormone: validation of a chemiluminescent assay and concentrations in cats with hypercortisolism, primary hypoadrenocorticism and other diseases.

OBJECTIVES: The aims of this study were to validate a commercially available chemiluminescent assay for measurement of feline plasma adrenocorticotropic hormone concentration (ACTH), to determine the normal reference interval (RI) of plasma ACTH in healthy cats, to assess plasma ACTH in cats with naturally occurring hypercortisolism (HC), primary hypoadrenocorticism (PH) and other diseases (OD), and to evaluate the effect of aprotinin on plasma ACTH degradation. METHODS: Forty healthy cats, 10 with HC, 11 with PH and 30 with OD, were included. The chemiluminescent enzyme immunometric assay was evaluated by measurement of intra-assay precision, interassay precision and linearity. The RI for plasma ACTH in healthy cats was established using robust methods. Plasma ACTH of samples collected with and without aprotinin, stored at 4°C and assayed over a 6-day period, was measured. RESULTS: The intra-assay coefficients of variance (CVs) ranged from 2.7% to 4.3% and interassay CVs from 3.3% to 10.7%. Dilution studies showed excellent accuracy (R2 >0.99). The RI for plasma ACTH in healthy cats was 32-370 pg/ml. Plasma ACTH was not significantly different between healthy cats and the OD group. Cats with pituitary-dependent hypercortisolism (PDH) and PH had significantly higher plasma ACTH than the other groups. Plasma ACTH did not show significant differences when samples collected with and without aprotinin were compared. CONCLUSIONS AND RELEVANCE: The Immulite chemiluminescent assay is a valid technique for measuring plasma ACTH in cats and the RI of plasma ACTH is quite wide. Owing to the low overlap between healthy or OD cats and cats with HC or PH, the measurement of plasma ACTH appears to be useful and should be included in the diagnostic work-up when HC or PH are suspected. Furthermore, the measurement of plasma ACTH may be an accurate test for differentiating PDH from adrenal-dependent hypercortisolism.

Analysis of peripheral blood chemiluminescence in horses.

The aim of this study was to analyze the chemiluminescence (CL) of peripheral blood in clinically healthy horses of different sexes and ages. The tests were carried out on 119 half- -breed horses, representing various forms of use (66 recreational horses and 53 sport horses). The test material was peripheral blood, which was collected under resting conditions, i.e. before physical activity related to the use of these animals. In the blood samples, spontaneous and stimulated CL with zymosan and phorbol myristate acetate were determined. It has been found that regular training effort increases the blood's pro-oxidative potential, which was demonstrated by significantly higher (p⟨0.05) CL values in sport horses compared to recreational animals. Analysis of the results did not show any statistically significant correlation between sex or age of the horses with chemiluminescence values in peripheral blood. The result of the research suggests the need to optimize the results of blood CL measurements, taking into account the number of neutrophils and the concentration of hemoglobin in the blood of tested animals. Analysis of non-optimized blood CL results may lead to premature conclusions.

Comparison of free thyroxine measurement by chemiluminescence and equilibrium dialysis following 131I therapy in hyperthyroid cats.

OBJECTIVES: The first objective was to assess correlation between free thyroxine (fT4) measurements by equilibrium dialysis (fT4ED; Antech Diagnostics) and a chemiluminescent enzyme immunoassay (fT4CEIA; IMMULITE 2000 Veterinary Free T4 [Siemens Healthcare Diagnostics Products]) in hyperthyroid, otherwise healthy, cats before (T0), and 1 month (T1) and 11-23 months (T2) after radioactive iodine (131I) therapy. The second objective was to determine correlation between thyroid status based on fT4 (by both techniques) and the gold standard, thyroid scintigraphy. METHODS: Thyroid status, including thyroid-stimulating hormone (TSH), total thyroxine (TT4) and fT4 serum concentrations, were assessed in 45 client-owned hyperthyroid cats before (T0), and 1 month (T1) and 11-23 months (T2) after 131I therapy. fT4 was determined by a chemiluminescent enzyme immunoassay (CEIA) and equilibrium dialysis (ED). Quantitative thyroid scintigraphy (with sodium 99m-Tc-pertechnetate) was performed at T2. RESULTS: Spearman correlation between fT4CEIA and fT4ED was 0.81, 0.88 and 0.79 at T0, T1 and T2, respectively. fT4CEIA was consistently lower than fT4ED, with a median difference of -5.4 pmol/l (P <0.001) and -4.9 pmol/l (P <0.0001) at T1 and T2, respectively. At T2, all cats were identified as euthyroid based on thyroid scintigraphy. None of the cats were identified as being hypothyroid, based on serum TT4 and TSH measurements. Nine of 22 (40.9%) cats had an fT4CEIA below the reference interval (RI) at T2, whereas only 2/22 (9.1%) cats had an fT4ED concentration below the RI at T2. CONCLUSIONS AND RELEVANCE: Good correlation exists between both assays at T1 and T2, but a significant systematic difference is noted at both time points. This could be an indication for reconsideration of the current RI, although further studies are warranted for assessing test accuracy (in otherwise healthy cats and cats with non-thyroidal illness). At this time, routine use of fT4CEIA after 131I therapy is not advised in feline patients.

The effect of freeze-thaw cycles on determination of immunoreactive plasma adrenocorticotrophic hormone concentrations in horses.

BACKGROUND: Determination of plasma adrenocotrophic hormone (ACTH) concentration (endogenous or thyrotropin-releasing hormone [TRH] stimulation test) is the most commonly used diagnostic test for pituitary pars intermedia dysfunction (PPID) in horses. Because ACTH is unstable, samples often are frozen to be shipped to laboratories or to allow for batch analysis of research samples. However, the effect of multiple freeze-thaw cycles on equine ACTH is unknown. OBJECTIVE: To determine the effects of multiple freeze-thaw cycles on immunoreactive ACTH concentration. ANIMALS: Twenty-eight horses ranging from 10 to 27 years of age were used. METHODS: Prospective study. Horses were divided into 4 groups: group 1, PPID-negative, without TRH stimulation; group 2, PPID-negative, with TRH stimulation; group 3, PPID-positive, without TRH stimulation; and group 4, PPID-positive, with TRH stimulation. Whole blood was collected from each horse at baseline or 30 minutes after TRH stimulation. Immunoreactive plasma ACTH concentration was determined using a chemiluminescence assay. Plasma samples then were frozen at -80°C >24 hours, thawed at 4°C and reanalyzed for 5 freeze-thaw cycles. Changes in plasma ACTH concentration were analyzed using a linear mixed-effect model. RESULTS: Significant effects of freeze-thaw cycles (P = .001) and PPID status (P = .04) on plasma ACTH concentration were observed, but no significant effect of TRH stimulation was identified. CONCLUSIONS AND CLINICAL IMPORTANCE: The plasma ACTH concentration is altered by freeze-thaw cycles, and the effect is observed sooner in horses with PPID. To diagnose PPID, multiple freeze-thaw cycles should be avoided when measuring plasma ACTH concentration.

Effect of EDTA on measurement of cortisol and thyroxine by chemiluminescent enzyme immunoassay in dogs.

The addition of ethylenediamine tetra-acetic acid (EDTA) to serum can affect the measurement of cortisol by chemiluminescent enzyme immunoassay (CEIA); addition of magnesium chloride (MgCl2) may reverse the effects. However, similar characteristics for thyroxine (T4) measurement are unknown. We measured cortisol and T4 in paired EDTA-anticoagulated plasma and serum samples from 50 dogs. Additionally, both hormones were measured in 15 samples of each type after the addition of MgCl2. Samples were collected under routine clinical conditions; therefore, specific EDTA concentrations in plasma samples were unknown. Cortisol and T4 values were significantly different comparing plasma and serum samples in the absence of MgCl2. For cortisol and T4, EDTA-plasma concentrations were 51.2% and 43.7% higher than serum, respectively (p < 0.001 for both). The addition of MgCl2 to plasma significantly decreased the measured cortisol concentrations (p < 0.001) but not T4 (p = 0.44). After addition of MgCl2, cortisol concentrations in EDTA-plasma were no longer significantly different from serum, whereas T4 concentrations in EDTA-plasma remained significantly different from serum. In the clinical setting in which tubes may be underfilled, use of EDTA-plasma significantly increases the measured concentration of cortisol and T4 obtained by CEIA. Addition of MgCl2 to EDTA-plasma can overcome the effects of EDTA when measuring cortisol, but not T4. Thus, T4 should not be measured in EDTA-plasma.

Comparison of immunofluorescence and chemiluminescence assays for measuring ACTH in equine plasma.

BACKGROUND: The chemiluminescence (CL) and immunofluorescence (IF) assays yield different results for basal adrenocorticotropin hormone concentrations [ACTH] in pony plasma. It is unclear whether this difference also occurs in basal samples from horses or samples from ponies following thyrotropin-releasing hormone (TRH) stimulation. OBJECTIVES: To compare the results of [ACTH] analysis by CL and IF methods in basal samples from horses and pony samples following TRH stimulation. STUDY DESIGN: Method comparison. METHODS: Plasma [ACTH] was measured concurrently using CL and IF methods in 12 ponies (basal and post-TRH stimulation) in November and basal samples from horses (n = 45; November and May). RESULTS: CL and IF methods yielded different results (P < .01). The median difference (CL-IF) (95% CI) for ponies was 5.9 (0.1-7.5) pg/mL at baseline and 227.9 (61-1001) pg/mL post TRH; and horses 1.9 (1.1-5.4) pg/mL in November and 9.4 (8.2-11.5) pg/mL in May, at baseline. Correlation was good in ponies at baseline (R = 0.80, P = .003) but not post-TRH, and good in horses in November and May (R = 0.68 and 0.71, P < .001). Bland-Altman analysis demonstrated moderate bias and wide 95% limits of agreement (95% LOA) in ponies at baseline (bias 5.5 pg/mL; 95% LOA -9.9 to 20.9 pg/mL) and horses in May (bias 10.6 pg/mL; 95% LOA -9 to 30.3 pg/mL) and very large bias and wide 95% LOA in ponies post-TRH (bias 477 pg/mL; 95% LOA -633 to 1587 pg/mL). Using CL cut-offs of >29 and >110 pg/mL, agreement was moderate (ƙ = 0.67) and very good (ƙ = 0.82) for binary classification of PPID in ponies at baseline and post-TRH; and good (ƙ = 0.73) for horses in November, but poor (ƙ = 0.40) in May. MAIN LIMITATIONS: Limited numbers of horses with [ACTH] above threshold values. CONCLUSIONS: The assays yielded different absolute values, particularly in post-TRH samples from ponies, suggesting TRH stimulates secretion of cross-reacting peptides other than ACTH. Agreement for binary classification for PPID was moderate to good, except in basal samples from horses in May.

Evaluation of two chemiluminescent assays compared with radioimmunoassay for serum progesterone measurement in bitches.

Serum progesterone (sP4) measurement is commonly used to determine the optimal time for mating in bitches, and to diagnose reproduction-related abnormalities. Radioimmunoassay (RIA) is the gold standard assay, but is becoming less available, and has several practical disadvantages. Chemiluminescence immunoassays (CLIA) are commonly used in human medicine for sP4 measurement, and are becoming more available in veterinary medicine. Our objective was to compare the sP4 results obtained by RIA and two CLIA systems (Immulite-Siemens [IS-CLIA] and Elecsys-Roche [ER-CLIA]) in the same sera in 60 client-owned healthy bitches at different estrous cycle stages. The agreement between the two CLIAs and RIA was examined using the Passing-Bablok regression and Bland Altman plots. Comparing sP4 concentrations measured by the IS-CLIA to the RIA yielded an intercept of 0.16 ng/mL (95% confidence interval [95%CI], 0.03-0.25) with a slope of 0.45 (95%CI, 0.44-0.47) and a median difference of -2.10 ng/mL (P < 0.0001) that was strongly correlated to the average of measurements (r = -0.97; P < 0.0001). Comparing sP4 concentrations measured by the ER-CLIA to the RIA yielded an intercept of -0.23 ng/mL (95%CI, -0.56 to -0.09) with a slope of 1.06 (95%CI, 1.00-1.12) and a median difference of -0.09 ng/mL (P = 0.9), that was weakly correlated to the average of measurements (r = 0.34; P = 0.018). The performance of the ER-CLIA was similar to the RIA, while the IS-CLIA showed significantly different results compared to the RIA. Our study supports the conclusion that sP4 results generated by the ER-CLIA can be used interchangeably with RIA results for clinical purposes, while IS-CLIA results require adjustment to RIA results for clinical practice.

Evaluation of an automated point-of-care test system for measuring thrombin-antithrombin complex in dogs.

OBJECTIVE: To evaluate the utility of the chemiluminescent enzyme immunoassay (CLEIA) method for point-of-care (POC) measurement of canine plasma thrombin-antithrombin complex (TAT) concentration. ASSESSMENT AND MAIN RESULTS: Plasma TAT concentration was measured in 54 healthy dogs and in 72 dogs with various diseases. A significant correlation was found between TAT concentration measured by CLEIA and that measured by an ELISA that was previously used in dogs. The upper limit of the reference value of TAT concentrations measured by CLEIA was determined to be 0.2 ng/mL based on the TAT concentration in 54 healthy dogs. TAT concentrations exceeded the reference interval in a portion of dogs when a hypercoagulable state may be present. CONCLUSIONS: Canine plasma TAT concentrations measured using CLEIA were correlated with that measured using ELISA. Hence, a POC testing instrument may be used for early detection of activation of thrombin generation in emergency and critical care settings.

Comparison of progesterone assay by chemiluminescence or radioimmunoassay for clinical decision-making in canine reproduction.

The Coat-A-Count® radioimmunoassay has been long and widely used to determine the concentration of progesterone in serum or plasma of bitches (progRIA), but was discontinued in 2014. The Immulite® 1000 LKPG1 chemiluminescence immunoassay has gained prominence since 2003 to determine the concentration of progesterone in serum of bitches, but the assay changed in 2012 (Immulite® 1000 LKPW1). This study assessed the feasibility of using Immulite® 1000 LKPW1 (progImm) to estimate the time of clinically relevant events during oestrus and compared progRIA and progImm 2 and 3 days after the first or only day of the luteinizing hormone surge (LH1). ProgImm first exceeded 5.1 nmol/L on the same day that progRIA first exceeded 6 nmol/L, a proxy for the occurrence of the LH surge, or the day before in 28 of 31 (90%) of oestrous periods. ProgImm first exceeded 13.6 nmol/L on the same day that progRIA first exceeded 16 nmol/L (a proxy for the day of ovulation) or the day before in 34 of 35 (97%) oestrous periods. ProgImm first exceeded 5.4 nmol/L on LH1 or the day before in 24 of 25 (95%) of oestrous periods. The median of progImm 2 days after LH1 was 1.2 nmol/L lower than the 10.7 nmol/L of progRIA (p = 0.001). The mean of progImm 3 days after LH1 was 2.2 nmol/L lower than the 19.0 nmol/L of progRIA (p 0.001). In conclusion, the days on which progImm first exceeded 5.1 nmol/L, 13.6 nmol/L and 5.4 nmol/L effectively estimate the days on which progRIA reached 6 nmol/L or 16 nmol/L or LH1.

Chemiluminescent ELISA with multi-epitope proteins to improve the diagnosis of canine visceral leishmaniasis.

Diagnosing canine visceral leishmaniasis (CVL) is difficult because clinical signs of the disease are non-specific and a many infected animals in endemic areas, as in Brazil, are asymptomatic. Serological tests are the most common diagnostic methods employed, but most have limitations. For this reason, the implementation of a rapid, sensitive, and specific diagnostic test for CVL has become increasingly important. In this study, we adapted a chemiluminescent enzyme-linked immunosorbent assay (CL ELISA), using two multi-epitope recombinant proteins (PQ10 and PQ20) and a crude Leishmania antigen produced using promastigotes of L. infantum, as antigens to detect CVL infection in animals from Belo Horizonte. To investigate cross-reactions, samples from dogs with other infections (babesiosis, ehrlichiosis and Trypanosoma cruzi) were tested. Assay performance validations were conducted to analyse parameters such as variability, reproducibility, and stability. CL ELISA sensitivity/specificity with PQ10 antigen was 93.1%/80.0%; with the PQ20 protein 93.1%/96.6%; and with the crude antigen 75%/73.3%. Inter-assay variability and inter-operator coefficient of variation were <7% and <15%, with PQ10 and PQ20, respectively. The accuracy of the CL ELISA was classified as excellent for PQ10 (AUC = 0.95) and PQ20 (AUC = 0.98) and moderate for the crude antigen (AUC = 0.77). The kappa score for qualitative agreement between two plate lots was excellent for PQ10 (0.89) and good for PQ20 (0.65). PQ20 remained more stable than PQ10. The CL ELISA with recombinant proteins is a promising tool to diagnose CVL.

Characterization of Monkeypox virus dissemination in the black-tailed prairie dog (Cynomys ludovicianus) through in vivo bioluminescent imaging.

Monkeypox virus (MPXV) is a member of the genus Orthopoxvirus, endemic in Central and West Africa. This viral zoonosis was introduced into the United States in 2003 via African rodents imported for the pet trade and caused 37 human cases, all linked to exposure to MPXV-infected black-tailed prairie dogs (Cynomys ludovicianus). Prairie dogs have since become a useful model of MPXV disease, utilized for testing of potential medical countermeasures. In this study, we used recombinant MPXV containing the firefly luciferase gene (luc) and in vivo imaging technology to characterize MPXV pathogenesis in the black-tailed prairie dog in real time. West African (WA) MPXV could be visualized using in vivo imaging in the nose, lymph nodes, intestines, heart, lung, kidneys, and liver as early as day 6 post infection (p.i.). By day 9 p.i., lesions became visible on the skin and in some cases in the spleen. After day 9 p.i., luminescent signal representing MPXV replication either increased, indicating a progression to what would be a fatal infection, or decreased as infection was resolved. Use of recombinant luc+ MPXV allowed for a greater understanding of how MPXV disseminates throughout the body in prairie dogs during the course of infection. This technology will be used to reduce the number of animals required in future pathogenesis studies as well as aid in determining the effectiveness of potential medical countermeasures.

A sensitive luminescence syncytium induction assay (LuSIA) based on a reporter plasmid containing a mutation in the glucocorticoid response element in the long terminal repeat U3 region of bovine leukemia virus.

BACKGROUND: Bovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease of cattle. Previously, we reported the luminescence syncytium induction assay (LuSIA), an assay for BLV infectivity based on CC81-BLU3G cells, which form syncytia expressing enhanced green fluorescent protein (EGFP) when co-cultured with BLV-infected cells. To develop a more sensitive LuSIA, we here focused on the glucocorticoid response element (GRE) within the U3 region of the BLV long terminal repeat (LTR). METHODS: We changed five nucleotide sites of the GRE in a pBLU3-EGFP reporter plasmid containing the BLV-LTR U3 region promoter by site-directed mutagenesis and we then constructed a new reporter plasmid (pBLU3GREM-EGFP) in which the EGFP reporter gene was expressed under control of the GRE-mutated LTR-U3 promoter. We also established a new CC81-derived reporter cell line harboring the GRE-mutated LTR-U3 promoter (CC81-GREMG). To evaluate the sensibility, the utility and the specificity of the LuSIA using CC81-GREMG, we co-cultured CC81-GREMG cells with BLV-persistently infected cells, free-viruses, white blood cells (WBCs) from BLV-infected cows, and bovine immunodeficiency-like virus (BIV)- and bovine foamy virus (BFV)-infected cells. RESULTS: We successfully constructed a new reporter plasmid harboring a mutation in the GRE and established a new reporter cell line, CC81-GREMG; this line was stably transfected with pBLU3GREM-EGFP in which the EGFP gene is expressed under control of the GRE-mutated LTR-U3 promoter and enabled direct visualization of BLV infectivity. The new LuSIA protocol using CC81-GREMG cells measures cell-to-cell infectivity and cell-free infectivity of BLV more sensitively than previous protocol using CC81-BLU3G. Furthermore, it did not respond to BIV and BFV infections, indicating that the LuSIA based on CC81-GREMG is specific for BLV infectivity. Moreover, we confirmed the utility of a new LuSIA based on CC81-GREMG cells using white blood cells (WBCs) from BLV-infected cows. Finally, the assay was useful for assessing the activity of neutralizing antibodies in plasma collected from BLV-infected cows. CONCLUSION: The new LuSIA protocol is quantitative and more sensitive than the previous assay based on CC81-BLU3G cells and should facilitate development of several new BLV assays.

A luminescence-based assay for evaluating bactericidal antibody to Borrelia burgdorferi in vaccinated horses' serum.

BACKGROUND: Current serological tests cannot discriminate between bactericidal Borrelia burgdorferi antibodies from others that are merely a response to Borrelia antigenic stimulation. OBJECTIVE: To develop a sensitive and convenient luminescence-based serum bactericidal assay (L-SBA) to identify serum borreliacidal activity. STUDY DESIGN: Prospective validation study and method comparison. METHODS: Serum samples were obtained either from archives of the Animal Health Diagnostic Center at Cornell University (N = 7) or from a vaccination trial (N = 238). Endogenous complement-inactivated serum sample was incubated with exogenic complement and B. burgdorferi ML23 pBBE22luc, which is able to process luciferin with luciferase and produce luminescence in viable Borrelia. After incubation, a light signal can be detected by using a luminometer to calculate the borreliacidal antibody titre. RESULTS: Components of the reaction mixture including spirochetes and complement from various sources and concentrations were tested to identify a reliable recipe for our complement-mediated L-SBA. We also applied this L-SBA on measuring bactericidal antibody activities and calculated the half inhibitory concentration (IC50 ) of serum samples from clinical collections. Furthermore, we analysed the L-SBA titres and anti-outer surface protein A (OspA) antibody levels from vaccinated horses using the multiplex assays and found that there is a relationship between results generated using these two different assays. The increases of L-SBA titres correlated with increases of anti-OspA antibody titre in sera (r = 0.423). MAIN LIMITATIONS: Immunoreactivity of commercial complement may differ from different batches. Clinical protection of borreliacidal antibody levels has not been determined. CONCLUSIONS: The L-SBA provided a sensitive and easy-operating platform for the evaluation of bactericidal antibody to B. burgdorferi, and we anticipated L-SBA would function well as an evaluation tool of vaccine efficiency in the future.

Analytical validation of a conventional cardiac troponin I assay for dogs and cats.

BACKGROUND: Cardiac troponins are gold-standard biomarkers of myocardial injury. There is a need for validation of assays with higher availability and lower costs in veterinary medicine. OBJECTIVES: The primary aim of the present study was to perform an analytical validation of the IMMULITE 2000 TnI assay for use in dogs and cats. A secondary aim was to evaluate its agreement with the previously validated and sensitive Siemens ADVIA Centaur TnI-Ultra assay. METHODS: Intra- and inter-assay variation, detection limits, the linearity under dilution, and a sample addition study (modified spike-and-recovery analysis) were investigated to assess analytical performance in 15 canine and 15 feline serum samples. Agreement between the assays was evaluated by correlation and Bland-Altman analyses including an additional 99 canine serum samples. RESULTS: Intra-assay variation of cTnI in canine and feline serum was 3.71% and 4.68%, while inter-assay variation was 5.88% and 6.54%, respectively. The assay performed with acceptable linearity within a clinically relevant range of serum cTnI concentrations. The sample addition study revealed insufficient recovery in the range of 71.9%-81.4% for dogs and 62.6%-75.7% for cats. This was considered to be due to a negative matrix effect. A significant correlation between the assays was found, and the Bland-Altman analysis showed acceptable agreement for a wide range of concentrations, but revealed a proportional error, with the IMMULITE TnI assay consistently measuring a higher concentration than the Centaur TnI-Ultra assay. This was relevant only at high serum cTnI concentrations. CONCLUSIONS: The IMMULITE TnI assay is considered acceptable for clinical use in dogs and cats.

Blood periovulatory progesterone quantification using different techniques in the dog.

Blood progesterone concentration is used in several procedures related to the reproduction in the bitch, such as ovulation monitoring, estimating time of parturition, or hypo-luteoidism management. Several techniques are available to evaluate blood progesterone concentration, such as the radioimmunoassay (RIA), the chemiluminescent immunoassay (CLIA), and the enzyme-linked immunosorbent assay (ELISA). The aim of this study was to compare the blood progesterone concentration using these three methods during the periovulatory period of 23 bitches. Vaginal cytology was used to classify cytologic estrus (CE) and cytologic diestrus (CD), and blood samples were collected once during proestrus and every other day between CE and CD. The samples were retrospectively classified in the different phases of the estrus based on CD. Pregnancy rate and gestational length were also recorded. A significant increase of the circulating progesterone during the progression of the estrus was recorded, and there were significant differences in the values when using the different methods, with lesser, intermediate, and greatest values with use of the RIA, CLIA, and ELISA, respectively. There was a high correlation (Pearson's correlation coefficient = 0.978) and substantial strength-of-agreement (Lin's concordance correlation coefficient = 0.966) between values obtained when using CLIA and RIA, while there was a high correlation (Pearson's correlation coefficient = 0.955) but poor strength-of-agreement (Lin's concordance correlation coefficient = 0.866) with use of the ELISA and RIA. The data reported in this study provide evidence that the method used for measuring the blood progesterone concentration during the periovulatory phase of the bitch significantly affected the progesterone values.

Development of a chemiluminescence immunoassay using recombinant non-structural epitope-based proteins to accurately differentiate foot-and-mouth disease virus-infected and vaccinated bovines.

The contamination of inactivated vaccine with non-structural proteins (NSPs) leads to a high false-positive rate, which is a substantial barrier to accurately differentiate foot-and-mouth disease virus (FMDV)-infected animals from vaccinated animals. To address this problem, a new chemiluminescence immunoassay (CLIA) method was developed to detect antibodies targeting the two recombinant epitope-based proteins located in 3A and 3B. The 3Aepitp-3Bepitp CLIA exhibited a diagnostic sensitivity of 94.0% and a diagnostic specificity of 97.5% for the detection of serum samples (naïve bovines, n = 52, vaccinated bovines, n = 422, infected bovines, n = 116) from animals with known status. The CLIA method also had a concordance rate of 88.1% with the PrioCHECK FMDV NSP ELISA based on the detection of 270 serum samples from the field. Importantly, the 3Aepitp-3Bepitp CLIA produced no false-positives when used to detect FMDV in samples from bovines that had been vaccinated up to five times, and it was demonstrated a low false-positive rate when the bovines had been vaccinated up to ten (2.15%) and fifteen times (5.93%). Therefore, the 3Aepitp-3Bepitp CLIA detects FMDV in samples from frequently vaccinated bovines with high accuracy and represents an alternative method to differentiate FMDV-infected and vaccinated bovines.