Equine Serum Research Articles

The Analysis of Phenylbutazone and Its Active Metabolite, Oxyphenbutazone, in Equine Tissues (Muscle, Kidney, and Liver), Urine, and Serum by LC-MS/MS.

This study reports the use of two validated LC with tandem MS (MS/MS) methods to study the residue depletion profile of phenylbutazone (PBZ) and its metabolite oxyphenbutazone (OXPBZ) from equine serum, urine, and muscle, kidney, and liver tissues. One LC-MS/MS method, with an LOQ of 1.0 ng/mL for PBZ and 2.0 ng/mL for OXPBZ, was used for the analysis of the two drugs in the biological fluids (equine urine and serum); the other LC-MS/MS method, with an LOQ of 0.5 ng/g for PBZ and OXPBZ, was used for the analysis of the drugs in the equine tissue samples. PBZ was administered intravenously to two horses dosed with 8.8 mg/kg PBZ once daily for 4 days and sacrificed humanely at a slaughter plant 7 days after the last drug administration. Urine, serum, and kidney, liver, and muscle tissues were collected from the two horses and shipped on ice to the laboratory and stored at -20°C until analysis. The concentrations of PBZ and OXPBZ residues in the biological fluid and tissue samples collected at slaughter were measured with the two validated LC-MS/MS methods using deuterated internal standards. The results demonstrate that the validated methods are fit for studying the depletion kinetics of PBZ residues in equine tissues and biological fluids.

Modulating the oxidative environment during mesenchymal stem cells chondrogenesis with serum increases collagen accumulation in agarose culture.

Chondrogenesis of mesenchymal stem cells (MSCs) is induced in culture conditions that have been associated with oxidative stress, although the extent to which the oxidative environment affects differentiation and extracellular matrix (ECM) accumulation is not known. The objectives of this study were to evaluate the oxidative environment during MSCs chondrogenesis in conventional serum-free medium, and the effect of serum-supplementation on intracellular reactive oxygen species (ROS) and chondrogenesis. Young adult equine MSCs were seeded into agarose and cultured in chondrogenic medium, with or without 5% fetal bovine serum (FBS), for up to 15 days. Samples were evaluated for intracellular ROS, the antioxidant glutathione, ECM and gene expression measures of chondrogenesis, and carbonylation as an indicator of oxidative damage. Intracellular ROS increased with time in culture, and was lower in medium supplemented with FBS. Glutathione decreased ∼12-fold during early chondrogenesis (p < 0.0001), and was not affected by FBS (p = 0.25). After 15 days of culture, FBS supplementation increased hydroxyproline accumulation ∼80% (p = 0.0002); otherwise, measures of chondrogenesis were largely unaffected. Protein carbonylation in chondrogenic MSCs cultures was not significantly different between serum-free and FBS cultures (p = 0.72). Supplementation with adult equine serum increased hydroxyproline accumulation by 45% over serum-free culture (p = 0.0006). In conclusion, this study characterized changes in the oxidative environment during MSC chondrogenesis, and suggested that lowering ROS may be an effective approach to increase collagen accumulation. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:506-514, 2018.

Seroprevalence of Toxoplasma gondii and Trichinella spiralis in Horses in Xinjiang, Northwestern China

Toxoplasma gondii and Trichinella spiralis are widely distributed in humans and animals throughout the world, but the data on prevalence of horses in China are limited. In this study, 409 equine serum samples from Xinjiang Uygur Autonomous Region, Northwestern China, were tested by enzyme-linked immunosorbent assay to investigate the antibodies against T. gondii and T. spiralis in horses of this region. Results showed that 22 samples (5.38%) were positive for T. gondii and 14 (3.42%) for T. spiralis. Two samples (0.49%) were both positive with T. gondii and T. spiralis. It was found that the prevalence of T. gondii in older horses (>15 years) was significantly higher than that in younger animals. There was no significant difference between the prevalence and the genders of the animals. To verify the results of seroprevalence for T. gondii, 10 samples of uterine secretions from aborting mares with positive T. gondii antibody, were tested by polymerase chain reaction assay. The internal transcribed spacer 1 gene specific for T. gondii was amplified with a ratio of 100% (10/10), which was consistent with the result of seroprevalence. The results of the present survey indicate that infection with T. gondii and T. spiralis in horses should not be ignored in Xinjiang, Northwestern China. Consumption of horse meat in this region may represent a potential source for human infection with T. gondii and T. spiralis.

Detection of West Nile Virus and other common equine viruses in three locations from the Leeward Islands, West Indies

Equines in the West Indies are used for recreational purposes, tourism industry, racing and agriculture or can be found in feral populations. Little is known in the Caribbean basin about the prevalence of some major equine infectious diseases, some with zoonotic potential, listed as reportable by the OIE.Our objective was to study the prevalence of antibodies for West Nile Virus (WNV), Equine Herpes Virus-1 and 4 (EHV-1 and EHV-4), Equine Influenza (EI), Equine Viral Arteritis (EVA) and Equine Infectious Anemia Virus (EIAV) using a retrospective serological convenience study. We used 180 equine serum samples, 140 from horses and 40 from donkeys in St. Kitts, Nevis, and Sint Eustatius, collected between 2006 and 2015 that were tested with ELISA kits and virus neutralization (for WNV and EVA).Combining ELISA with virus neutralization testing, 25 (13.8%) equine sera were WNV positive (a mixture of indigenous and imported equines) and 3 sera (1.6%) showed doubtful results. For EHV-1, 41 equines (23.7%), mean age 6.7 years, were seropositive. For EHV-4, 138 equines were found seropositive (82.8%), mean age 6.3 years. For EI, 49 equines (27.2%), mean age 7.5 years, were seropositive on ELISA, some previously vaccinated horses. No antibodies against EAV were found on virus neutralization testing, although one animal (0.6%), was EAV positive on ELISA. All samples were EIAV negative.The seroprevalence for EHV-1 and EHV-4 is similar to other parts of the world. For the first time in the study location serologic evidence of antibodies against WNV and EI is reported. This was found in both indigenous and imported animals, highlighting the need for developing proper surveillance plans based on complementary methods of virus detection. Further studies will be needed to define the prevalence, rates of transmission, characterize local virus strains, and study their impact on these populations.

Characterization of equine vitamin D-binding protein, development of an assay, and assessment of plasma concentrations of the protein in healthy horses and horses with gastrointestinal disease.

OBJECTIVE To purify and characterize equine vitamin D-binding protein (VDBP) from equine serum and to evaluate plasma concentrations of VDBP in healthy horses and horses with gastrointestinal injury or disease. ANIMALS 13 healthy laboratory animals (8 mice and 5 rabbits), 61 healthy horses, 12 horses with experimentally induced intestinal ischemia and reperfusion (IR), and 59 horses with acute gastrointestinal diseases. PROCEDURES VDBP was purified from serum of 2 healthy horses, and recombinant equine VDBP was obtained through a commercial service. Equine VDBP was characterized by mass spectrometry. Monoclonal and polyclonal antibodies were raised against equine VDBP, and a rocket immunoelectrophoresis assay for equine VDBP was established. Plasma samples from 61 healthy horses were used to establish working VDBP reference values for study purposes. Plasma VDBP concentrations were assessed at predetermined time points in horses with IR and in horses with naturally occurring gastrointestinal diseases. RESULTS The working reference range for plasma VDBP concentration in healthy horses was 531 to 1,382 mg/L. Plasma VDBP concentrations were significantly decreased after 1 hour of ischemia in horses with IR, compared with values prior to induction of ischemia, and were significantly lower in horses with naturally occurring gastrointestinal diseases with a colic duration of < 12 hours than in healthy horses. CONCLUSIONS AND CLINICAL RELEVANCE Plasma VDBP concentrations were significantly decreased in horses with acute gastrointestinal injury or disease. Further studies and the development of a clinically relevant assay are needed to establish the reliability of VDBP as a diagnostic and prognostic marker in horses.

Modeling the development of proteolytic phenotypes in multi-species oral biofilms

ABSTRACTIn chronic periodontitis, subgingival biofilms induce an inflammatory response leading to periodontal tissue destruction which may cause tooth loss. The response includes exudation of fluid (GCF) from the gingival pocket, giving rise to a protein-rich micro-environment in the biofilm. We hypothesize that proteolytic activity in biofilms is a virulence factor contributing to sustained inflammation.To study the influence of GCF on proteolytic activity in a periodontitis-associated biofilm, a multi-species consortium (Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra and Streptococcus constellatus) was grown in 10% equine serum (to model GCF) or BHI (control) for 2-5 days. Cell-associated and secreted proteolytic activity was investigated with zymography, fluorometry or fluorescent substrates in combination with fluorescence in situ hybridization (FISH).After 2 days, streptococci dominated the consortium in BHI whereas in serum diversity was maintained over 5 days. The serum consortium also developed proteolytic activity, which was absent in BHI. Zymography revealed an array of secreted proteases. FISH revealed proteolytic activity associated with the cell surface of P. gingivalis and F. nucleatum but not P. micra or S. constellatus.Thus, serum favored survival of P. gingivalis and F. nucleatum as well as production of proteases that can act as virulence factors in chronic periodontitis.

The relationship between the variants in the 5'-untranslated regions of equine chorionic gonadotropin genes and serum equine chorionic gonadotropin levels.

ObjectiveAn experiment was conducted to study the association between the single nucleotide polymorphisms (SNPs) in 5′-untranslated regions (5′-UTR) of equine chorionic gonadotropin (eCG) genes and the serum eCG levels.MethodsSNPs in 5′-UTR of eCG genes were screened across 10 horse breeds, including 7 Chinese indigenous breeds and 3 imported breeds using iPLEX chemistry, and the association between the serum eCG levels of 174 pregnant Da’an mares and their serum eCG levels (determined with ELISA) was analyzed.ResultsFour SNPs were identified in the 5′-UTR of the eCGα gene, and one of them was unique in the indigenous breeds. There were 2 SNPs detected at the 5′ end of the eCGβ subunit gene, and one of them was only found in the Chinese breeds. The SNP g.39948246T>C at the 5′-UTR of eCGα was associated significantly with eCG levels of 75-day pregnant mare serum (p<0.05) in Da’an mares. Prediction analysis on binding sites of transcription factors showed that the g.39948246T>C mutation causes appearance of the specific binding site of hepatocyte nuclear factor 3 forkhead homolog 2 (HFH-2), which is a transcriptional repressor belonging to the forkhead protein family of transcription factors.ConclusionThe SNP g.39948246T>C at the 5′-UTR of eCGα is associated with eCG levels of 75-day pregnant mare serum (p<0.05).

Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay

Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse.Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observed. The new IgA mAb 84-1 was used in combination with the previously characterized anti-equine IgA mAb BVS2 for the development and validation of a fluorescent bead-based assay to quantify total IgA in equine serum and various secretions. The IgA assay's linear detection ranged from 64pg/ml to 1000ng/ml. For the quantification of IgA in serum or in secretions an IgA standard was purified from serum or nasal wash fluid (secretory IgA), respectively. The different standards were needed for accurate IgA quantification in the respective samples taking the different signal intensities of monomeric and dimeric IgA on the florescent bead-based assay into account.IgA was quantified by the bead-based assay established here in different equine samples of healthy adult individuals. In serum the median total IgA was 0.45mg/ml for Thoroughbred horses (TB, n=10) and 1.16mg/ml in Icelandic horses (ICH, n=12). In nasopharyngeal secretions of TB (n=7) 0.13mg/ml median total IgA was measured, and 0.25mg/ml for ICH (n=12). Saliva of ICH (n=6) contained a median of 0.15mg/ml, colostrum of Warmbloods (n=8) a median of 1.89mg/ml IgA. Compared to IgG1 and IgG4/7 quantified in the same samples, IgA appeared as the major immunoglobulin isotype in nasopharyngeal secretions and saliva while it is a minor isotype in serum and colostrum.The newly developed monoclonal antibodies against equine IgA and the resulting bead-based assay for quantification of total IgA can notably improve the evaluation of mucosal immunity in horses.

Fn14 Expression is Lower in Female Compared to Male Skeletal Muscle, Independent of Adult Age

BackgroundTweak, a pro‐inflammatory cytokine, and its receptor, Fn14, are recognized as important inflammatory mediators. In skeletal muscle (SkM), elevated TWEAK signaling is associated with reduced insulin sensitivity, reduced mitochondrial biogenesis and SkM atrophy. Further, TWEAK signaling is dysregulated with aging, underlying SkM deterioration and pathological remodeling. Several studies suggest a relationship between estrogen and TWEAK signaling, such that estrogen increases TWEAK expression while suppressing Fn14 expression. Sex‐specific differences in SkM TWEAK and Fn14 have not be identified, but are likely relevant for SkM health in aging.ObjectivesWe sought to evaluate whether SkM of young females has lower Fn14 and higher TWEAK expression compared to young males, and whether Fn14 and TWEAK changes with age in both sexes.MethodsSkM biopsies were collected from the vastus lateralis of young (n=5) and old females (n=6) and young (n=6) and old males (n=4). RNA was extracted from biopsy tissue using TRIzol reagent. Gene expression was measured using qPCR. In a follow‐up experiment human myogenic progenitor cells (MPCs) were isolated from young females (YF‐MPC, n=5) and young males (YM‐MPC, n=6). Cells were cultured in Ham's F12 supplemented with 20% FBS and 5ng/ml bFGF (GM). Once cells reached 75% confluence, all cultures were switched to differentiation media [(DM), Ham's F12 + 2% HI equine serum]. MPCs were cultured in DM for 72 hours and then continued in DM for an additional 48 hours with or without 10ng/ml TNFα. RNA was isolated and gene expression measured using qPCR.ResultsAt the whole tissue level, females had a 2.86 fold lower Fn14 expression (p&lt;0.05) and 1.34 fold higher TWEAK expression (p&lt;0.05) compared males, independent of age. In response to TNFα treatment, differentiated YF‐MPC experienced a 1.7 fold increase in Fn14 expression (p&lt;0.05) while no change in Fn14 expression was observed in differentiated YM‐MPC. TNFα treatment did not affect TWEAK expression in either differentiated YF‐ or YM‐MPC (p&gt;0.05).ConclusionAs hypothesized, females have lower expression of Fn14 and higher expression of TWEAK in skeletal muscle, likely driven, in part, through estrogen‐related mechanisms. However, differences in Fn14 and TWEAK were not differential with age in the female SkM. The young female SkM was collected when the participants had low levels of estrogen, which could partially explain why we observed no difference with age in the females. Intriguingly, TNFα induced Fn14 in differentiated YF‐MPCs, but had no effect in differentiated YM‐MPCs. The significance of this difference for pathological remodeling of skeletal muscle, especially during regeneration, requires further elucidation.Support or Funding InformationCornell University

Tetanus in adults: results of the multicenter ID-IRI study.

Tetanus is an acute, severe infection caused by a neurotoxin secreting bacterium. Various prognostic factors affecting mortality in tetanus patients have been described in the literature. In this study, we aimed to analyze the factors affecting mortality in hospitalized tetanus patients in a large case series. This retrospective multicenter study pooled data of tetanus patients from 25 medical centers. The hospitals participating in this study were the collaborating centers of the Infectious Diseases International Research Initiative (ID-IRI). Only adult patients over the age of 15 years with tetanus were included. The diagnosis of tetanus was made by the clinicians at the participant centers. Izmir Bozyaka Education and Research Hospital's Review Board approved the study. Prognostic factors were analyzed by using the multivariate regression analysis method. In this study, 117 adult patients with tetanus were included. Of these, 79 (67.5%) patients survived and 38 (32.5%) patients died. Most of the deaths were observed in patients >60 years of age (60.5%). Generalized type of tetanus, presence of pain at the wound area, presence of generalized spasms, leukocytosis, high alanine aminotransferase (ALT) and C-reactive protein (CRP) values on admission, and the use of equine immunoglobulins in the treatment were found to be statistically associated with mortality (p < 0.05 for all). Here, we describe the prognostic factors for mortality in tetanus. Immunization seems to be the most critical point, considering the advanced age of our patients. A combination of laboratory and clinical parameters indicates mortality. Moreover, human immunoglobulins should be preferred over equine sera to increase survival.

Design, synthesis and evaluation of novel ferulic acid-O-alkylamine derivatives as potential multifunctional agents for the treatment of Alzheimer's disease

A series of novel ferulic acid-O-alkylamines derivatives were designed, synthesized, and evaluated as multitarget-directed ligands against Alzheimer's disease. In vitro studies displayed that all the synthesized target compounds showed impressive inhibitory activity against butyrylcholinesterase (BuChE), significant inhibition/disaggregation of self-induced β-amyloid (Aβ) aggregation and acted as potential antioxidants. Particularly, compound 7f, one of the most potent BuChE inhibitor (IC50 value of 0.021 μM for equine serum BuChE, 8.63 μM for ratBuChE and 0.07 μM for human serum BuChE), was found to be a good acetylcholinesterase (AChE) inhibitor (IC50 = 2.13 μM for electric eel AChE, 1.8 μM for ratAChE and 3.82 μM for human erythrocytes AChE), and the result of molecular docking provided an explanation for its selective BuChE inhibitory activity. Compound 7f also had noteworthy inhibitory effects on self-induced Aβ1-42 aggregation (50.8 ± 0.82%) and was found to disaggregate self-induced Aβ1-42 aggregation (38.7 ± 0.65%), which was further elucidated by the transmission electron microscopy. Meanwhile, compound 7f showed the modest antioxidant activity (0.55 eq of Trolox), good protective effect against H2O2-induced PC12 cell injury, with low toxicity. Moreover, compound 7f could cross the blood-brain barrier (BBB) in vitro. Significantly, compound 7f did not exhibit any acute toxicity in mice at doses up to 1000 mg/kg, and the step-down passive avoidance test showed this compound significantly reversed scopolamine-induced memory deficit in mice. Taken together, the results indicated that compound 7f is a very promising multifunctional agent in the treatment of Alzheimer's disease, particularly the advanced stages of AD.

Use of polyvalent equine anti-viper serum to treat delayed coagulopathy due to suspected Sistrurus miliarius streckeri envenomation in two children

Background: Western Pygmy Rattlesnake (WPR) envenomation reportedly causes refractory and persistent coagulopathy when treated with CroFab® (Crotalidae Polyvalent Immune Fab). We report two cases where polyvalent equine anti-viper serum (AntivipmynTRI®) was used to treat recurrent coagulopathy in children.Case details: The first patient was a 16-month-old male who was bitten by a confirmed WPR. The patient received a total of 18 vials of CroFab®. His labs normalized, swelling gradually improved, and the child was discharged to home. On day 5, the child returned to the emergency department with a great deal of inguinal tenderness. Labs were obtained and the child’s INR was >13.1, while the fibrinogen was <60 mg/dL and the d-dimer was 11.72 mg/L. A decision was made to administer Antivipmyn TRI®, and the child received a total of 10 vials. Lab values significantly improved: INR 1.2, fibrinogen 93 mg/dL, and d-dimer 4.21 mg/L. The second patient was a 20-month-old male who presented following snake envenomation. The child was administered a total of 22 vials of CroFab® over approximately 70 h following envenomation. Physical exam continued to improve, however, lab results showed an increasing INR 1.98, decreasing platelet count 124 × 103 per μL, fibrinogen <60 mg/dL, and d-dimer >20 ug/mL. A total of 15 vials of Antivipmyn TRI® were administered to this patient. Following this administration, labs and clinical exam both significantly improved. Labs revealed INR 1.16, fibrinogen 110 mg/dL, d-dimer 3.2 mg/L and platelet count 215 × 103/μL.Discussion: CroFab® is still the first-line treatment for children bitten by a WPR, but in some cases patients develop a recurrent coagulopathy. The rapid response demonstrated by Antivipmyn TRI® leads us to conclude that this is a potential therapy for this clinical situation.

3,4-Dihydroquinazoline derivatives inhibit the activities of cholinesterase enzymes

A series of 3,4-dihydroquinazoline derivatives consisting of the selected compounds from our chemical library on the diversity basis and the new synthetic compounds were in vitro tested for their inhibitory activities for both acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase (BChE, from equine serum) enzymes. It was discovered that most of the compounds displayed weak AChE and strong BuChE inhibitory activities. In particular, compound 8b and 8d were the most active compounds in the series against BChE with IC50 values of 45nM and 62nM, as well as 146- and 161-fold higher affinity to BChE, respectively. To understand the excellent activity of these compounds, molecular docking simulations were performed to get better insights into the mechanism of binding of 3,4-dihydroquinazoline derivatives. As expected, compound 8b and 8d bind to both catalytic anionic site (CAS) and peripheral site (PS) of BChE with better interaction energy values than AChE, in agreement with our experimental data. Furthermore, the non-competitive/mixed-type inhibitions of both compounds further confirmed their dual binding nature in kinetic studies.

Novel bipharmacophoric inhibitors of the cholinesterases with affinity to the muscarinic receptors M1 and M2.

A set of hybrid compounds composed of the fragment of allosteric modulators of the muscarinic receptor, i.e. W84 and naphmethonium, and the well-known AChE inhibitor tacrine on the one hand, and the skeletons of the orthosteric muscarinic agonists, iperoxo and isox, on the other hand, were synthesized. The two molecular moieties were connected via a polymethylene linker of varying length. These bipharmacophoric compounds were investigated for inhibition of AChE (from electric eel) and BChE (from equine serum) as well as human ChEs in vitro and compared to previously synthesized dimeric inhibitors. Among the studied hybrids, compound 10-C10, characterized by a 10 carbon alkylene linker connecting tacrine and iperoxo, proved to be the most potent inhibitor with the highest pIC50 values of 9.81 (AChE from electric eel) and 8.75 (BChE from equine serum). Docking experiments with compounds 10-C10, 7b-C10, and 7a-C10 helped to interpret the experimental inhibitory power against AChE, which is affected by the nature of the allosteric molecular moiety, with the tacrine-containing hybrid being much more active than the naphthalimido- and phthalimido-containing analogs. Furthermore, the most active AChE inhibitors were found to have affinity to M1 and M2 muscarinic receptors. Since 10-C10 showed almost no cytotoxicity, it emerged as a promising lead structure for the development of an anti-Alzheimer drug.

The effect of sex and time of day on testosterone concentrations in equine saliva and serum

In terms of exercise, testosterone is important for the growth and maintenance of skeletal muscle mass. Sampling saliva could be a non-invasive alternative to blood sampling for the quantification of testosterone levels in horses. The objective of this study was to compare testosterone concentrations in saliva and serum (sampled simultaneously) from horses of different sexes and at different times throughout the day. A total of 67 warmblood riding horses (21 geldings, 22 mares and 24 stallions) were included in the study. Saliva and blood samples were collected in the morning (06:00-08:00), at midday (11:00-13:00) and in the evening (17:00-19:00). The results demonstrated a weak correlation between saliva and serum testosterone concentrations (rs=0.25, P=0.04). Stallions had higher serum testosterone concentrations than mares and geldings (P&lt;0.001), but there was no significant effect of sex on salivary testosterone concentrations. The time of day did not affect the concentration of testosterone in either saliva or serum. In conclusion, our results indicate that saliva samples cannot be recommended for measuring testosterone levels in horses. However, further research is needed to identify the disturbing factors.

Amino derivatives of platanic acid act as selective and potent inhibitors of butyrylcholinesterase

A set of thirtyfive 30-norlupan derivatives (2–36) was prepared from the natural triterpenoid platanic acid (PA), and the hydroxyl group at C-3, the carboxyl group at C-17 and the carbonyl group at C-20 were modified. These derivatives were tested for their inhibitory activity for the enzymes acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase (BChE, from equine serum) using Ellman's assay. Extra enzyme kinetic studies were performed. The most active compound was (3β, 20R)-3-acetyloxy-20-amino-30-norlupan-28-oate (32) showing a Ki value of 0.01 ± 0.003 μM for BChE. This compound proved to be a selective (FB = 851), mixed-type inhibitor for BChE.

Development and application of a UHPLC-MS/MS method for the simultaneous determination of 17 steroidal hormones in equine serum.

A new, fast and simple analytical method that is able to identify and quantify simultaneously 17 steroid hormones and metabolites (pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone, androsterone, androstenedione, dehydroepiandrosterone, dehydroepiandrosterone sulfate, testosterone, cortisol, corticosterone, aldosterone, 11-deoxycortisol, 11-deoxycorticosterone, dihydrotestosterone, estrone and estradiol) has been developed in equine serum using the ultra-high-performance liquid chromatography-tandem mass spectrometry technique. A total of 400 µl of sample was deproteinized with 1000 µl of acetonitrile, evaporated, restored with 50 µl of a solution of 25% methanol and injected in ultra-high-performance liquid chromatography-tandem mass spectrometry triple quadrupole. The recovery percentage obtained by spiking the matrix at two different concentrations with a standard mixture of steroid hormones was in all cases higher than 85.60% and with the percentage of coefficient of variation lower than 8.37%. The range of the correlation coefficients of the calibration curves of the analyzed compounds was 0.9922-0.9986, and the limits of detection and limits of quantification were in the range of 0.002-2 and 0.0055-5.5 ng ml-1 , respectively. The detected limit of quantification for testosterone (i.e. 50 pg ml-1 ) is twofold lower with respect to its threshold admitted in geldings plasma (100 pg ml-1 free testosterone). The high sensitivity and the quantitative aspect of the method permitted to detect most of the steroids in equine serum. Once validated, the method was used to quantify 17 steroid hormones in mare, stallion and gelding serum samples. The main steroids detected were corticosterone (range 37.25-51.26 ng ml-1 ) and cortisol (range 32.57-52.24 ng ml-1 ), followed by 17-OH-pregnenolone, dihydrotestosterone and pregnenolone. Copyright © 2016 John Wiley & Sons, Ltd.

Mineral Status and Interrelationship in Soil, Forage, and Blood Serum of Horses in the Rainy and Dry Seasons

The objective was to evaluate the content of P, Ca, Mg, K, Na, Cu, Fe, Zn, Se, and Mn in soil, forage, and serum of horses in several production units (PU) during rainy and dry seasons and predict their concentration in serum from their content in soil and forage. Soil and pastures were sampled in the dry (November–December) and in rainy seasons (June–July), and blood samples were collected from the jugular vein of 76 horses in both seasons at four PU. The experimental design was a completely random design within a 4 × 2 (PU × season) factorial arrangement of treatments. Concentration of minerals in soil differed (P < .05) among PU, and contents of P, Ca, Mg, and K were low; Zn and Fe were high; and Cu and Mn were adequate. Mineral concentrations in forage differed among PU and season, and among PU within season (interaction P < .05). Contents of Ca, Mg, Na, Zn, and Cu were low; Fe was high; and P, K, Se, and Mn adequate. The mineral concentration in equine blood serum differed (P < .05) among PU and season. Overall, there were deficiencies of P, Ca, Mg, Na, Cu, and Se, but adequate amounts of K, Zn, and Fe. There are imbalances of minerals in soil and forages which effected their concentration inequine blood.

Albumin-stabilized fluorescent silver nanodots

Ligand-stabilized Ag nanoclusters (NCs) possess many attractive features including high fluorescence quantum yield, large absorption cross-section, good photostability, large Stokes shift and two-photon absorption cross sections. While plenty of fluorescent clusters have been synthesized on various polymer templates, only a few studies have been reported on the fluorescent Ag clusters on peptides and proteins. We study silver NCs synthesized on different protein matrices, including bovine serum albumin, human serum albumin, egg albumin, equine serum albumin, and lysozyme. Our results show that red-emitting Ag NCs can effectively be stabilized by the disulfide bonds in proteins and that the looser structure of the denatured protein favors formation of the clusters.

Seroprevalence and Risk Factors Associated With Corynebacterium pseudotuberculosis Detectable Antibodies in Equids in Alabama

A cross-sectional serological survey was carried out to screen the equine population of the nonendemic state of Alabama for the presence of detectable antibody titers against Corynebacterium pseudotuberculosis. A second objective was to determine the association of detectable titers with risk factors such as exposure to ruminants or previous travel to endemic states. A total of 342 equine serum samples from 40 Alabama counties were analyzed using the synergistic hemolysis inhibition test (SHI). The prevalence of detectable antibody titers (≥1:8) was 52.5% (95% confidence interval [CI], 47%–57.9%). Titers ≥1:128 were detected in 2.63% (95% CI, 1.2%–4.9%), and titers ≥1:512 were detected in 0.3% (95% CI, 0%–1.6%) of the sampled population. In the final generalized linear model, age (P < .001), breed (P = .023), and contact with cattle (P = .05) were associated with increasing SHI titers. Contact with goats was associated in the initial but not in the final analysis (P = .19). Previous travel was not associated with increasing SHI titer (P = .97). The results demonstrated a high prevalence of low detectable titers and low prevalence of titers ≥ 1:128 in a nonendemic population. Further evaluation of SHI cutoff titers and accuracy is warranted to reduce the risk of a false positive diagnosis.