Feline parathyroid hormone: validation of hormonal assays and dynamics of secretion

Validated assays for quantification of intact parathyroid hormone (I-PTH) are no longer available. Moreover, the third-generation PTH assay that only detects the whole PTH molecule (W-PTH) has never been tested in cats. The work presented here is aimed to validate a commercially available assay for measurement of I-PTH and W-PTH in cats and to study the dynamics of PTH secretion in healthy cats. Our results show that both assays are reliable for the measurement of feline PTH. In healthy adult cats W-PTH concentration (15.1 ± 1.6 pg/mL) was greater (P < 0.001) than I-PTH concentration (9.1 ± 0.7 pg/mL). The dynamics of PTH secretion in response to changes in extracellular calcium (Ca(2+)) were investigated in 13 cats by studying PTH-Ca(2+) curves. PTH-Ca(2+) curves were obtained by intravenous infusion of disodium ethylenediaminetetraacetic acid and CaCl(2). PTH was measured using both I-PTH and W-PTH assays. During hypocalcemia a sigmoidal curve that was similar when measured with I-PTH or W-PTH was obtained. The maximal PTH concentration in response to hypocalcemia was greater with W-PTH (179.6 ± 41.9 pg/mL) than with I-PTH (67.6 ± 10.5 pg/mL; P = 0.01). However, hypercalcemia resulted in an equivalent PTH inhibition, with both assays yielding PTH concentrations as follows: W-PTH = 4.0 ± 0.4 pg/mL and I-PTH = 4.9 ± 0.3 pg/mL (NS). Parameters of the feline PTH-Ca(2+) curve are similar to what has been previously reported in dogs.     

An unusual case of generalized soft-tissue mineralization in a suckling foal

An atypical case of severe soft-tissue mineralization in a 3-week-old foal from a herd of Andalusian horses is described. The herd clinical history and the laboratory findings were compatible with a diagnosis of secondary hyperparathyroidism due to a mineral imbalance in the diet (low calcium and high phosphorus intake). Mares showed a marked increase in serum parathyroid hormone (PTH) approximately 10 times normal levels. Serum PTH was marginally elevated in foals. Clinical signs (unthriftiness, painful joints, lameness in one or more limbs, and stiff gait) were more pronounced in foals than in mares. Two foals died and necropsy of one of them revealed extensive soft-tissue mineralization of arterial walls and pulmonary parenchyma. Clinical signs in mares and foals resolved by 4 weeks after diet adjustment.     

Long-term effects of intermittent equine parathyroid hormone fragment (ePTH-1-37) administration on bone metabolism in healthy horses

Intermittent administration of parathyroid hormone (PTH) is an anabolic therapy for osteoporotic conditions in humans. This study evaluated the effects of equine PTH fragment (ePTH-1-37) administration on bone metabolism in 12 healthy horses. Six horses each were treated once daily for 120days with subcutaneous injections of 0.5μg/kg ePTH-1-37 or placebo. Blood was collected to determine ionized calcium (Ca(++)), total Ca (Ca(T)), inorganic phosphorus, serum equine osteocalcin (eOC), carboxy-terminal telopeptide of type I collagen (ICTP), bone-specific alkaline phosphatase, and carboxy-terminal cross-linked telopeptide of type I collagen. Bone mineral density (BMD) was determined with dual X-ray absorptiometry of the metacarpus and calcaneus. Significantly higher blood Ca(++) and plasma Ca(T) concentrations were measured 5h after ePTH-1-37 administration compared to placebo. Higher serum eOC concentrations were found for ePTH-1-37 treatment at days 90 (P<0.05) and 120 (P=0.05). Significantly higher serum ICTP levels were observed with ePTH-1-37 treatment at days 60 and 90. For both study groups, BMD increased significantly in the calcaneus. Long-term use of ePTH-1-37 seemed to have no negative effects on bone metabolism in healthy horses. The absence of undesirable side effects is the premise to ensure safety for further clinical investigations in horses with increased bone resorption processes.     

Comparison of serum parathyroid hormone and ionized calcium and magnesium concentrations and fractional urinary clearance of calcium and phosphorus in healthy horses and horses with enterocolitis

OBJECTIVE: To evaluate calcium balance and parathyroid gland function in healthy horses and horses with enterocolitis and compare results of an immunochemiluminometric assay (ICMA) with those of an immunoradiometric assay (IRMA) for determination of serum intact parathyroid hormone (PTH) concentrations in horses. ANIMALS: 64 horses with enterocolitis and 62 healthy horses. PROCEDURES: Blood and urine samples were collected for determination of serum total calcium, ionized calcium (Ca2+) and magnesium (Mg2+), phosphorus, BUN, total protein, creatinine, albumin, and PTH concentrations, venous blood gases, and fractional urinary clearance of calcium (FCa) and phosphorus (FP). Serum concentrations of PTH were measured in 40 horses by use of both the IRMA and ICMA. RESULTS: Most (48/64; 75%) horses with enterocolitis had decreased serum total calcium, Ca2+, and Mg2+ concentrations and increased phosphorus concentrations, compared with healthy horses. Serum PTH concentration was increased in most (36/51; 70.6%) horses with hypocalcemia. In addition, FCa was significantly decreased and FP significantly increased in horses with enterocolitis, compared with healthy horses. Results of ICMA were in agreement with results of IRMA. CONCLUSIONS AND CLINICAL RELEVANCE: Enterocolitis in horses is often associated with hypocalcemia; 79.7% of affected horses had ionized hypocalcemia. Because FCa was low, it is unlikely that renal calcium loss was the cause of hypocalcemia. Serum PTH concentrations varied in horses with enterocolitis and concomitant hypocalcemia. However, we believe low PTH concentration in some hypocalcemic horses may be the result of impaired parathyroid gland function.     

Heparinised blood ionised calcium concentrations in horses with colic or diarrhoea compared to normal subjects

Our objectives were to 1) establish ionised calcium (ICa), C-terminal PTH and biologically active PTH (intact molecule) concentrations in blood from normal horses, 2) examine the stability of ionised calcium and acid-base values in stored equine heparinised blood and serum and 3) check the applicability of the formulas based on these parameters in certain disease states. Mean +/- s.d. % ionised calcium in heparinised blood of normal Warmbloods was 51 +/- 2.7 (n = 20) of total calcium, range 1.45-1.75 mmol/l (n = 15) at Michigan State University and 1.43-1.69 mmol/l (n = 20) at Utrecht University. Mean +/- s.d. EDTA plasma concentration for intact +/PTH in normal horses measured 0.6 +/- 0.3 pmol/l (n = 11). Both mean serum and the heparinised blood ionised calcium concentrations changed (not significantly) after 102 h storage at room temperature. Six cycles of freezing and thawing did not affect serum ionised calcium concentration significantly. Ionised calcium concentration and pH in heparinised blood of 20 normal Warmbloods were used to calculate the regression equation for the prediction of the adjusted ionised calcium concentration to a pH of 7.4. The linear regression equation found was: adjusted plasma ICa at pH 7.4 mmol/l = -6.4570 + 0.8739 x (measured pH) + 0.9944 x (measured ICa mmol/l). By means of this formula, mean adjusted ionised calcium concentration in heparinised blood calculated was 100% of the actual value given by the analyser in the normal horses. When using this formula in horses with colic or diarrhoea, mean adjusted ionised calcium concentration was underestimated by 0.2 and 0.3%, respectively. Furthermore, to adjust the measured ionised calcium concentration in heparinised blood to a pH of 7.4 in healthy as well as in 2 groups of diseased horses 2 formulas with a good prediction are now available.     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand.

METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3).

RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017].

CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

Viruses associated with outbreaks of equine respiratory disease in New Zealand

AIM: To identify viruses associated with respiratory disease in young horses in New Zealand. METHODS: Nasal swabs and blood samples were collected from 45 foals or horses from five separate outbreaks of respiratory disease that occurred in New Zealand in 1996, and from 37 yearlings at the time of the annual yearling sales in January that same year. Virus isolation from nasal swabs and peripheral blood leukocytes (PBL) was undertaken and serum samples were tested for antibodies against equine herpesviruses (EHV-1, EHV-2, EHV-4 and EHV-5), equine rhinitis-A virus (ERAV), equine rhinitis-B virus (ERBV), equine adenovirus 1 (EAdV-1), equine arteritis virus (EAV), reovirus 3 and parainfluenza virus type 3 (PIV3). RESULTS: Viruses were isolated from 24/94 (26%) nasal swab samples and from 77/80 (96%) PBL samples collected from both healthy horses and horses showing clinical signs of respiratory disease. All isolates were identified as EHV-2, EHV-4, EHV-5 or untyped EHV. Of the horses and foals tested, 59/82 (72%) were positive for EHV-1 and/or EHV-4 serum neutralising (SN) antibody on at least one sampling occasion, 52/82 (63%) for EHV-1-specific antibody tested by enzyme-linked immunosorbent assay (ELISA), 10/80 (13%) for ERAV SN antibody, 60/80 (75%) for ERBV SN antibody, and 42/80 (53%) for haemagglutination inhibition (HI) antibody to EAdV-1. None of the 64 serum samples tested were positive for antibodies to EAV, reovirus 3 or PIV3. Evidence of infection with all viruses tested was detected in both healthy horses and in horses showing clinical signs of respiratory disease. Recent EHV-2 infection was associated with the development of signs of respiratory disease among yearlings [relative risk (RR)=2.67, 95% CI=1.59-4.47, p=0.017]. CONCLUSIONS: Of the equine respiratory viruses detected in horses in New Zealand during this study, EHV-2 was most likely to be associated with respiratory disease. However, factors other than viral infection are probably important in the development of clinical signs of disease.     

Serum concentrations of calcium, phosphorus, magnesium and calciotropic hormones in donkeys

OBJECTIVE: To provide reference values for serum biochemical variables that are used for evaluation of mineral metabolism in donkeys and compare values with those in horses. ANIMALS: 18 donkeys and 18 horses. PROCEDURES: Total calcium (tCa), total magnesium (tMg), and inorganic phosphorus (P) concentrations were measured in serum samples via spectrophotometry. Ionized calcium (iCa) and magnesium (iMg) concentrations were quantified with selective electrodes. By use of a micropartition system, tCa and tMg were fractionated to separate protein-bound (pCa, pMg) and ultrafiltrable fractions. Complexed calcium (cCa) and magnesium (cMg) concentrations were calculated by substracting ionized fractions from ultrafiltrable fractions. Parathyroid hormone (PTH) and calcitriol (CTR) concentrations were measured via radioimmunoassay. RESULTS: Serum tCa concentration in donkeys (3.37 +/- 0.21 mmol/L) was composed of pCa (1.59 +/- 0.21 mmol/L [47.0 +/- 4.2%]), iCa (1.69 +/- 0.04 mmol/L [50.4 +/- 3.0%]), and cCa (0.09 +/- 0.08 mmol/L [2.6 +/- 2.9%]). Serum tMg concentration (1.00 +/- 0.08 mmol/L) was fractioned in pMg (0.23 +/- 0.08 mmol/L [23.4 +/- 8.1%]), iMg (0.59 +/- 0.04 mmol/L [58.8 +/- 5.1%]), and cMg (0.18 +/- 0.08 mmol/L [17.8 +/- 7.2%]). Serum concentrations of P (1.14 +/- 0.30 mmol/L), PTH (20.4 +/- 21.2 pg/mL), and CTR (13.4 +/- 5.9 pg/mL) were determined. CONCLUSIONS AND CLINICAL RELEVANCE: Serum variables of mineral metabolism in donkeys were within reference ranges for horses. However, when compared with horses, donkeys had higher iCa, cMg, and CTR and lower pMg and PTH concentrations.     

Rhodococcus (Corynebacterium) equi: bactericidal capacity of neutrophils from neonatal and adult horses

The capacity of hematogenous polymorphonuclear neutrophilic leukocytes (PMNL) to kill Rhodococcus equi was compared in horses of various ages. A radioisotope bactericidal assay was used to determine the capacity of PMNL to kill R equi. Assays were conducted on PMNL from horses in 3 groups: group I, 13 foals with a mean age of 3.3 days; group II, 10 group-I foals at a mean age of 35.7 days; and group III, adult dams of group-I foals. Bacteria were obtained from the lungs of a foal with R equi pneumonia and opsonized with fresh adult equine serum that contained R equi specific antibody. The mean peak percentage of R equi killed by PMNL was 78.9 for group I, 90.1 for group II, and 87.9 for group III. There was no significant difference (P greater than 0.05) among groups; however, 15% of foals in group I (2 foals) had a mean peak percentage of 30.5 killed, which was significantly (P less than 0.05) lower than the percentage for other foals in group I. The results of our investigation indicated that the capacity of PMNL to kill opsonized R equi is similar in neonatal, young, and adult horses. However, some neonatal foals have a substantially lower capacity to kill R equi, which may be an important factor in the pathogenesis of R equi infections.     

High Seroprevalence Against Lawsonia intracellularis Among Adult Horses in Belgium

Lawsonia intracellularis (LI) is an obligate intracellular gram-negative rod causing equine proliferative enteropathy (EPE). Occasional cases of EPE have been reported in foals living in Belgium, but the seroprevalence of equine LI in this country is unknown. The target population included clinically healthy adult horses, whose blood samples were collected and analyzed for specific IgG antibodies against LI using a blocking enzyme-linked immunosorbent assay test. The results were expressed as percentage of inhibition (PI). Samples that had a PI <20% were judged as negative, those between 20 and 30% as inconclusive, and those >30% were considered positive. A total of 356 blood samples were analyzed with 352 horses (98.8%) testing positive, 2 horses (0.6%) testing negative, and 2 horses (0.6%) showing inconclusive results. The large percentage of seropositive samples obtained in this study confirms a widespread exposure of Belgian horses to LI.     

Calcium Regulating Hormones and Serum Calcium and Magnesium Concentrations in Septic and Critically Ill Foals and their Association with Survival

Background: Disorders of calcium regulation are frequently found in humans with critical illness, yet limited information exists in foals with similar conditions including septicemia. The purpose of this study was to determine whether disorders of calcium exist in septic foals, and to determine any association with survival.
Hypothesis: Blood concentrations of ionized calcium (Ca²⁺) and magnesium (Mg²⁺) will be lower in septic foals with concomitant increases in parathyroid hormone (PTH), calcitonin (CT), and parathyroid-related peptide (PTHrP) compared with healthy foals. The magnitude of these differences will be negatively associated with survival.
Animals: Eighty-two septic, 40 sick nonseptic, and 24 healthy foals of ≤7 days were included.
Methods: Prospective, observational study. Blood was collected at initial examination for analysis. Foals with positive blood culture or sepsis score ≥14 were considered septic. Foals with disease other than sepsis and healthy foals were used as controls. Hormone concentrations were measured with validated immunoassays.
Results: Septic foals had decreased Ca²⁺ (5.6 versus 6.1 mg/dL, P < .01) and increased serum PTH (16.2 versus 3.2 pmol/L, P < .05), and phosphorus concentrations (7.1 versus 6.3 mg/dL, P < .01). No differences in serum Mg²⁺, PTHrP, and CT concentrations were found. Nonsurviving septic foals (n = 42/82) had higher PTH concentrations (41.1 versus 10.7 pmol/L, P < .01) than survivors (n = 40/82).
Conclusions and Clinical Importance: Septic foals were more likely to have disorders of calcium regulation compared with healthy foals, where hyperparathyroidemia was associated with nonsurvival.     

Equine haptoglobin: isolation, characterization, and the effects of ageing, delivery and inflammation on its serum concentration.

Haptoglobin (Hp) was isolated from equine serum by ammonium sulphate precipitation, anion-exchange chromatography and gel filtration. Equine Hp which migrated to the alpha 2-globulin region in electrophoresis, contained 2 fractions with molecular weights (NW) of 108,000 and 105,000, and each fraction consisting of 2 subunits. Quantitative measurement of Hp in equine serum was performed by the single radial immunodiffusion method using anti-equine Hp serum. In clinically normal horses, the highest concentration of serum Hp was found in newborn foals and a high value was maintained until 12 months of age. The concentration then decreased with age. Normal Hp values were 5.25 +/- 2.36 mg/ml in foals (less than or equal to 12 months old), 2.19 +/- 1.54 mg/ml in adult horses (greater than or equal to 18 months old) and 3.62 +/- 0.81 mg/ml in all horses. Serum Hp concentration in mares during the perinatal period in comparison with the normal adult female was high for 4 months pre-partum, a passing increase at delivery, and then decreased at 2 weeks post-partum returning to normal within 1 month of delivery. In horses with experimentally-induced inflammation, serum Hp concentration began to increase immediately after treatment and reached the highest value, 1.5 to 9 times higher than those of pre-treatment at 2 to 5 days, then decreased within 4 weeks. It was also elevated in most cases of horses with clinically inflammatory signs.     

Retrospective Study: Incidence of transfusion reactions to commercial equine plasma

Objective – To report on the incidence of transfusion reactions to commercial equine plasma in a hospital‐based population of horses, to characterize these reactions and report on outcome. Design – Retrospective study. Setting – University teaching hospital. Animals – Client‐owned horses referred to the University of Wisconsin. Interventions – Intravenous administration of 2 commercial equine plasma products when clinically indicated. Measurements and Main Results – Medical records of 107 horses that received plasma transfusions between 2003 and 2008 were evaluated. Transfusion reactions were recorded in 6 of 107 transfusions. All individuals were administered plasma from 1 commercial source. Foals <30 days of age received a hypergammaglobulinemic product and all adults received a lower IgG concentration product. No reactions were recorded in adults. In foals (<30 d) reactions were recorded in 6 of 69 cases (8.7%), all of which occurred in neonates <7 days of age (6/62; [9.7%]). The most frequent reactions were fever (4/6), tachycardia (2/6), tachypnea (2/6), and colic (2/6). All affected foals survived the reaction. There were no statistically significant differences (P<0.05) in any of the variables examined between those foals that did and those that did not experience transfusion reactions. Conclusion – The incidence of transfusion reactions was 8.7% in foals and 0% in adult horses in our referral population. Five of 6 foals responded to medical therapy and eventually received the clinically indicated transfusion. No transfusion related mortality occurred.     

Failure of low-dose recombinant human IL-2 to support the survival of virus-specific CTL clones infused into severe combined immunodeficient foals: lack of correlation between in vitro activity and in vivo efficacy

Although CTL are important for control of lentiviruses, including equine infectious anemia virus (EIAV), it is not known if CTL can limit lentiviral replication in the absence of CD4 help and neutralizing antibody. Adoptive transfer of EIAV-specific CTL clones into severe combined immunodeficient (SCID) foals could resolve this issue, but it is not known whether exogenous IL-2 administration is sufficient to support the engraftment and proliferation of CTL clones infused into immunodeficient horses. To address this question we adoptively transferred EIAV Rev-specific CTL clones into four EIAV-challenged SCID foals, concurrent with low-dose aldesleukin (180,000U/m2), a modified recombinant human IL-2 (rhuIL-2) product. The dose was calculated based on the specific activity on equine PBMC in vitro, and resulted in plasma concentrations considered sufficient to saturate high affinity IL-2 receptors in humans. Despite specific activity on equine PBMC that was equivalent to recombinant equine IL-2 and another form of rhuIL-2, aldesleukin did not support the engraftment and expansion of infused CTL clones, and control of viral load and clinical disease did not occur. It was concluded that survival of Rev-specific CTL clones infused into EIAV-challenged SCID foals was not enhanced by aldesleukin at the doses used in this study, and that in vitro specific activity did not correlate with in vivo efficacy. Successful adoptive immunotherapy with CTL clones in immunodeficient horses will likely require higher doses of rhuIL-2, co-infusion of CD4+ T lymphocytes, or administration of equine IL-2.     

The use of a neutralizing monoclonal antibody to detect infections of equine herpesvirus type 2 (EHV-2).

A blocking enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to equine herpesvirus 2 in serum samples of horses. By measuring the binding to a single epitope, this blocking ELISA gives a good picture of the antibody status in the animal. The test is based on a monoclonal antibody with neutralizing activity and had a sensitivity of 94% and a specificity of 100%. Antibodies due to newly acquired infection in foals were successfully detected with this blocking ELISA.     

Calcium homeostasis and intact plasma parathyroid hormone during exercise and training in young Standardbred horses

Physical exercise is known to affect calcium homeostasis in horses, but there is little information on the hormonal regulation of calcium metabolism during exercise. In order to evaluate the effects of exercise and training on calcium homeostasis and intact plasma parathyroid hormone, 7 untrained Standardbred horses were studied in a 6 week training programme. These horses were accustomed to running on the treadmill 3 weeks before onset of training and were exercised on a high-speed treadmill with an initial incremental standardised exercise test (SET 1: 6 incremental steps of 5 min duration each; first step 5 m/s, increase 1 m/s). SET 1 was followed by a lactate-guided training programme (6 weeks in total) with 2 types of exercise in alternating order with a day of rest after each work day: high-speed exercise (HSE) of 15 min duration, starting at VLa4, continuous increase in speed every 60 s by 0.3 m/s (14 incremental steps); and low-speed exercise (LSE) at a constant velocity at VLa2.5, duration approximately 60-90 min. The whole training programme consisted of 8 HSE and 8 LSE sessions. HSE and LSE were calculated to require the same energy expenditure. A final SET (SET 2) finished the training programme. Blood samples for lactate, plasma total calcium [Ca], blood ionised calcium [Ca2+], blood pH, plasma inorganic phosphorus [P(i)] and plasma intact parathyroid hormone [PTH] were collected before, during and after SETs 1 and 2, before and after the first and eighth HSE and LSE. During SETs 1 and 2, HSEs 1 and 8 there was a decrease in ionised Ca2+ and pH and a rise in lactate, intact PTH and P(i). LSEs 1 and 8 resulted in an increase in pH, whereas lactate, ionised Ca2+, total Ca, P(i) and intact PTH were not affected. No changes in calcium metabolism were detected during training. Results of this study suggest that intact PTH is a mediator in counter-regulation of exercise-induced hypocalcaemia.     

A prospective study of the roles of clostridium difficile and enterotoxigenic Clostridium perfringens in equine diarrhoea

Faecal samples from adult horses and from foals with diarrhoea or with normal faeces were evaluated for the presence of Clostridium difficile, C. difficile toxins, C. perfringens enterotoxin (CPE) and C. perfringens spore counts. Clostridium difficile was isolated from 7/55 horses (12.7%) and 11/31 foals (35.5%) with colitis, but from 1/255 normal adults (0.4%) and 0/47 normal foals (P<0.001). Clostridium difficile toxins A and/or B were detected in 12/55 diarrhoeic adults (21.8%) and 5/30 diarrhoeic foals (16.7%) but in only 1/83 adults (1.2%) and 0/21 foals with normal faeces (P<0.001 and P<0.05, respectively). Clostridium perfringens enterotoxin was detected in 9/47 diarrhoeic adults (19%) and 8/28 diarrhoeic foals (28.6%), but was not detected in 47 adult horses (P<0.002) or 4 foals (P = 0.22) with normal faeces. The positive predictive value of isolation of C. perfringens with respect to the presence of CPE was only 60% in adult horses and 64% in foals. There was no association between total C. perfringens spore count and CPE in the faeces. The overall mortality rate from colitis was 22% for adult horses and 18% for foals. Clostridium difficile toxin-positive adult horses with colitis were less likely to survive than C. difficile-negative horses with colitis (P = 0.03). This study provides further evidence that C. difficile and enterotoxigenic C. perfringens are associated with equine enterocolitis.     

Development of a solid-phase assay for measurement of sulfated glycosaminoglycan concentrations in equine synovial fluid

OBJECTIVE: To develop a new 1,9-dimethylmethylene blue (DMMB) assay for measurement of sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF) by use of membrane technology and to compare the assay's ability to measure sGAG concentrations with that of 2 other established DMMB assays. SAMPLE POPULATION: 25 samples of SF collected from affected joints of 14 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses. PROCEDURE: A solid-phase DMMB assay was developed to measure sGAG concentrations in SE Results for the assay were then compared with results obtained by use of the direct spectrophotometric method (ie, Famdale method) and microplate DMMB assay. RESULTS: The solid-phase assay and direct spectrophotometric assay measured the same sGAG concentrations in identical equine SF, but those concentrations differed significantly from results obtained by use of the microplate DMMB assay. All other aspects of the solid-phase DMMB assay were comparable to both the direct spectrophotometric and microplate DMMB assays. CONCLUSIONS AND CLINICAL RELEVANCE: The new solid-phase assay can be used interchangeably with the direct spectrophotometric method to measure sGAG concentrations in equine SF samples, but it cannot be interchanged with the microplate DMMB assay. Results can be rapidly obtained with the solid-phase assay. Also, the solid-phase assay can detect nanogram quantities of sGAGs in SF, circumvent the problem of premature precipitation of sGAG-dye complexes, and provide quantitative or qualitative results. The solid-phase assay may replace other DMMB assays for measuring sGAG concentrations in SF obtained from horses.     

Hypertrophy and physiological death of equine chondrocytes in vitro

REASON FOR PERFORMING STUDY: Equine osteochondrosis results from a failure of endochondral ossification during skeletal growth. Endochondral ossification involves chondrocyte proliferation, hypertrophy and death. Until recently no culture system was available to study these processes in equine chondrocytes. OBJECTIVE: To optimise an in vitro model in which equine chondrocytes can be induced to undergo hypertrophy and physiological death as seen in vivo. METHODS: Chondrocytes isolated from fetal or older (neonatal, growing and mature) horses were cultured as pellets in 10% fetal calf serum (FCS) or 10% horse serum (HS). The pellets were examined by light and electron microscopy. Total RNA was extracted from the pellets, and quantitative PCR carried out to investigate changes in expression of a number of genes regulating endochondral ossification. RESULTS: Chondrocytes from fetal foals, grown as pellets, underwent hypertrophy and died by a process morphologically similar to that seen in vivo. Chondrocytes from horses age >5 months did not undergo hypertrophy in pellet culture. They formed intramembranous inclusion bodies and the cultures included cells of osteoblastic appearance. Pellets from neonatal foals cultured in FCS resembled pellets from older horses, however pellets grown in HS underwent hypertrophy but contained inclusion bodies. Chondrocytes from fetal foals formed a typical cartilage-like tissue grossly and histologically, and expressed the cartilage markers collagen type II and aggrecan mRNA. Expression of Sox9, collagen type II, Runx2, matrix metalloproteinase-13 and connective tissue growth factor mRNA increased at different times in culture. Expression of fibroblast growth factor receptor-3 and vascular endothelial growth factor mRNA decreased with time in culture. CONCLUSIONS: Freshly isolated cells from fetal growth cartilage cultured as pellets provide optimal conditions for studying hypertrophy and death of equine chondrocytes. POTENTIAL RELEVANCE: This culture system should greatly assist laboratory studies aimed at elucidating the pathogenesis of osteochondrosis.     

Clostridium difficile: prevalence in horses and environment,and antimicrobial susceptibility

REASONS FOR PERFORMING STUDY: Clostridium difficile has been associated with acute colitis in mature horses. OBJECTIVES: To survey C. difficile colonisation of the alimentary tract with age, occurrence of diarrhoea and history of antibiotic therapy; and to study the occurrence and survival of C. difficile in the environment and antimicrobial susceptibility of isolated strains. METHODS: A total of 777 horses of different breeds, age and sex were studied. Further, 598 soil samples and 434 indoor surface samples were examined. Antimicrobial susceptibility of 52 strains was investigated by Etest for 10 antibiotics. RESULTS: In horses that developed acute colitis during antibiotic treatment, 18 of 43 (42%) were positive to C. difficile culture and 12 of these (28%) were positive in the cytotoxin B test. Furthermore, C. difficile was isolated from a small number of diarrhoeic mature horses (4 of 72 [6%]) with no history of antibiotic treatment, but not from 273 healthy mature horses examined or 65 horses with colic. An interesting new finding was that, in normal healthy foals age < 14 days, C. difficile was isolated from 1/3 of foals (16 of 56 [29%]). All older foals (170) except one were negative. Seven of 16 (44%) nondiarrhoeic foals treated with erythromycin or gentamicin in combination with rifampicin were also excretors of C. difficile. On studfarms, 14 of 132 (11%) outdoor soil samples were positive for C. difficile in culture, whereas only 2 of 220 (1%) soil samples from farms with mature horses were positive for C. difficile (P = < 0.001). By PCR, it was demonstrated that strains from the environment and healthy foals can serve as a potential reservoir of toxigenic C. difficile. The experimental study conducted here found that C. difficile survived in nature and indoors for at least 4 years in inoculated equine faeces. The susceptibility of 52 strains was investigated for 10 antibiotics and all were susceptible to metronidazole (MIC < or = 4 mg/l) and vancomycin (MIC < or = 2 mg/l). CONCLUSIONS: C. difficile is associated with acute colitis in mature horses, following antibiotic treatment. Furthermore, C. difficile was isolated from 1 in 3 normal healthy foals age < 14 days. POTENTIAL RELEVANCE: Strains from healthy foals and the environment can serve as a potential reservoir of toxigenic C. difficile.