Effects of clarithromycin, azithromycin and rifampicin on terbutaline-induced sweating in foals.

BACKGROUND: Erythromycin (ERY) induces anhidrosis in foals. Azithromycin (AZI) and clarithromycin (CLA), often combined with rifampicin (RIF), are commonly used to treat Rhodococcus equi infections, but effects on sweating have not been investigated. OBJECTIVE: To determine the effects of AZI, CLA and RIF on sweat responses in normal foals. STUDY DESIGN: Each experiment was a blinded, duplicated, six foal × three period counterbalanced within subjects design (12 foals/experiment). METHODS: Antimicrobials were given orally for 5 days. In Experiment 1, ERY, AZI and CLA were given. In Experiment 2, ERY, RIF and ERY/RIF combination were used. Quantitative intradermal terbutaline sweat tests were performed daily for 3 days before and 1, 2, 5, 9, 24, and 39 days after treatment. Data were analysed by repeated measures analysis of variance procedures. Significance was P≤0.05. RESULTS: In Experiment 1, all macrolides suppressed sweating although CLA and AZI were less potent than ERY. In Experiment 2, significant sweat suppression occurred in foals given ERY with or without RIF, but there was no effect of RIF alone. Rifampicin reduced sweat suppression by ERY on Day 1 of treatment but not thereafter. MAIN LIMITATIONS: Because ERY blood concentrations were not measured, effects of RIF on ERY-induced anhidrosis could not definitively be ascribed to altered ERY bioavailability. CONCLUSIONS: All macrolides commonly used to treat R. equi pneumonia, i.e. ERY, AZI and CLA, induce anhidrosis in foals. The potent anti-sudorific effect of ERY is delayed, but not substantially affected by concurrent RIF administration.

Factors associated with outcome in 94 hospitalised foals diagnosed with neonatal encephalopathy.

REASONS FOR PERFORMING STUDY: Neonatal encephalopathy is the most common neurological abnormality identified in neonatal foals, but its clinical course has been rarely characterised. OBJECTIVES: To describe factors associated with nonsurvival in a population of foals diagnosed with neonatal encephalopathy. STUDY DESIGN: Retrospective cross-sectional clinical study. METHODS: Cases were selected from equine neonatal (≤14 days of age) admissions between 1996 and 2007. Multivariable logistic regression was used to identify clinical parameters, laboratory variables and therapeutic interventions associated with nonsurvival. RESULTS: A total of 94 foals were included in the study. Median age at admission was 12 h (range 0-96 h). The most frequently identified clinical signs included abnormal udder seeking (59%), abnormal suckle (55%), inability to stand (42%), abnormal gastrointestinal motility (37%), abnormal consciousness (34%) and seizure activity (22%). Overall, 75 (79.8%) foals survived to be discharged from the hospital and 19 foals died or were subjected to euthanasia. Variables significantly associated with nonsurvival in the multivariable model were serum total calcium concentration, serum activity of alkaline phosphatase, recumbency, number of concurrent diseases, and use of vasopressors/inotropes. The model correctly classified 92.0% of cases. CONCLUSIONS: Overall survival was good and similar to previous reports. Vasopressors/inotropes were the only therapeutic intervention associated with nonsurvival, suggesting that persistent hypotension is associated with nonsurvival in the current population. Foals with concurrent disease, high total calcium and low alkaline phosphatase at admission, and that were recumbent or required treatment with vasopressors/inotropes during hospitalisation, were significantly less likely to survive.

Spinal Cord Hamartomatous Myelodysplasia in 2 Horses With Clinical Neurologic Deficits.

Two horses euthanized for neurologic deficits were diagnosed with hamartomatous myelodysplasia of the spinal cord. One was a 5-week-old Holsteiner colt exhibiting spasms of muscle rigidity in the extensor muscles of the limbs and epaxial muscles, and the other was a 3-year-old Thoroughbred colt exhibiting progressive ataxia and hypermetria in the pelvic limbs. Each had focal disorganization of the white and gray matter of the spinal cord forming a mass interspersed with neurons, glial cells, and disoriented axon bundles. In the Holsteiner colt, the mass was at the level of C5 and included islands of meningeal tissue contiguous with the leptomeninges. The mass occluded the central canal forming hydromyelia cranial to the occlusion. In the Thoroughbred colt, the mass was at the level of L1 on the dorsal periphery of the spinal cord and did not involve the central canal.

Equine Protozoal Myeloencephalitis: An Updated Consensus Statement with a Focus on Parasite Biology, Diagnosis, Treatment, and Prevention.

Equine protozoal myeloencephalitis (EPM) remains an important neurologic disease of horses. There are no pathognomonic clinical signs for the disease. Affected horses can have focal or multifocal central nervous system (CNS) disease. EPM can be difficult to diagnose antemortem. It is caused by either of 2 parasites, Sarcocystis neurona and Neospora hughesi, with much less known about N. hughesi. Although risk factors such as transport stress and breed and age correlations have been identified, biologic factors such as genetic predispositions of individual animals, and parasite-specific factors such as strain differences in virulence, remain largely undetermined. This consensus statement update presents current published knowledge of the parasite biology, host immune response, disease pathogenesis, epidemiology, and risk factors. Importantly, the statement provides recommendations for EPM diagnosis, treatment, and prevention.

Macrolide-induced hyperthermia in foals: Role of impaired sweat responses.

REASONS FOR PERFORMING STUDY: The mechanism of hyperthermia, a potentially fatal adverse effect of erythromycin treatment of foals, is unknown. OBJECTIVES: To determine the cause of erythromycin-associated hyperthermia. It was hypothesised that the normal sweat response of foals is impaired by treatment with erythromycin. STUDY DESIGN: Blinded, crossover study in 10 healthy pony foals. METHODS: Foals kept in stalls were given either erythromycin (25 mg/kg bwt orally, 3 times daily) or control for 10 days then turned out for a further 10 days. Quantitative intradermal terbutaline sweat tests were performed on Days 1 (baseline), 3, 10 and 20. The effects on terbutaline-induced sweating of erythromycin, terbutaline concentration and treatment day were analysed by repeated-measures ANOVA with Bonferroni-corrected pairwise post hoc comparisons. Peak temperatures were compared by Wilcoxon's signed rank test and proportions by McNemar's related samples test. Significance was set at P<0.05. RESULTS: There were significant 2-factor interactions for treatment × terbutaline after baseline, treatment × day at every terbutaline concentration, and day × terbutaline for erythromycin (P<0.001) but not control (P = 0.9) treatment. Sweating was significantly reduced from baseline in erythromycin-treated foals at all subsequent days. Erythromycin-treated foals produced less sweat at all time-points than did control-treated foals (P<0.05). Peak rectal temperatures of erythromycin-treated foals were significantly higher (P = 0.02) than those of controls. During the first 3 days outside more erythromycin-treated than control-treated foals required treatment for hyperthermia (6 vs. 0; P = 0.03). CONCLUSIONS: We believe drug-induced anhidrosis is the likely cause of hyperthermia in some foals treated with erythromycin.

Accurate antemortem diagnosis of equine protozoal myeloencephalitis (EPM) based on detecting intrathecal antibodies against Sarcocystis neurona using the SnSAG2 and SnSAG4/3 ELISAs.

BACKGROUND: Recent work demonstrated the value of antigen-specific antibody indices (AI and C-value) to detect intrathecal antibody production against Sarcocystis neurona for antemortem diagnosis of equine protozoal myeloencephalitis (EPM). OBJECTIVES: The study was conducted to assess whether the antigen-specific antibody indices can be reduced to a simple serum : cerebrospinal fluid (CSF) titer ratio to achieve accurate EPM diagnosis. ANIMALS: Paired serum and CSF samples from 128 horses diagnosed by postmortem examination. The sample set included 44 EPM cases, 35 cervical-vertebral malformation (CVM) cases, 39 neurologic cases other than EPM or CVM, and 10 non-neurologic cases. METHODS: Antibodies against S. neurona were measured in serum and CSF pairs using the SnSAG2 and SnSAG4/3 (SnSAG2, 4/3) ELISAs, and the ratio of each respective serum titer to CSF titer was determined. Likelihood ratios and diagnostic sensitivity and specificity were calculated based on serum titers, CSF titers, and serum : CSF titer ratios. RESULTS: Excellent diagnostic sensitivity and specificity was obtained from the SnSAG2, 4/3 serum : CSF titer ratio. Sensitivity and specificity of 93.2 and 81.1%, respectively, were achieved using a ratio cutoff of ≤100, whereas sensitivity and specificity were 86.4 and 95.9%, respectively, if a more rigorous cutoff of ≤50 was used. Antibody titers in CSF also provided good diagnostic accuracy. Serum antibody titers alone yielded much lower sensitivity and specificity. CONCLUSIONS AND CLINICAL IMPORTANCE: The study confirms the value of detecting intrathecal antibody production for antemortem diagnosis of EPM, and they further show that the antigen-specific antibody indices can be reduced in practice to a simple serum : CSF titer ratio.

Cytotoxic activity of extracts from Hypochaeris radicata.

Pasture-associated stringhalt is an acquired equine disease characterized by peripheral neuropathy and hyperflexion of the pelvic limbs. The disease occurs most commonly during periods of drought in horses grazing pastures heavily contaminated by Hypochaeris radicata. We hypothesized that stringhalt is caused by neurotoxins elaborated by H. radicata in response to the stress of drought conditions. Supernates were collected from H. radicata that were stressed (or not) by immersion in copper chloride solution, then extracted with ethyl acetate and dried. Dilutions of extracts from stressed (SE) and control, unstressed (UE) plants were incubated with myelinating spinal cord cultures (MSCC) established from fetal Swiss mice, and with spinal ganglion cultures (SGC) and dermal fibroblast cultures derived from neonatal mouse tissues. Cytotoxicity in culture monolayers was evaluated both morphologically by microscopy and by release of lactate dehydrogenase activity into culture supernates. Three different SGC preparations were exposed to a single H. radicata extract and single preparations of fibroblasts and MSCC were exposed to three different extracts. Repin, a plant-derived sesquiterpene lactone neurotoxin, was included as a positive control. Significant dose-dependent cytotoxicity was seen within 24 h in all three culture types when incubated with SE or repin. Complete morphologic destruction of culture monolayers was induced by the highest concentrations tested of SE (100 µg/mL) and repin (30 µg/mL). Cytotoxic effect of SE was significantly greater than that of UE for all three cell types and was not due to copper contamination of the extract. This study has identified a cytotoxic activity in leaf exudates of H. radicata that was upregulated by the model stressor, copper chloride.

Ethnic differences in time trends in asthma prevalence in New Zealand: ISAAC Phases I and III.

SETTING: The International Study of Asthma and Allergies in Childhood (ISAAC) Phase III survey, New Zealand. OBJECTIVE: To assess the prevalence of asthma symptoms and time trends by ethnicity between ISAAC Phase I (1992-1993) and Phase III (2001-2003). DESIGN: Information on asthma symptoms and environmental exposures was collected in children aged 6-7 years (n = 10,873) and adolescents aged 13-14 years (n = 13,317). RESULTS: In children, the prevalence of current wheeze was 28.5% in Maori (prevalence odds ratio [POR] = 1.49, 95%CI 1.32-1.68), and 25.2% in Pacific Islanders (POR 1.28, 95%CI 1.07-1.54) compared with 20.7% in Europeans/Pakeha. In adolescents, 29.9% of Maori (POR = 1.13, 95%CI 1.03-1.23) and 20.8% of Pacific Islanders (POR 0.74, 95%CI 0.62-0.87) experienced current wheeze compared to 28.6% of Europeans/Pakeha. Between Phases I and III, the prevalence of current wheeze increased significantly by 0.49%/year in Pacific Islanders, increased non-significantly by 0.12%/year in Maori, and decreased significantly by 0.25%/year in Europeans/Pakeha children. In adolescents, the prevalence of current wheeze increased by 0.05%/year in Pacific Islanders and decreased by 0.33%/year in Europeans/Pakeha and by 0.07%/year in Maori. CONCLUSION: Ethnic differences in asthma symptom prevalence in New Zealand have increased. The reasons for this are unclear, but may reflect inequalities in access to health services.

Uncertainty of sweat chloride testing: does the right hand know what the left hand is doing?

Although analytical variation in sweat electrolyte testing can be easily estimated, there is limited data on total variation. This study aims to evaluate the total variation of the sweat test by measuring the difference between sweat electrolyte values in specimens obtained simultaneously from two sites. Chloride is recommended in published guidelines as the only discriminant for the diagnosis of cystic fibrosis, and sodium may be measured as a guide to the adequacy of collection and analysis. Both are reported here. Sweat was collected in patients by the Gibson Cooke method from two sites simultaneously. Coefficient of variation in this laboratory is 4.1 and 5% for chloride and sodium, respectively. 295 patients had sufficient sweat collected from both sites for analysis. The values for chloride and sodium were compared between the two sites. The total coefficient of variation (CV(t)) calculated for the whole group between the two sites was 20.2% for chloride and 16.9% for sodium, and the standard deviations 4.3 mmol/L and 4.8 mmol/L, respectively. In patients with intermediate chloride concentrations; in different age groups; and when those tests with a difference between sodium and chloride concentration of more than 15 were excluded, minimal differences in these figures were observed. Use of strictly defined cut-off points to discriminate between normal and intermediate electrolyte values, and between intermediate and raised electrolyte values, does not reflect the variation in sweat electrolyte content found within an individual patient. This has important implications for reporting.

Detection of treatable neonatal liver disease by expanded newborn screening.

Two neonates were identified at age 48 h by expanded newborn screening, with abnormal methionine and tyrosine concentrations, which were confirmed on repeat samples. Evidence of previously unsuspected liver disease was found at recall, and there was radiological and biochemical evidence of severe liver disease with hepatic synthetic failure. After inborn errors of metabolism (IEMs) were excluded, both were considered to have neonatal haemochromatosis, on the basis of raised ferritin, iron saturation, and very high α-fetoprotein and confirmed by a mildly hyperferritinaemic sibling in the first case, and raised ferritin and iron saturation in the second. However, it was not feasible to obtain tissue confirmation as the requirement for early therapy precluded biopsy. The babies were treated with antioxidants and iron-chelating agents, and the coagulopathy and hypoalbuminaemia were corrected. Both made a complete recovery and remain well after follow-up. Newborn screening programmes could consider advising clinicians, when tyrosine and methionine values are elevated, that once IEMs are excluded liver disease from other causes must be sought. Neonatal haemochromatosis is an example of one such disease that is potentially treatable.

Quantitative intradermal terbutaline sweat test in horses.

The aim of the current study was to quantify sweating responses to intradermal terbutaline in normal horses. Seven Thoroughbred horses were used. Terbutaline (10-fold dilutions from 1000-0.001 mg/l) and a saline control were injected intradermally (0.1 ml/site) and sweat collected for 30 min into absorbent pads taped over each injection site. Tests were performed monthly for 11 successive months and temperature, relative humidity and dewpoint were measured at the time of testing. There was no significant effect (P

Naturally occurring Sarcocystis infection in domestic cats (Felis catus).

Equine protozoal myeloencephalitis is an important neurological disease of horses in the United States. Consequently, there is an active research effort to identify hosts associated with the primary causative agent, Sarcocystis neurona. The purpose of this study was to determine whether the domestic cat (Felis catus) is a natural host for S. neurona. Muscle sections from 50 primarily free-roaming domestic cats were examined for the presence of sarcocysts. Serum from cats in this group and another group of 50 free-roaming cats were evaluated for the presence of S. neurona antibody. Sarcocysts were found in five of 50 (10%) cats, and S. neurona antibody in five of 100 (5%) cats. Morphological, molecular (including ribosomal RNA genes), and biological characterisation of these sarcocysts showed that they were not S. neurona or S. neurona-like. Sarcocysts found in the cats were identified morphologically as Sarcocystis felis, a common parasite of wild felids. The life cycle of S. felis is not known, and prior to this study, no molecular marker for S. felis existed. Although cats were found to be infected with S. felis sarcocysts, serological data provided evidence of possible infection with S. neurona as well. Further work is needed to determine the role of the domestic cat in the life cycle of S. neurona.

The striped skunk (Mephitis mephitis) is an intermediate host for Sarcocystis neurona.

Striped skunks, initially negative for antibodies to Sarcocystis neurona, formed sarcocysts in skeletal muscles after inoculation with S. neurona sporocysts collected from a naturally infected Virginia opossum (Didelphis virginiana). Skunks developed antibodies to S. neurona by immunoblot and muscles containing sarcocysts were fed to laboratory-reared opossums which then shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0 x 7.5 microm and each contained four sporozoites and a residuum. Sarcocysts from skunks and sporocysts from opossums fed infected skunk muscle were identified as S. neurona using PCR and DNA sequence analysis. A 2-month-old, S. neurona-naive pony foal was orally inoculated with 5 x 10(5) sporocysts. Commercial immunoblot for antibodies to S. neurona performed using CSF collected from the inoculated pony was low positive at 4 weeks p.i., positive at 6 weeks p.i., and strong positive at 8 weeks p.i. Gamma-interferon gene knockout mice inoculated with skunk/opossum derived sporocysts developed serum antibodies to S. neurona and clinical neurologic disease. Merozoites of S. neurona present in the lung, cerebrum, and cerebellum of mice were detected by immunohistochemistry using polyclonal antibodies to S. neurona. Based on the results of this study, the striped skunk is an intermediate host of S. neurona.

The nine-banded armadillo (Dasypus novemcinctus) is naturally infected with Sarcocystis neurona.

Sarcocysts were dissected from the tongue of a nine-banded armadillo (Dasypus novemcinctus). DNA was extracted and characterised by PCR amplification followed by restriction fragment length polymorphism analysis and nucleotide sequencing. A total of 1879 nucleotides were compared; the sarcocyst DNA sequence was identical to that reported for Sarcocystis neurona. DNA was extracted from the sarcocysts of five more nine-banded armadillos. A 254-nucleotide sequence was determined for each and found to be identical to S. neurona. Western blot techniques for detection of anti-S. neurona antibody were developed for use with armadillo plasma and samples from 19 wild-caught and 17 captive-raised armadillos were examined. Whereas all of the 19 wild-caught armadillos had antibodies to S. neurona, only one of 17 captive-raised armadillos did. These results suggest that the nine-banded armadillo are naturally infected with S. neurona.

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host for Sarcocystis neurona.

The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host of at least three species of Sarcocystis, Sarcocystis dasypi, Sarcocystis diminuta, and an unidentified species; however, life cycles of these species have not been determined. Following feeding of armadillo muscles containing sarcocysts to the Virginia opossum (Didelphis virginiana), the opossums shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0x7.5 microm and each contained four sporozoites and a residual body. Sporocysts were identified as Sarcocystis neurona using PCR and DNA sequencing. A 2-month-old foal that was negative for S. neurona antibodies in the CSF was orally inoculated with 5x10(5) sporocysts. At 4 weeks post-infection, the foal had a 'low positive' result by immunoblot for CSF antibodies to S. neurona and by week 6 had a 'strong positive' CSF result and developed an abnormal gait with proprioceptive deficits and ataxia in all four limbs. Based on the results of this study, the nine-banded armadillo is an intermediate host of S. neurona.

Immunoconversion against Sarcocystis neurona in normal and dexamethasone-treated horses challenged with S. neurona sporocysts.

Equine protozoal myeloencephalitis is a common neurologic disease of horses in the Americas usually caused by Sarcocystis neurona. To date, the disease has not been induced in horses using characterized sporocysts from Didelphis virginiana, the definitive host. S. neurona sporocysts from 15 naturally infected opossums were fed to horses seronegative for antibodies against S. neurona. Eight horses were given 5x10(5) sporocysts daily for 7 days. Horses were examined for abnormal clinical signs, and blood and cerebrospinal fluid were harvested at intervals for 90 days after the first day of challenge and analyzed both qualitatively (western blot) and quantitatively (anti-17kDa) for anti-S. neurona IgG. Four of the challenged horses were given dexamethasone (0.1mg/kg orally once daily) for the duration of the experiment. All challenged horses immunoconverted against S. neurona in blood within 32 days of challenge and in CSF within 61 days. There was a trend (P = 0.057) for horses given dexamethasone to immunoconvert earlier than horses that were not immunosuppressed. Anti-17kDa was detected in the CSF of all challenged horses by day 61. This response was statistically greater at day 32 in horses given dexamethasone. Control horses remained seronegative throughout the period in which all challenged horses converted. One control horse immunoconverted in blood at day 75 and in CSF at day 89. Signs of neurologic disease were mild to equivocal in challenged horses. Horses given dexamethasone had more severe signs of limb weakness than did horses not given dexamethasone; however, we could not determine whether these signs were due to spinal cord disease or to effects of systemic illness. At necropsy, mild-moderate multifocal gliosis and neurophagia were found histologically in the spinal cords of 7/8 challenged horses. No organisms were seen either in routinely processed sections or by immunohistochemistry. Although neurologic disease comparable to naturally occurring equine protozoal myeloencephalitis (EPM) was not produced, we had clear evidence of an immune response to challenge both systemically and in the CNS. Broad immunosuppression with dexamethasone did not increase the severity of histologic changes in the CNS of challenged horses. Future work must focus on defining the factors that govern progression of inapparent S. neurona infection to EPM.

Viability of Sarcocystis neurona sporocysts and dose titration in gamma-interferon knockout mice.

Gamma-interferon knockout mice have become the model animal used for studies on Sarcocystis neurona. In order to determine the viability of S. neurona sporocysts and to evaluate the course of the disease in these mice, sporocysts were collected from opossums (Didelphis virginiana), processed, and stored for varying periods of time. Gamma-interferon knockout mice were then inoculated orally with different isolates at different doses. These animals were observed daily for clinical signs until they died or it appeared necessary to humanely euthanize them. 15 of 17 (88%) mice died or showed clinical signs consistent with neurologic disease. The clinical neurologic symptoms observed in these mice appeared to be similar to those observed in horses. 15 of 17 (88%) mice were euthanized or dead by day 35 and organisms were observed in the brains of 13 of 17 (77%) mice. Dose appeared not to effect clinical signs, but did effect the amount of time in which the course of disease was completed with some isolates. The minimum effective dose in this study was 500 orally inoculated sporocysts. Efforts to titrate to smaller doses were not attempted. Direct correlation can be made between molecularly characterized S. neurona sporocysts and their ability to cause neurologic disease in gamma-interferon knockout mice.

Development of Sarcocystis falcatula in its intermediate host, the Brown-headed Cowbird (Molothrus ater).

Sporocysts of Sarcocystis falcatula obtained from experimentally infected Virginia opossums (Didelphis virginiana) were inoculated orally to 60 wild-caught Brown-headed Cowbirds (Molothrus ater). Another 30 Brown-headed Cowbirds were not challenged and served as uninfected controls. Two inoculated and one control cowbird were necropsied every 2 weeks and the pectoral and thigh muscles were examined grossly for cyst development. Stained histologic sections of pectoral muscle, thigh muscle, and lung were examined by light microscopy and presence, density, and size of sarcocysts were determined. Sarcocysts were present by 6 weeks post-inoculation (PI) and were still growing at 40 weeks PI. The sarcocysts from birds 40 weeks post-infection were infective to an opossum. The morphology of the sarcocyst wall by transmission electron microscopy substantiated the identification as S. falcatula. Lung sections were examined for the presence of schizonts, but were seen only at 2 weeks PI. This evaluation was complicated by the presence of unidentified microfilariae. These birds are migratory and the continued growth and development of muscle cysts would allow them to be a source of infection at both extremes of their geographic range, regardless of which end of the migration at which they were infected.