Molecular detection of Sarcocystis neurona in cerebrospinal fluid from 210 horses with suspected neurologic disease.

An ante-mortem diagnosis of equine protozoal myeloencephalitis (EPM) is presently based on clinical presentation, immunodiagnostics performed on serum and cerebrospinal fluid (CSF), and ruling out other neurological disorders. Molecular techniques introduce a novel and promising approach for the detection of protozoal agents in CSF. Hypothesizing that real-time PCR (rtPCR) can be a useful complement to EPM diagnostics, 210 CSF samples from horses suspected of neurological disease with EPM included as a differential diagnosis were tested using rtPCR to detect Sarcocystis neurona DNA and immunodiagnostics targeting antibodies against the same pathogen, performed on serum and CSF samples. Molecular and immunological results were compared with respect to origin of the horse, time of the year, signalment, clinical signs and treatment history. Twenty-five horses tested positive in CSF for S. neurona by rtPCR only, while 30 horses had intrathecally-derived antibodies to S. neurona only (serum to CSF ratio ≤ 64 by indirect fluorescent antibody test - IFAT), and 13 horses tested rtPCR-positive in CSF with evidence of intrathecally-derived antibodies to S. neurona. Previous treatment for EPM was the only variable presenting statistical difference between the two testing modalities, highlighting that animals with history of anti-protozoal treatment were more likely to test positive solely in IFAT, while horses without treatment were more likely to test positive by rtPCR only. The results support the use of molecular diagnosis for EPM caused by S. neurona as a complement to immunodiagnostics. The use of rtPCR in CSF for the detection of S. neurona may improve the diagnostic work-up of neurologic disease suspected horses, especially in animals without previous anti-protozoal treatment.

Characterisation of the cerebrospinal fluid of horses with West Nile virus neuroinvasive disease.

West Nile virus (WNV) is a zoonotic arbovirus transmitted by mosquitoes between wild birds (natural hosts) and other vertebrates. Horses and humans are incidental, dead-end hosts, but can develop severe neurological disorders. Owing to the close contact of cerebrospinal fluid (CSF) with the extracellular fluid of the brain, the analysis of CSF composition can reflect central nervous system (CNS) impairments enabling the diagnosis and understanding of various neurodegenerative CNS disorders. Our objective was to compare the findings from the CSF samples of horses with neuroinvasive WNV infection with those of healthy controls. We compared findings from fifteen CSF samples of 13 horses with acute WNV encephalomyelitis with those of 20 healthy controls. Protein, particular enzymes and ions, glucose and lactate showed abnormal levels in a significant number of WNV cases. None of the six horses with elevated glucose concentrations survived. Rather neutrophilic than mononuclear pleocytosis was identified with WNV infection. Neutrophils probably play a role in the development of inflammatory response and brain damage. Although elevated glucose levels reliably predicted the outcome, they might be the consequence of increased plasma levels and reflect general stress rather than CNS pathophysiology. The CSF findings of WNV encephalomyelitis patients are non-specific and variable but facilitate the differential diagnosis.

Comparative accuracy and precision of two commercial laboratory analyzers for the quantification of ammonia in cerebrospinal fluid.

BACKGROUND: Hyperammonemia is one of the contributing factors of hepatic encephalopathy (HE). Although blood ammonia concentrations are frequently measured in patients suspected of HE, systemic levels do not necessarily reflect the amount of ammonia in the central nervous system. Measuring ammonia in cerebrospinal fluid (CSF) can help to understand HE better and potentially improve the diagnosis and follow-up of patients with HE. OBJECTIVES: The objectives of this technical report were to evaluate the accuracy and precision of two commercial blood ammonia analyzers (Catalyst Dx, CatDX and Pocket Chem BA, PocBA) to measure CSF ammonia concentrations. METHODS: A pool of normal equine CSF was spiked with concentrated ammonia, and a series of six spiked samples were measured in parallel with both CatDx and PocBA. RESULTS: CatDx and PocBA data correlated excellently with but differed significantly from the spiked ammonia concentrations. These differences were smaller when ammonia CSF concentrations were measured with the PocBA than with the CatDx. In addition, values obtained with the PocBA were more precise than those measured with the CatDx, especially for low ammonia concentrations. CONCLUSION: This in-house comparative study shows that ammonia concentrations in spiked equine CSF correlate well with those measured by two commercial blood ammonia analyzers. Nevertheless, concentrations obtained with the PocBA are more accurate and more precise than those obtained with the CatDx, making the former device the preferred choice for clinical veterinary applications.

Viruses in Horses with Neurologic and Respiratory Diseases.

Metagenomics was used to identify viral sequences in the plasma and CSF (cerobrospinal fluid) of 13 horses with unexplained neurological signs and in the plasma and respiratory swabs of 14 horses with unexplained respiratory signs. Equine hepacivirus and two copiparvoviruses (horse parvovirus-CSF and a novel parvovirus) were detected in plasma from neurological cases. Plasma from horses with respiratory signs contained the same two copiparvoviruses plus equine pegivirus D and respiratory swabs contained equine herpes virus 2 and 5. Based on genetic distances the novel copiparvovirus qualified as a member of a new parvovirus species we named Eqcopivirus. These samples plus another 41 plasma samples from healthy horses were tested by real-time PCRs for multiple equine parvoviruses and hepacivirus. Over half the samples tested were positive for one to three viruses with eqcopivirus DNA detected in 20.5%, equine hepacivirus RNA and equine parvovirus-H DNA in 16% each, and horse parvovirus-CSF DNA in 12% of horses. Comparing viral prevalence in plasma none of the now three genetically characterized equine parvoviruses (all in the copiparvovirus genus) was significantly associated with neurological and respiratory signs in this limited sampling.

Analysis of neurofilament concentration in healthy adult horses and utility in the diagnosis of equine protozoal myeloencephalitis and equine motor neuron disease.

Neurofilaments (NFs) are structural proteins of neurons that are released in significant quantities in the cerebrospinal fluid and blood as a result of neuronal degeneration or axonal damage. Therefore, NFs have potential as biomarkers for neurologic disorders. Neural degeneration increases with age and has the potential to confound the utility of NFs as biomarkers in the diagnosis of neurologic disorders. We investigated this relationship in horses with and without neurological diagnosis. While controlling for horse type (draft, pleasure, and racing), we evaluated the relationship between serum heavy-chain phosphorylated neurofilaments (pNF-H) and age, sex, and serum vitamin E concentrations. Serum pNF-H concentrations increased by 0.002 ng/ml for each year increase in age. There were significant differences in the serum pNF-H concentration among the type of activity performed by the horse. The highest serum pNF-H concentration was found in horses performing heavy work activity (racehorse) and with lower serum pNF-H concentration found among light (pleasure riding) and moderate (draft) activity. There was no significant association between the pNF-H concentration and sex or vitamin E concentration. Serum pNF-H concentration was elevated among horses afflicted with EMND and EPM when compared with control horses without evidence of neurologic disorders. Accordingly, serum pNF-H concentration can serve as a useful biomarker to complement the existing diagnostic work-up of horses suspected of having EPM or EMND.

Melarsomine hydrochloride (Cymelarsan®) fails to cure horses with Trypanosoma equiperdum OVI parasites in their cerebrospinal fluid.

The aim of this study was to evaluate the ability of melarsomine hydrochloride (Cymelarsan®) to cure horses suffering from a nervous form of dourine, a sexually-transmitted disease caused by Trypanosoma equiperdum. The recently described experimental model for assessing drug efficacy against horse trypanosomosis allowed us to obtain eight horses (Welsh pony mares) infected by T. equiperdum with parasites in their cerebrospinal fluid. The Cymelarsan® treatment evaluated consisted of the daily administration of 0.5 mg/kg of Cymelarsan® over 7 days. Two control horses remained untreated, three horses received the treatment 36 days p.i. and three horses received the treatment 16 days p.i. Following treatment, we observed parasite clearance in blood, stabilization of rectal temperature and a relative improvement in the mean packed cell volume levels for all treated horses. However, live parasites were later observed again in the CSF of all treated horses. Our results indicate the inability of Cymelarsan® to reach Trypanozoon located in the central nervous system of infected horses and thus discourage the use of Cymelarsan® to treat animals suffering from a nervous form of dourine.

Phosphorylated neurofilament H (pNF-H) as a potential diagnostic marker for neurological disorders in horses.

The current study aimed at the investigating the potential use of phosphorylated neurofilament H (pNF-H) as a diagnostic biomarker for neurologic disorders in the horse. Paired serum and cerebrospinal fluid (CSF) samples (n=88) and serum only (n=30) were obtained from horses diagnosed with neurologic disorders and clinically healthy horses as control. The neurologic horses consisted of equine protozoal myeloencephalitis (EPM) (38 cases) and cervical vertebral malformation (CVM) (23 cases). Levels of pNF-H were determined using an ELISA. The correlation between CSF and serum concentrations of pNF-H was evaluated using Spearman's Rank test and the significance of the difference among the groups was assessed using a nonparametric test. Horses had higher pNF-H levels in the CSF than serum. Horses afflicted with EPM had significantly higher serum pNF-H levels in comparison to controls or CVM cases. The correlation between CSF and serum pNF-H levels was poor in both the whole study population and among subgroups of horses included in the study. There was significant association between the likelihood of EPM and the concentrations of pNF-H in either the serum or CSF. These data suggest that pNF-H could be detected in serum and CSF samples from neurologic and control horses. This study demonstrated that pNF-H levels in serum and CSF have the potential to provide objective information to help in the early diagnosis of horses afflicted with neurologic disorders.

Cartilage oligomeric matrix protein neoepitope in the synovial fluid of horses with acute lameness: A new biomarker for the early stages of osteoarthritis.

BACKGROUND: Clinical tools to diagnose the early changes of osteoarthritis (OA) that occur in the articular cartilage are lacking. OBJECTIVES: We sought to identify and quantify a novel cartilage oligomeric matrix protein (COMP) neoepitope in the synovial fluid from the joints of healthy horses and those with different stages of OA. STUDY DESIGN: In vitro quantitative proteomics and assay development with application in synovial fluids samples obtained from biobanks of well-characterised horses. METHODS: Articular cartilage explants were incubated with or without interleukin-1ß for 25 days. Media were analysed via quantitative proteomics. Synovial fluid was obtained from either normal joints (n = 15) or joints causing lameness (n = 17) or with structural OA lesions (n = 7) and analysed for concentrations of the COMP neoepitope using a custom-developed inhibition enzyme-linked immunosorbent assay (ELISA). Explants were immunostained with polyclonal antibodies against COMP and the COMP neoepitopes. RESULTS: Semitryptic COMP peptides were identified and quantified in cell culture media from cartilage explants. A rabbit polyclonal antibody was raised against the neoepitope of the N-terminal portion of one COMP fragment (sequence SGPTHEGVC). An inhibition ELISA was developed to quantify the COMP neoepitope in synovial fluid. The mean concentration of the COMP neoepitope significantly increased in the synovial fluid from the joints responsible for acute lameness compared with normal joints and the joints of chronically lame horses and in joints with chronic structural OA. Immunolabelling for the COMP neoepitope revealed a pericellular staining in the interleukin-1ß-stimulated explants. MAIN LIMITATIONS: The ELISA is based on polyclonal antisera rather than a monoclonal antibody. CONCLUSIONS: The increase in the COMP neoepitope in the synovial fluid from horses with acute lameness suggests that this neoepitope has the potential to be a unique candidate biomarker for the early molecular changes in articular cartilage associated with OA.

Detection and DNA quantification of Enterococcus casseliflavus in a foal with septic meningitis.

CASE DESCRIPTION A 3-month-old 180-kg (396-lb) Hanoverian colt was examined because of fever, lethargy, inappetence, drooping of the left ear, and stiff neck posture. Initial treatment included empirical antimicrobial treatment and NSAIDs. CLINICAL FINDINGS Initial findings were consistent with CNS anomalies. Endoscopy revealed hyperemia, ecchymosis, and some mucopurulent exudate in the right guttural pouch. Hematologic findings were consistent with neutrophilic inflammation. On the third day of hospitalization, severe neurologic signs were observed. Computed tomography of the skull revealed a comminuted fracture of the axial aspect of the right mandibular condyle. Examination of CSF revealed turbidity, xanthochromia, and intracellular and extracellular cocci, consistent with septic meningitis. After DNA extraction from blood and CSF, sequenced products from a PCR assay for the bacterial 16S rRNA gene were 99% identical to Enterococcus casseliflavus. Microbial culture of CSF and blood samples yielded bacteria with Enterococcus spp morphology; antimicrobials were selected on the basis of susceptibility testing that identified the isolate as vancomycin resistant. A quantitative PCR assay was used to estimate Enterococcus DNA concentrations in CSF and blood. TREATMENT AND OUTCOME Treatment for E casseliflavus meningitis, including trimethoprim-sulfadiazine and ampicillin sodium administration, resulted in resolution of clinical signs. Culture of CSF and blood samples after 12 days of the targeted treatment yielded no growth. CLINICAL RELEVANCE To the authors' knowledge, this was the first report of E casseliflavus meningitis in a horse. Treatment was successful; vancomycin-resistant enterococci can be a clinical problem and may potentially be zoonotic.

Blood and Cerebrospinal Fluid α-Tocopherol and Selenium Concentrations in Neonatal Foals with Neuroaxonal Dystrophy.

BACKGROUND: Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (NAD/EDM) is a neurodegenerative disorder affecting genetically predisposed foals maintained on α-tocopherol (α-TP)-deficient diet. OBJECTIVE: Intramuscular α-TP and selenium (Se) administration at 4 days of age would have no significant effect on serum or cerebrospinal fluid (CSF) α-TP in healthy foals. Serum and CSF α-TP, but not Se, would be significantly decreased in NAD/EDM-affected foals during first year of life. ANIMALS: Fourteen Quarter horse foals; 10 healthy foals supplemented with 0.02 mL/kg injectable α-TP and Se (n = 5) or saline (n = 5) at 4 days of age and 4 unsupplemented NAD/EDM-affected foals. METHODS: Complete neurologic examinations were performed, blood and CSF were collected before (4 days of age) and after supplementation at 10, 30, 60, 120, 180, 240, and 360 days of age. Additional blood collections occurred at 90, 150, 210, and 300 days. At 540 days, NAD/EDM-affected foals and 1 unsupplemented healthy foal were euthanized and necropsies performed. RESULTS: Significant decreases in blood, CSF α-TP and Se found in the first year of life in all foals, with most significant changes in serum α-TP from 4-150 days. Dam α-TP and Se significantly influenced blood concentrations in foals. Injection of α-TP and Se did not significantly increase CSF Se, blood or CSF α-TP in healthy foals. NAD/EDM-affected foals had significantly lower CSF α-TP through 120 days. CONCLUSIONS AND CLINICAL IMPORTANCE: Injection of α-TP and Se at 4 days of age does not significantly increase blood or CSF α-TP. Despite all 14 foals remaining deficient in α-TP, only the 4 genetically predisposed foals developed NAD/EDM.

Ultrasound-guided atlanto-occipital puncture for cerebrospinal fluid analysis on the standing horse.

The atlanto-occipital site (AO) is convenient for retrieving an adequate volume and quality of cerebrospinal fluid (CSF) in the diagnosis of neurological disease in horses. However, general anaesthesia is not always possible for horses displaying severe neurological signs, or for economical reasons. The objectives of the present work were to determine the feasibility and safety of ultrasound-guided CSF puncture at the AO site on the standing horse. Seven horses (six healthy and one mildly ataxic) were sedated with acepromazine (0.02 mg/kg bodyweight intravenously or 0.04 mg/kg bodyweight intramuscularly) and detomidine (0.01 mg/kg bodyweight intravenously), and placed in stocks or in a recovery stall with the head kept on a headstand. Puncture was performed by ultrasonographic guidance with a parasagittal technique, as previously described, using a 20 g, 3.5 inch spinal needle. In all horses, no adverse reaction was observed when crossing the dura mater and 20 ml of CSF was rapidly retrieved without any blood contamination. Ultrasound-guided CSF puncture can be performed easily at the AO site on a healthy standing horse. Regarding the potential risk of this procedure, safety measures and close observation are essential. Further studies on a larger amount of ataxic horses are also required before considering this technique as an alternative option for CSF puncture.

Meningitis and meningoencephalomyelitis in horses: 28 cases (1985-2010).

OBJECTIVE: To describe clinical and clinicopathologic findings and outcome of horses with meningitis and meningoencephalomyelitis. DESIGN: Retrospective case series. ANIMALS: 28 horses. PROCEDURES: Medical records of horses admitted to the hospital during a 25-year period were reviewed. Horses with a definitive diagnosis of meningitis or meningoencephalomyelitis were included in this study. Information extracted from the medical records included signalment, history, reason for admission, clinical signs, results of clinicopathologic testing and diagnostic procedures, treatment, outcome, and necropsy findings. RESULTS: 22 horses had confirmed infectious disease (19 bacterial, 2 parasitic, and 1 fungal), 4 had suspected infectious disease on the basis of CSF cytologic examination findings, and 2 had noninfectious meningitis or meningoencephalomyelitis. Trauma of the head and vertebral column with disruption of the blood-brain barrier and local ascending or hematogenous spread were the most common routes of infection. Common neurologic signs included abnormal mental status, cranial nerve deficits, vestibular dysfunction, ataxia, tetraparesis, and apparent neck pain. Common hematologic abnormalities included leukocytosis, neutrophilia, lymphopenia, and hyperfibrinogenemia. Cytologic examination of CSF samples revealed moderate to marked suppurative inflammation. Mortality rate was 96.4%. Microbial culture of CSF yielded bacterial growth in 15 of 23 horses (before death [2 horses], after death [11], and both [2]). CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that meningitis and meningoencephalomyelitis are uncommon disorders in horses. Infectious disease was more common than noninfectious disease. Local trauma, ascending infection, or hematogenous spread of infection were the most common causes of meningitis or meningoencephalomyelitis. Neurologic deficits, neutrophilia, lymphopenia, hyperfibrinogenemia, and CSF with neutrophilic pleocytosis were common findings in affected horses.

Ultrasound-guided cervical centesis to obtain cerebrospinal fluid in the standing horse.

Horses with intracranial lesions and severe ataxia are not good anesthesia candidates; however, only one method to obtain cerebrospinal fluid (CSF) from the cervical region in a standing horse has been reported. This method is not performed routinely due to the difficulty for sample acquisition. Our hypothesis is that standing cervical centesis can be performed in horses without complication. Ultrasound-guided centesis of the CSF between C1 and C2 in 11 clinically normal horses and two horses with neurologic signs were performed. Horses were sedated and ultrasound was used to identify the subarachnoid space and spinal cord between C1 and C2. With ultrasound guidance, a needle was introduced into the dorsal aspect of the subarachnoid space using a lateral approach. Ten milliliters of CSF was obtained and analyzed. Two normal horses in this study had moderate red blood cell contamination in the CSF (940 and 612 RBC/microl). One horse had 11 RBC/microl and the remaining horses had < 4 RBC/microl. The total procedure time was approximately 2 min. No reaction was observed and no complications were detected up to 48 h after the procedure. Ultrasound-guided centesis between C1 and C2 is a rapid procedure that causes minimal to no reaction in standing, sedated horses used in this study. The use of ultrasound to guide a standing C1-2 centesis of the subarachnoid space provides an additional route to obtain CSF for analysis in the equine patient.

Effect of intermittent oral administration of ponazuril on experimental Sarcocystis neurona infection of horses.

OBJECTIVE: To evaluate the effect of intermittent oral administration of ponazuril on immunoconversion against Sarcocystis neurona in horses inoculated intragastrically with S neurona sporocysts. ANIMALS: 20 healthy horses that were seronegative for S neurona-specific IgG. PROCEDURES: 5 control horses were neither inoculated with sporocysts nor treated. Other horses (5 horses/group) each received 612,500 S neurona sporocysts via nasogastric tube (day 0) and were not treated or were administered ponazuril (20 mg/kg, PO) every 7 days (beginning on day 5) or every 14 days (beginning on day 12) for 12 weeks. Blood and CSF samples were collected on day - 1 and then every 14 days after challenge for western blot assessment of immunoconversion. Clinical signs of equine protozoal myeloencephalitis (EPM) were monitored, and tissues were examined histologically after euthanasia. RESULTS: Sera from all challenged horses yielded positive western blot results within 56 days. Immunoconversion in CSF was detected in only 2 of 5 horses that were treated weekly; all other challenged horses immunoconverted within 84 days. Weekly administration of ponazuril significantly reduced the antibody response against the S neurona 17-kd antigen in CSF. Neurologic signs consistent with EPM did not develop in any group; likewise, histologic examination of CNS tissue did not reveal protozoa or consistent degenerative or inflammatory changes. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of ponazuril every 7 days, but not every 14 days, significantly decreased intrathecal anti-S neurona antibody responses in horses inoculated with S neurona sporocysts. Protocols involving intermittent administration of ponazuril may have application in prevention of EPM.

Effects of blood contamination of cerebrospinal fluid on results of indirect fluorescent antibody tests for detection of antibodies against Sarcocystis neurona and Neospora hughesi.

The purpose of this study was to determine the effect of blood contamination of cerebrospinal fluid (CSF) on the results of indirect fluorescent antibody tests (IFATs) for Sarcocystis neurona and Neospora hughesi. The in vitro study used antibody-negative CSF collected from non-neurologic horses immediately after euthanasia and blood samples from 40 healthy horses that had a range of IFAT antibody titers against S. neurona and N. hughesi. Serial dilutions of whole blood were made in seronegative CSF to generate blood-contaminated CSF with red blood cell (RBC) concentrations ranging from 10 to 100,000 RBCs/microl. The blood-contaminated CSF samples were then tested for antibodies against both pathogens using IFAT. Blood contamination of CSF had no detectable effect on IFAT results for S. neurona or N. hughesi at any serologic titer when the RBC concentration in CSF was <10,000 RBCs/microl. At concentrations of 10,000-100,000 RBCs/microl of CSF, positive CSF results (IFAT titer >or=5) for S. neurona and N. hughesi were detected only when the corresponding serum titers were >or=160 and >or=80, respectively. The IFAT performed on CSF is reliable for testing horses for equine protozoal myeloencephalitis caused by S. neurona or N. hughesi, even when blood contamination causes the RBC concentration in CSF to be up to 10,000 RBCs/microl.

Ultrasound-assisted collection of cerebrospinal fluid from the lumbosacral space in equids.

OBJECTIVE: To describe ultrasonographic landmarks for use in collection of CSF from the lumbosacral region in equids. DESIGN: Prospective study. ANIMALS: 37 equids (27 with neurologic disease and 10 with nonneurologic disease). PROCEDURES: Standing equids (n = 17) were sedated with detomidine hydrochloride (0.006 to 0.01 mg/kg [0.003 to 0.005 mg/lb], IV) followed by butorphanol tartrate (0.01 mg/kg, IV) and restrained with a nose twitch for collection of CSF. The CSF was collected from 20 laterally recumbent equids (10 sedated and 10 immediately after euthanasia). Anatomic landmarks were identified ultrasonographically. Height at the dorsal point of the shoulders, body weight, depth of the spinal needle, number of attempts to collect CSF, and cytologic evaluation of CSF were recorded. RESULTS: Lumbosacral puncture cranial to the cranial border of the most superficial location of both tuber sacrale along the midline was consistently successful for CSF collection (35/37 equids). Two horses had anatomic abnormalities that precluded CSF collection. Mean number of attempts to collect CSF per animal was 1.1. Height and body weight were strongly correlated with needle depth for CSF collection. Pelvic and sacral displacement was observed in several laterally recumbent animals, which resulted in discrepancies of the midline between the cranial and caudal aspects of the vertebral column. In most equids, the spinal needle was aligned on the midline of the caudal aspect of the vertebral column. CONCLUSIONS AND CLINICAL RELEVANCE: Ultrasonography was a useful aid for collection of CSF from the lumbosacral space and decreased the risk of repeated trauma and contamination in equids.