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Topic:Equine Protozoal Myeloencephalitis

Equine Protozoal Myeloencephalitis (EPM) is a neurological disease affecting horses, caused by the protozoan parasites Sarcocystis neurona or, less commonly, Neospora hughesi. The disease occurs when these parasites infect the central nervous system, leading to a range of neurological symptoms. Horses with EPM may exhibit signs such as ataxia, muscle weakness, and incoordination. Diagnosis can be challenging and typically involves a combination of clinical assessment, serological testing, and sometimes cerebrospinal fluid analysis. This page compiles peer-reviewed research studies and scholarly articles that explore the pathogenesis, diagnostic approaches, and management strategies for Equine Protozoal Myeloencephalitis in horses.
Evaluation and comparison of an indirect fluorescent antibody test for detection of antibodies to Sarcocystis neurona, using serum and cerebrospinal fluid of naturally and experimentally infected, and vaccinated horses.
The Journal of parasitology    May 29, 2004   Volume 90, Issue 2 379-386 doi: 10.1645/GE-3263
Duarte PC, Daft BM, Conrad PA, Packham AE, Saville WJ, MacKay RJ, Barr BC, Wilson WD, Ng T, Reed SM, Gardner IA.The objectives of this study were to evaluate the accuracy of the indirect fluorescent antibody test (IFAT) using serum and cerebrospinal fluid (CSF) of horses naturally and experimentally infected with Sarcocystis neurona, to assess the correlation between serum and CSF titers, and to determine the effect of S. neurona vaccination on the diagnosis of infection. Using receiver-operating characteristic analysis, the areas under the curve for the IFAT were 0.97 (serum) and 0.99 (CSF). Sensitivity and specificity were 83.3 and 96.9% (serum, cutoff 80) and 100 and 99% (CSF, cutoff 5), respectively...
Cell-mediated immune responses in horses with equine protozoal myeloencephalitis.
The Journal of parasitology    May 29, 2004   Volume 90, Issue 2 428-430 doi: 10.1645/GE-3289RN
Spencer JA, Ellison SE, Guarino AJ, Blagburn BL.Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome seen in horses from the Americas and is mainly caused by Sarcocystis neurona. Cell-mediated immune responses to mitogens have been shown to be reduced in horses with EPM, although it is not known whether the parasite causes this immunosuppression or if the immunosuppression is required for disease manifestation. Recently, a 29-kDa surface antigen from S. neurona merozoites was identified as being highly immunodominant on Western blot. This antigen has been sequenced and cloned, and the expressed protein has been named SnSAG1. Is...
Sarcocystis neurona-specific immunoglobulin G in the serum and cerebrospinal fluid of horses administered S neurona vaccine.
Journal of veterinary internal medicine    February 10, 2004   Volume 18, Issue 1 98-103 doi: 10.1892/0891-6640(2004)18<98:snigit>2.0.co;2
Witonsky S, Morrow JK, Leger C, Dascanio J, Buechner-Maxwell V, Palmer W, Kline K, Cook A.A vaccine against Sarcocystis neurona, which induces equine protozoal myeloencephalitis (EPM), has received conditional licensure in the United States. A major concern is whether the immunoglobulin G (IgG) response elicited by the vaccine will compromise the use of Western blotting (WB) as a diagnostic tool in vaccinated horses with neurologic disease. Our goals were to determine if vaccination (1) causes seroconversion: (2) causes at least a transient increase in S neurona-specific IgG in the cerebrospinal fluid (CSF); and (3) induces an IgG response that can be differentiated from that induc...
Assessing the agreement of Western blot test results for paired serum and cerebrospinal fluid samples from horses tested for antibodies to Sarcocystis neuronaf.
Veterinary parasitology    August 26, 2003   Volume 115, Issue 3 233-238 doi: 10.1016/s0304-4017(03)00224-3
Rossano MG, Kaneene JB, Schott HC, Sheline KD, Mansfield LS.Equine protozoal myeloencephalitis (EPM) is a neurological disease of equids that is caused by infection of the central nervous system with Sarcocystis neurona. Veterinarians diagnose EPM by performing a neurological examination and by ordering Western blot tests for antibodies to S. neurona in the blood and/or cerebrospinal fluid (CSF). The negative predictive value of the Western blot test is generally accepted to be high for both serum and CSF. If the agreement between serum and CSF test results is strong, serum tests could be used to substitute for CSF tests in some cases. The purpose of t...
Clotrimazole, ketoconazole, and clodinafop-propargyl as potent growth inhibitors of equine Babesia parasites during in vitro culture.
The Journal of parasitology    July 26, 2003   Volume 89, Issue 3 604-606 doi: 10.1645/0022-3395(2003)089[0604:CKACAP]2.0.CO;2
Bork S, Yokoyama N, Matsuo T, Claveria FG, Fujisaki K, Igarashi I.The antifungal agents clotrimazole (CLT) and ketoconazole (KC) and the herbicide clodinafop-propargyl (CP) inhibit growth of Plasmodium sp., Toxoplasma sp., and Trypanosoma sp. In the present study, we evaluated these drugs against the in vitro growth of the equine protozoan parasites Babesia equi and B. caballi. Clotrimazole (IC50: 2 and 17 microM), KC (IC50: 6 and 22 microM), and CP (IC50: 450 and 354 microM) were effective growth inhibitors. Interestingly, intraerythrocytic KC-treated Babesia sp. were observed to be in immediate contact with the plasma fraction of the blood in electron micr...
First case report of Sarcocystis neurona-induced equine protozoal myeloencephalitis in Japan.
The Journal of veterinary medical science    July 18, 2003   Volume 65, Issue 6 757-759 doi: 10.1292/jvms.65.757
Katayama Y, Wada R, Kanemaru T, Sasagawa T, Uchiyama T, Matsumura T, Anzai T.Equine protozoal myeloencephalitis developed in a three-year-old male Thoroughbred racehorse imported from the United States. The animal showed astasia five days after the onset of ataxia. Histopathologically, focal nonpurulent myelitis accompanied by hemorrhage and perivascular infiltration was observed in the fourth and fifth cervical spinal cord. Immunohistochemically, shizonts were occasionally observed and were positive for anti-Sarcocystis neurona (S. neurona) antiserum. S. neurona-specific antibodies were detected in the serum and cerebrospinal fluid by Western blot. This is the first e...
Comparison of a serum indirect fluorescent antibody test with two Western blot tests for the diagnosis of equine protozoal myeloencephalitis. Duarte PC, Daft BM, Conrad PA, Packham AE, Gardner IA.A serum indirect fluorescent antibody test (IFAT) was compared with a Western blot (WB) and a modified Western blot (mWB) for diagnosis of equine protozoal myeloencephalitis (EPM). Using receiver-operating characteristic (ROC) analysis, the area under the curve of the IFAT was greater than the areaunder the curves of the WB and the mWB (P = 0.025 and P = 0.044, respectively). There was no statistically significant difference between the areas under the curves of the WBs (P > 0.05). On the basis of an arbitrarily chosen cut-off titer for a positive test result of 1:80 for the IFAT and interpret...
A herd-level analysis of risk factors for antibodies to Sarcocystis neurona in Michigan equids.
Preventive veterinary medicine    January 28, 2003   Volume 57, Issue 1-2 7-13 doi: 10.1016/s0167-5877(02)00192-7
Rossano MG, Kaneene JB, Marteniuk JV, Banks BD, Schott HC, Mansfield LS.Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by infection of the central nervous system with the protozoan parasite Sarcocystis neurona. A herd-level analysis of a cross-sectional study of serum antibodies to S. neurona in Michigan equids was conducted, using data collected in 1997 for study that included 1121 equids from 98 Michigan horse farms. Our objective was to identify specific herd-level risk factors associated with seropositivity. We tested associations between herd seroprevalence and various farm-management practices (including feed-s...
Experimental induction of equine protozoan myeloencephalitis (EPM) in the horse: effect of Sarcocystis neurona sporocyst inoculation dose on the development of clinical neurologic disease.
The Journal of parasitology    January 23, 2003   Volume 88, Issue 6 1164-1170 doi: 10.1645/0022-3395(2002)088[1164:EIOEPM]2.0.CO;2
Sofaly CD, Reed SM, Gordon JC, Dubey JP, Ogleebee MJ, Njoku CJ, Grover DL, Saville WJ.The effect of inoculation dose of Sarcocystis neurona sporocysts on the development of clinical neurologic disease in horses was investigated. Twenty-four seronegative weanling horses were subjected to the natural stress of transport and then randomly assigned to 6 treatment groups of 4 horses each. Horses were then immediately inoculated with either 10(2), 10(3), 10(4), 10(5), or 10(6) S. neurona sporocysts or placebo using nasogastric tube and housed indoors. Weekly neurologic examinations were performed by a blinded observer. Blood was collected weekly for antibody determination by Western ...
Reactivity against Sarcocystis neurona and Neospora by serum antibodies in healthy French horses from two farms with previous equine protozoal myeloencephalitis-like cases.
Veterinary parasitology    January 14, 2003   Volume 111, Issue 1 1-7 doi: 10.1016/s0304-4017(02)00346-1
Pitel PH, Lindsay DS, Caure S, Romand S, Pronost S, Gargala G, Mitchell SM, Hary C, Thulliez P, Fortier G, Ballet JJ.Sarcocystis neurona is considered a leading cause of equine protozoal myeloencephalitis (EPM), a common infectious neurological disease in horses in the Americas. EPM-like cases associated with S. neurona peptide reactive antibodies in Western blots were recently described in Normandy, France. In this report, antibodies reacting with S. neurona merozoites were detected using an agglutination assay at titers ranging from 50 to 500 in sera from 18/50 healthy horses from two farms with a previous EPM-like case. Higher values were found in older animals. Four out of six horses which traveled or st...
Sensitivity and specificity of western blot testing of cerebrospinal fluid and serum for diagnosis of equine protozoal myeloencephalitis in horses with and without neurologic abnormalities.
Journal of the American Veterinary Medical Association    October 9, 2002   Volume 221, Issue 7 1007-1013 doi: 10.2460/javma.2002.221.1007
Daft BM, Barr BC, Gardner IA, Read D, Bell W, Peyser KG, Ardans A, Kinde H, Morrow JK.To determine sensitivity and specificity of western blot testing (WBT) of CSF and serum for diagnosis of equine protozoal myeloencephalitis (EPM) in horses with and without neurologic abnormalities. Methods: Prospective investigation. Methods: 65 horses with and 169 horses without neurologic abnormalities. Methods: CSF and serum from horses submitted for necropsy were tested for Sarcocystis neurona-specific antibody with a WBT. Results of postmortem examination were used as the gold standard against which results of the WBT were compared. Results: Sensitivity of WBT of CSF was 87% for horses w...
Antigen-specific antibodies in cerebrospinal fluid after intramuscular injection of ovalbumin in horses.
Journal of veterinary internal medicine    September 27, 2002   Volume 16, Issue 5 588-592 doi: 10.1892/0891-6640(2002)0162.3.co;2
Furr M.Eighteen normal horses were assigned to 1 of 3 treatment groups to investigate the effects of IM or intrathecal (IT) administration of ovalbumin on serum and cerebrospinal fluid (CSF) antibody production. Horses of group 1 were injected intramuscularly with ovalbumin and adjuvant, while horses in treatment groups 2 and 3 received ovalbumin intrathecally or intravenously, followed by IM injection as in group 1. Serum and CSF antibody titers were tested in group I every 30 days for 4 months, while serum and CSF were collected in group 2 and 3 horses at postvaccination day 60. Horses of group 1 (...
Clinical diagnosis of equine protozoal myeloencephalitis (EPM).
Journal of veterinary internal medicine    September 27, 2002   Volume 16, Issue 5 618-621 doi: 10.1892/0891-6640(2002)0162.3.co;2
Furr M, MacKay R, Granstrom D, Schott H, Andrews F.The research article is about the study and in-depth understanding of diagnosing equine protozoal myeloencephalitis (EPM), a disease that impacts horses’ neurological functions. Introduction The main focus of the research […]
Detection of antibodies against Sarcocystis neurona in cerebrospinal fluid from clinically normal neonatal foals.
Journal of the American Veterinary Medical Association    July 20, 2002   Volume 220, Issue 2 208-211 doi: 10.2460/javma.2002.220.208
Cook AG, Maxwell VB, Donaldson LL, Parker NA, Ward DL, Morrow JK.To determine whether antibodies against Sarcocystis neurona could be detected in CSF from clinically normal neonatal (2 to 7 days old) and young (2 to 3 months old) foals. Methods: Prospective study. Methods: 15 clinically normal neonatal Thoroughbred foals. Methods: Serum and CSF samples were obtained from foals at 2 to 7 days of age and tested for antibodies against S. neurona by means of western blotting. Serum samples from the mares were also tested for antibodies against S. neurona. Additional CSF and blood samples were obtained from 5 foals between 13 and 41 days after birth and between ...
Folate deficiency during treatment with orally administered folic acid, sulphadiazine and pyrimethamine in a horse with suspected equine protozoal myeloencephalitis (EPM).
Equine veterinary journal    July 11, 2002   Volume 34, Issue 3 311-316 doi: 10.2746/042516402776186128
Piercy RJ, Hinchcliff KW, Reed SM.No abstract available
Effect of sulfadiazine and pyrimethamine on selected physiologic and performance parameters in athletically conditioned thoroughbred horses during an incremental exercise stress test.
Veterinary therapeutics : research in applied veterinary medicine    June 7, 2002   Volume 3, Issue 1 49-63 
Colahan PT, Bailey JE, Johnson M, Rice BL, Chou CC, Cheeks JP, Jones GL, Yang M.Following the regimen used to treat equine protozoal myeloencephalitis, sulfadiazine (20 mg/kg) and pyrimethamine (1mg/kg) were administered orally once daily to 12 physically conditioned Thoroughbred horses for 4 consecutive days. The horses were randomly assigned to two test groups in a crossover design, with each horse serving as its own control. A stepwise exercise stress test was conducted to exhaustion. No effect on athletic performance was observed, and only marginal effects were noted in some hematologic and serochemical measurements, including decreased total white blood cell counts, ...
Reduced levels of nitric oxide metabolites in cerebrospinal fluid are associated with equine protozoal myeloencephalitis.
Clinical and diagnostic laboratory immunology    May 3, 2002   Volume 9, Issue 3 605-610 doi: 10.1128/cdli.9.3.605-610.2002
Njoku CJ, Saville WJ, Reed SM, Oglesbee MJ, Rajala-Schultz PJ, Stich RW.Equine protozoal myeloencephalitis (EPM) is a disease of horses that is primarily associated with infection with the apicomplexan Sarcocystis neurona. Infection with this parasite alone is not sufficient to induce the disease, and the mechanism of neuropathogenesis associated with EPM has not been reported. Nitric oxide (NO) functions as a neurotransmitter, a vasodilator, and an immune effector and is produced in response to several parasitic protozoa. The purpose of this work was to determine if the concentration of NO metabolites (NO(x)(-)) in the cerebrospinal fluid (CSF) is correlated with...
Characterization of monoclonal antibodies developed against Sarcocystis neurona.
Parasitology research    March 8, 2002   Volume 88, Issue 6 501-506 doi: 10.1007/s00436-002-0602-y
Marsh AE, Hyun C, Barr BC, Tindall R, Lakritz J.Equine protozoal myeloencephalitis (EPM), caused by a protozoal parasite infection of the central nervous system, is the most commonly diagnosed neurologic disease of horses in North America. In specific regions of the United States approximately 50% of the horse population is seropositive to Sarcocystis neurona. However, not all seropositive horses develop clinical signs. Detailed clinical examination, along with cerebrospinal fluid antibody evaluation are often used to diagnose EPM. Postmortem evaluation of the brain stem and spinal cord for histopathologic lesions compatible with nonsuppura...
Detection of Sarcocystis neurona antibodies in French horses with neurological signs.
International journal for parasitology    February 19, 2002   Volume 32, Issue 4 481-485 doi: 10.1016/s0020-7519(01)00370-8
Pitel PH, Pronost S, Gargala G, Anrioud D, Toquet MP, Foucher N, Collobert-Laugier C, Fortier G, Ballet JJ.No abstract available
Molecular characterisation of a major 29 kDa surface antigen of Sarcocystis neurona.
International journal for parasitology    January 29, 2002   Volume 32, Issue 2 217-225 doi: 10.1016/s0020-7519(01)00324-1
Ellison SP, Omara-Opyene AL, Yowell CA, Marsh AE, Dame JB.A gene encoding a major 29 kDa surface antigen from Sarcocystis neurona, the primary causative agent of equine protozoal myeloencephalitis (EPM), was cloned, sequenced, and expressed as a recombinant protein. A cDNA library was prepared in the expression vector lambda ZAP from polyA+mRNA isolated from S. neurona merozoites cultivated in vitro. Random sequencing of 96 clones identified a clone of an abundant transcript having a translated amino acid sequence with 30% identity to the 31-kDa surface antigen of Sarcocystis muris cyst merozoites. Southern blot analysis indicated that the correspond...
Lymphocyte responses and immunophenotypes in horses with Sarcocystis neurona infection.
Equine veterinary journal    January 5, 2002   Volume 33, Issue 7 726-729 doi: 10.2746/042516401776249255
Tornquist SJ, Boeder LJ, Mattson DE, Cebra CK, Bildfell RJ, Hamir AN.No abstract available
Transforming growth factor beta concentrations and interferon gamma responses in cerebrospinal fluid of horses with equine protozoal myeloencephalitis.
Equine veterinary journal    January 5, 2002   Volume 33, Issue 7 721-725 doi: 10.2746/042516401776249408
Furr M, Pontzer C.The following experiment was performed to test the hypothesis that transforming growth factor beta (TGF-beta) concentration varies in the cerebrospinal fluid and serum of horses with EPM and to determine if cerebrospinal fluid (CSF) alters the interferon-gamma (IFN-gamma) rersponse of equine peripheral blood mononuclear cells (PBMCs). The concentration of transforming growth factor-beta (TGF-beta2) was investigated in the serum and cerebrospinal fluid (CSF) of 18 horses (9 normal, 9 affected with equine protozoal myeloencephalitis [EPM]). The TGF-beta2 assay was validated in a group of 6 norma...
Risk factors associated with the presence of Sarcocystis neurona sporocysts in opossums (Didelphis virginiana).
Veterinary parasitology    January 5, 2002   Volume 102, Issue 3 179-184 doi: 10.1016/s0304-4017(01)00549-0
Rickard LG, Black SS, Rashmir-Raven A, Hurst G, Dubey JP.Sarcocystis neurona is the most important cause of equine protozoal myeloencephalitis (EPM) in horse in the Americas. The only known definitive host for this parasite in the United States is the opossum (Didelphis virginiana); however, despite the importance of the disease, the epidemiology of the parasite in the definitive host is poorly understood. To begin addressing these data gaps, potential risk factors were evaluated for their association with the presence of sporocysts of S. neurona in opossums live-trapped in March 1999 and November 1999 to May 2000. Sporocysts of S. neurona were foun...
Mice lacking the gene for inducible or endothelial nitric oxide are resistant to sporocyst induced Sarcocystis neurona infections.
Veterinary parasitology    January 5, 2002   Volume 103, Issue 4 315-321 doi: 10.1016/s0304-4017(01)00555-6
Rosypal AC, Lindsay DS, Duncan R, Ahmed SA, Zajac AM, Dubey JP.Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome in horses from the Americas and is usually caused by infection with the apicomplexan parasite, Sarcocystis neurona. Little is known about the role of immunobiological mediators to this parasite. Nitric oxide (NO) is important in resistance to many intracellular parasites. We, therefore, investigated the role of inducible and endothelial NO in resistance to clinical disease caused by S. neurona in mice. Groups of interferon-gamma gene knockout (IFN-gamma-KO) mice, inducible nitric oxide synthase gene knockout (iNOS-KO) mice, endo...
Lymphocyte phenotype subsets in the cerebrospinal fluid of normal horses and horses with equine protozoal myeloencephalitis.
Veterinary therapeutics : research in applied veterinary medicine    October 1, 2001   Volume 2, Issue 4 317-324 
Furr M, Pontzer C, Gasper P.The percentages of T-lymphocytes, lymphocyte subsets CD4+ and CD8+ T-cells, and lymphocyte adhesion molecule CD11a/CD18 were determined in the peripheral blood and cerebrospinal fluid (CSF) of seven normal horses and four horses with equine protozoal myeloencephalitis (EPM) using flow cytometry. There was a greater percentage of CD5+ cells in the CSF (79.0%) than in peripheral blood (67.0%), although this did not achieve statistical significance. Furthermore, the lymphocyte population in CSF comprises a significantly greater (P = .01) percentage of CD8+ T-cells, resulting in a decrease of the ...
Suspected protozoal myeloencephalitis in a two-month-old colt.
The Veterinary record    September 18, 2001   Volume 149, Issue 9 269-273 doi: 10.1136/vr.149.9.269
Gray LC, Magdesian KG, Sturges BK, Madigan JE.A two-month-old Appaloosa colt developed neurological signs shortly after birth involving deficits affecting cranial nerves IV, VII, VIII, IX, X and XII, and possibly nerve VI. The most likely differential diagnoses were congenital anomalies, meningoencephalitides, trauma or nutritional causes. The foal was investigated by the analysis of cerebrospinal fluid (CSF), electromyelography (EMG), brain auditory evoked responses, magnetic resonance imaging (MRI), peripheral nerve biopsy, and Western blot analysis for the presence of intrathecal antibodies to Sarcocystis neurona, the causative agent o...
Safety of ponazuril 15% oral paste in horses.
Veterinary therapeutics : research in applied veterinary medicine    July 1, 2001   Volume 2, Issue 3 223-231 
Kennedy T, Campbell J, Selzer V.Ponazuril, a triazine-derivative compound, is proposed as a treatment for equine protozoal myeloencephalitis caused by Sarcocystis neurona. Ponazuril 15% oral paste was administered to 24 horses at 0, 10, or 30 mg/kg body weight for either 28 or 56 days, representing zero, two, and six times the proposed dosage rate and one and two times the recommended duration of treatment, respectively. Serum chemistry analysis, coagulation profile, and hematology measurements were performed weekly and necropsy evaluations, including histopathology, were conducted for all animals at the end of the study. Mi...
The effects of pyrantel tartrate on Sarcocystis neurona merozoite viability.
Veterinary therapeutics : research in applied veterinary medicine    July 1, 2001   Volume 2, Issue 3 268-276 
Kruttlin EA, Rossano MG, Murphy AJ, Vrable RA, Kaneene JB, Schott HC, Mansfield LS.Sarcocystis neurona is the etiologic agent of equine protozoal myeloencephalitis, a neurologic disease of horses. The present study was designed to test the hypothesis that pyrantel tartrate can kill S. neurona merozoites growing in equine dermal cell culture. Sarcocystis neurona merozoites were exposed to a range of concentrations of pyrantel tartrate or sodium tartrate ranging from 0.001 to 0.01 M. Merozoites were then placed onto equine dermal cell cultures and incubated for 2 weeks to check for viability. At 1 and 2 weeks after inoculation, plaque counts were compared between treatments an...
Efficacy of ponazuril 15% oral paste as a treatment for equine protozoal myeloencephalitis.
Veterinary therapeutics : research in applied veterinary medicine    July 1, 2001   Volume 2, Issue 3 215-222 
Furr M, Kennedy T, MacKay R, Reed S, Andrews F, Bernard B, Bain F, Byars D.Equine protozoal myeloencephalitis (EPM) is a neurologic disease of horses most commonly caused by the protozoan parasite Sarcocystis neurona. Until recently the only treatment option was the combination of a sulfonamide with pyrimethamine. The present study was performed to assess the efficacy of ponazuril, an anticoccidial triazine-based compound, as a treatment for naturally occurring EPM. One hundred one horses with EPM were randomly allocated to treatment with ponazuril 15% oral paste at either 5 or 10 mg/kg body weight for 28 consecutive days. Horses were evaluated clinically and by anal...
Cerebrospinal fluid and serum concentrations of ponazuril in horses.
Veterinary therapeutics : research in applied veterinary medicine    July 1, 2001   Volume 2, Issue 3 232-237 
Furr M, Kennedy T.Ponazuril was administered orally to 10 adult horses at 5 mg/kg body weight, once a day for 28 days. Blood was collected once a week from each horse from Days 0 through 35, daily from Days 35 through 42, and on Day 49. Cerebrospinal fluid (CSF) was also collected once a week from Day 0 through Day 49. Concentrations of ponazuril in the serum and CSF were determined, and pharmacokinetic calculations were performed. Ponazuril was readily absorbed following oral administration; and after 7 days of dosing, the serum concentration was 4.33 +/- 1.10 mg/L, and the mean CSF concentration was 0.162 +/-...