Determination of immunoglobulin A concentrations in the serum and cerebrospinal fluid of dogs: an estimation of its diagnostic value in canine steroid-responsive meningitis-arteritis

Previous studies on canine steroid-responsive meningitis-arteritis (SRMA) suggested that elevation of immunoglobulin A (IgA) concentrations in both serum and cerebrospinal fluid (CSF) is specific for SRMA throughout the different disease stages. Recent studies however have raised concerns about the value of this test. The purpose of this study was to investigate the diagnostic value of IgA concentration testing in paired CSF and serum samples. IgA concentrations of 525 paired canine CSF and serum samples were evaluated. Samples were obtained from dogs with SRMA (n=311) and dogs with miscellaneous conditions (n=214) such as other central nervous system (CNS) inflammatory diseases (n=34), CNS tumours (n=46), idiopathic epilepsy (n=42), intervertebral disc disease (n=46) and non-CNS diseases (n=46). Serum IgA concentrations were significantly higher in dogs with untreated SRMA compared to those with other diseases. IgA CSF concentrations were significantly higher in dogs with SRMA compared to other disease categories, with the exception of inflammatory CNS disease. The sensitivity for IgA concentrations in serum and CSF was 91% with a specificity of 78%. Analysis of 525 paired samples confirmed that IgA concentrations were higher in dogs with SRMA. Calculation of the diagnostic value of IgA concentration confirmed that the test is highly sensitive for SRMA. Testing paired CSF and serum samples for IgA is still recommended for the diagnosis of suspected cases of SRMA.     

Agarose gel electrophoresis of cerebrospinal fluid proteins of dogs after sample concentration using a membrane microconcentrator technique

BACKGROUND: Cerebrospinal fluid (CSF) is produced in the cerebral ventricles through ultrafiltration of plasma and active transport mechanisms. Evaluation of proteins in CSF may provide important information about the production of immunoglobulins within the central nervous system as well as possible disturbances in the blood-brain barrier. OBJECTIVE: The objective of this study was to measure the concentration and fractions of protein in CSF samples using a membrane microconcentrator technique followed by electrophoresis, and to compare the protein fractions obtained with those in serum. METHODS: CSF samples from 3 healthy dogs and 3 dogs with canine distemper virus infection were concentrated using a membrane microconcentrator having a 0.5 to 30,000 d nominal molecular weight limit (Ultrafree, Millipore, Billerica, MA, USA). Protein concentration was determined before and after concentration. Agarose gel electrophoresis was done on concentrated CSF samples, serum, and serial dilutions of one of the CSF samples. RESULTS: Electrophoretic bands were clearly identified in densitometer tracings in CSF samples with protein concentrations as low as 1.3 g/dL. The higher CSF protein concentration in dogs with distemper was mainly the result of increased albumin concentration. CONCLUSION: The microconcentrating method used in this study enables characterization of the main protein fractions in CSF by routine electrophoresis and may be useful for interpreting the underlying cause of changes in CSF protein concentrations.     

Blood–brain‐barrier disruption in chronic canine hypothyroidism

Background: Central nervous system (CNS) manifestations of hypothyroidism have been associated with cerebrovascular complications. Reports of cerebrospinal fluid (CSF) abnormalities are rare in hypothyroid dogs. Objective: The aim of this study was to determine if chronic hypothyroidism causes blood–brain‐barrier (BBB) abnormalities that are detectable using indirect CSF biomarkers. Methods: The study included 18 normal, euthyroid, female mixed‐breed dogs. Hypothyroidism was induced by ¹³¹iodine administration in 9 dogs; 9 served as untreated controls. Evaluations included physical and neurologic examination, complete CSF analysis, serum and CSF protein electrophoresis, measurement of plasma vascular endothelial growth factor (VEGF) and serum S‐100B concentrations, and calculation of CSF albumin quota (AQ) and were conducted at baseline and 6, 12, and 18 months after induction of hypothyroidism. Data were analyzed using repeated measures ANOVA. Results: At baseline, differences between groups were not detected for any variable. Throughout the study, controls dogs remained free of neurologic disease and had test variables that remained within reference intervals. Two hypothyroid dogs developed CNS signs during the study, and evidence of cerebrovascular disease was found at necropsy. At 12 and 18 months, the CSF total protein, VEGF, S‐100B, and fractional albumin concentrations, and AQ were significantly higher (P<.04) in hypothyroid dogs than controls. Among test variables assayed in serum or plasma, the only significant difference was a higher S‐100B concentration in hypothyroid dogs (P=.003) at 18 months. Conclusions: BBB integrity is disrupted in chronic hypothyroidism. Significant increases in CSF concentrations of VEGF and S100‐B in hypothyroid dogs indicate dysfunction in both endothelial and glial elements of the BBB.     

Evaluation of nephelometry for albumin measurement in serum and cerebrospinal fluid: experiences with an indwelling subarachnoidal catheter system for repetitive cerebrospinal fluid collection in horses.

The measurement of albumin concentrations in cerebrospinal fluid (CSF) and serum for albumin quotient (AQ) calculations in normal horses was performed by 2 methods: 1) total protein measurement, followed by electrophoresis of the samples to obtain an albumin percentage; and 2) albumin immunoprecipitation quantitated by nephelometry. The results of both methods correlated well, and nephelometry was chosen to determine the albumin concentrations in CSF samples obtained from an indwelling subarachnoidal catheter for daily sampling. Because the use of an indwelling catheter to collect repetitive CSF samples is a novel technique, routine cytological CSF analysis was performed along with daily clinical evaluation to ascertain the well-being of the horses. The catheters were placed in 2 horses for periods of 14 and 17 days. One horse exhibited pleocytosis on cytological evaluation of CSF on 2 occasions for a 1-2-day duration; however, the AQ showed a significant increase on only 1 occasion. The other horse had a normal cell count in CSF but showed 2 sudden changes in the AQ value; however, these values remained within the 95% confidence interval for AQ in horses. Albumin quotient values of the second horse were consistently below the lower range of the confidence interval. Results from this study indicate that nephelometry can be used for albumin determination in serum and CSF samples from horses. Furthermore, an indwelling subarachnoidal catheter system can provide serial CSF samples in horses, thus obviating the need for repetitive centesis for serial CSF sampling.     

Proteome analysis of cerebrospinal fluid in healthy beagles and canine encephalitis

We performed proteomics analysis of the cerebrospinal fluid (CSF) of healthy dogs and dogs with meningoencephalitis of unknown etiology (MUE). By comparing two-dimensional electrophoreses (2DE), an upregulated spot was found in MUE dogs. This protein was identified as a neuron-specific enolase (NSE) by analysis with MALDI-TOF mass spectrometry. In comparing dot blots using an antibody against NSE, the NSE levels in the CSF of MUE dogs was significantly higher than that of the controls. NSE is a diagnostic marker of neuroendocrine tumors, brain injury and spinal cord trauma in humans. It seems that the NSE concentration in the CSF is increased by cellular destruction in canine encephalitis. Though elevation of NSE may not be specific in canine encephalitis because the NSE level was increased in other CNS diseases, further study including measurement with serum is necessary.     

硝化应激在神经型犬瘟热疾病发展中的临床意义

为了研究硝化应激在神经型犬瘟热疾病发展中的作用,并探讨其对该病的诊断价值,选取犬瘟热临床病例113例,其中有神经症状组(NCD)共37例,无神经症状组(NNS)共76例,并设25例无犬瘟热健康犬作为对照组(CG)。就诊时分别采取血液和脑脊液,测定一氧化氮(NO)、神经元型一氧化氮合成酶(nNOS)和神经元特异性烯醇化酶(NSE)水平。结果显示:NCD组2种组织液中的NO、nNOS和NSE升高,显著高于NNS组(P<0.05)与CG组(P<0.01);ROC曲线分析发现,2种组织液中的NO、nNOS和NSE对神经型犬瘟热的诊断具有良好的灵敏度和特异性,血浆中以nNOS最佳,截断值为230.1 ng/mL,脑脊液中以NSE最佳,截断值为3.67 ng/mL;NSE对神经型犬瘟热具有一定预后价值,血浆中NSE<2.07的病例死亡率显著低于NSE≥2.07的病例。结果表明:神经型犬瘟热疾病发展中伴有硝化应激和神经损伤,血浆与脑脊液中的NO、nNOS和NSE具有对神经型犬瘟热的诊断及预测作用。     

Canine borreliosis: a laboratory diagnostic trial

The aim of this study was to investigate samples from dogs suggestive of active canine borreliosis (group A) by culture and PCR and the detection of antibodies against Borrelia burgdorferi sensu lato in order to confirm a presumptive clinical diagnosis of canine borreliosis by laboratory results. Criteria for such a diagnosis were: history of tick exposure, lameness, neurological signs, nephropathy, lethargy, anorexia, and fever. A total of 302 samples comprising EDTA blood, urine, synovial fluid, cerebrospinal fluid, and tissue (skin, synovial membrane, kidney) from 98 dogs (26 with arthritis, 46 with neurological signs, 21 with nephropathy, 5 with non-specific symptoms) were collected and examined. Moreover, 55 healthy dogs (group B) and 236 dogs with symptoms or injuries unlikely to be associated with borreliosis (group C) were included in this study. Blood serum samples collected from all individuals (n=389) were analysed by ELISA. Twenty-one (21%) out of 98 dogs from group A, 4 (7%) out of 55 from group B and 15 (6%) out of 236 dogs from group C were positive for antibodies against B. burgdorferi sensu lato. The seroprevalences between groups A, B and C differed significantly. None of the corresponding samples investigated by PCR and culture were positive for spirochetal DNA or viable spirochetes. Borrelia afzelii was grown from one EDTA-blood sample but the corresponding blood serum sample remained antibody-negative. Consequently, the etiologic role of B. afzelii in this case is unclear. In approximately 40% of the presumptive canine borreliosis cases, other lesions have been found to be responsible for clinical signs. This study affirms that a definitive diagnosis of canine borreliosis cannot be made by clinical symptoms and serology based on a single consultation. Moreover, this study clearly revealed that the diagnostic sensitivity is enhanced by a thorough consideration and exclusion of other diseases.     

Reference intervals for the activity of lactate dehydrogenase and its isoenzymes in the serum and cerebrospinal fluid of healthy canines

Background

Lactate dehydrogenase (LD) exists as 5 isoenzymes (LD‐1 through LD‐5) that are expressed throughout the body and can be detected in both serum and cerebrospinal fluid (CSF). LD and its isoenzymes have been relatively unstudied in veterinary medicine, although studies in human medicine have demonstrated that changes in total LD activity and atypical isoenzyme patterns can indicate disease processes, including neurologic abnormalities.

Objectives

The purpose of this study was to establish RIs for LD and its isoenzymes in the serum and CSF of clinically healthy dogs. By establishing a definitive RI for this enzyme in healthy canines, further study of the clinical and diagnostic usefulness of LD can be undertaken.

Methods

Serum and atlantoaxial CSF were collected from clinically healthy dogs. Total LD activity was measured spectrophotometrically immediately after collection. Isoenzyme distributions were also determined within 8 hours of collection using the QuickGel LD Isoenzyme technique and a densitometric scanner.

Results

The median serum total LD in healthy canines was 69.0 U/L (n = 41; range: 21.0‐217.0 U/L), while the median CSF total LD was 10.0 U/L (n = 40; range: 6.0‐19.3 U/L). LD‐5 is the predominant isoenzyme in canine serum (n = 40), contributing over half of the total enzyme activity. Conversely, in canine CSF (n = 42), LD‐1 is the predominant isoenzyme, followed by LD‐2 and LD‐3.

Conclusions

Knowledge of the distribution and concentration of LD in the serum and CSF of healthy dogs will set the foundation for future studies of canine LD as a potentially clinically useful biomarker.     

Autoantibodies against structures of the central nervous system in steroid responsive meningitis-arteriitis in dogs

Steroid-responsive meningitis-arteriitis (SRMA) is a disease of dogs familiar in small animal practice for decades. A combined evaluation of IgA in serum and cerebrospinal fluid (CSF) is an important diagnostic tool. It is suspected that immunpathological mechanisms are involved in the pathogenesis of SRMA because of the marked response to steroids. Excessive production of IgA seems to play a central role and might be caused by an immune reaction to self-antigens of the central nervous system (CNS). To test this hypothesis, we analyzed CSF samples from 55 dogs with SRMA using the western blot method. After blotting canine brain tissue, IgA, IgM and IgG of the CSF samples were tested for their binding to CNS antigens. We also evaluated CSF samples from 45 dogs with other brain diseases, including different encephalitides and intracranial tumors, and from healthy dogs as controls. Positive reactions (mostly IgA) were observed in the CSF samples from dogs with SRMA, different encephalitides and brain tumors (a total of 8% positive samples). The occurrence of autoantibodies against CNS structures was significantly higher in the control group "other brain diseases" than in the SRMA group (p = 0.0135). There was no significant difference in the number of positive samples between dogs with SRMA and the negative control group (healthy dogs, p = 0.1535). Despite the small number of positive samples, only dogs with abnormal findings in the CSF analysis also had autoantibodies in the CSF. There was no significant correlation between the occurrence of autoantibodies and levels of IgA, protein content and cell counts in the cerebrospinal fluid. However, there was a certain trend toward positive reactions in CSF samples with high protein content. The occurrence of autoantibodies in dogs with SRMA thus seems to be an epiphenomenona rather than the cause of the disease.     

Evaluation of the ADVIA 120 for analysis of canine cerebrospinal fluid

BACKGROUND: Conventional techniques for canine cerebrospinal fluid (CSF) analysis require large sample volumes and are labor intensive and subject to operator variability. Objective: The purpose of this study was to evaluate the ADVIA120 CSF assay for analysis of canine CSF samples. METHODS: CSF samples collected from 36 healthy control dogs and 17 dogs with neurologic disease were processed in parallel using the automated assay and established manual methods using a hemocytometer and cytocentrifugation. Results for WBC (total nucleated cell) count, RBC count, and differential nucleated cell percentages were compared using Spearman rank correlation coefficients and Bland-Altman bias plots. RESULTS: Correlation coefficients for WBC and RBC counts were 0.57 and 0.83 for controls, and 0.92 and 0.94 for ill dogs, respectively. Coefficients for the percentages of neutrophils, lymphocytes, and monocytes were 0.53, 0.26, and 0.12 for controls and 0.77, 0.92, and 0.70 for dogs with neurologic disease. When data were combined (n=53), correlation coefficients were 0.86 and 0.91 for WBC and RBC counts, and 0.63, 0.43, and 0.30 for neutrophil, lymphocyte, and monocyte percentages. A 9.5% positive bias and 7.0% negative bias were obtained for the ADVIA 120 CSF assay for lymphocytes and macrophages in dogs with neurologic disease with Bland-Altman analysis. A 12.2% positive bias was found for lymphocyte percentage in dogs with neurologic disease. CONCLUSIONS: Manual and automated CSF assays had moderate to excellent correlation for WBC and RBC concentrations, but results were more variable for differential cell percentages. The ADVIA assay may be more useful for assessment of canine CSF with adjustment of cell differentiation algorithms.     

Study of the effect of total serum protein and albumin concentrations on canine fructosamine concentration.

The relationship among serum fructosamine concentration and total serum protein and albumin concentrations were evaluated in healthy and sick dogs (diabetics and dogs with insulinoma were not included). Fructosamine was determined using a commercial colorimetric nitroblue tetrazolium method applied to the Technicon RA-500 (Bayer). Serum fructosamine concentration was not correlated to total protein in normoproteinemic (r = 0.03) and hyperproteinemic dogs (r = 0.29), but there was a high correlation (r = 0.73) in hypoproteinemic dogs. Similar comparison between serum fructosamine and albumin concentrations showed middle correlation (r = 0.49) in normoalbuminemic dogs and high degree of correlation (r = 0.67) in hypoalbuminemic dogs. These results showed the importance of recognizing serum glucose concentration as well as total serum protein and albumin concentrations in the assay of canine serum fructosamine concentration.     

Assessing the agreement of Western blot test results for paired serum and cerebrospinal fluid samples from horses tested for antibodies to Sarcocystis neuronaf

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 this study was to assess the agreement of the results of 181 paired serum and CSF Western blot antibody tests on equine samples submitted to the Michigan State University Animal Health Diagnostic Laboratory. The agreement of the paired serum and CSF results was assessed for three possible test outcomes--negative, positive or suspect. An additional analysis was performed in which samples reported as suspect were reclassified as negative. The kappa statistic for negative, positive and suspect samples was 0.469. The kappa statistic for the analysis in which the suspect results were reclassified as negative was 0.474. In addition, 29% (33/112) CSF samples from seropositive horses were negative. Our results demonstrate that the level of agreement is only moderate in diagnostic samples. This supports the practice of testing CSF of seropositive horses suspected of having EPM.     

Detection of oligoclonal bands in cerebrospinal fluid from German Shepherd dogs with degenerative myelopathy by isoelectric focusing and immunofixation

BACKGROUND: Detection of intrathecal IgG synthesis is important in evaluating inflammatory diseases in the central nervous system. Isoelectric focusing (IEF) is currently the most sensitive method to demonstrate intrathecal IgG synthesis and may have diagnostic value for German Shepherd degenerative myelopathy (GSDM). OBJECTIVE: The objective of this study was to adapt a modified IEF and immunofixation method for the detection of oligoclonal bands in cerebrospinal fluid (CSF) from dogs with GSDM. METHODS: Serum and lumbar CSF samples were collected from 6 German Shepherd dogs clinically diagnosed with GSDM. Samples were also collected from 6 clinically healthy dogs for comparison. The concentration of IgG was measured by quantitative ELISA and the concentration of total protein was measured by the Bradford protein assay. The presence of oligoclonal bands was evaluated by use of modified IEF followed by immunofixation. RESULTS: The concentrations of total protein and IgG, and the IgG/total protein ratio in CSF samples, were not significantly different between GSDM patients and control dogs. Four GSDM patients had oligoclonal bands in their CSF based on IEF-immunofixation. No oligoclonal bands were found in CSF from control dogs. CONCLUSION: The results suggest that the detection of oligoclonal bands by IEF-immunofixation may have diagnostic value for GSDM, and support the idea that humoral immune responses may play a role in the pathogenesis of GSDM.     

Low concentrations of cerebrospinal fluid GABA correlate to a reduced response to phenobarbital therapy in primary canine epilepsy

In this study, we investigated whether pretreatment cerebrospinal fluid (CSF) neurotransmitter concentrations of gamma-aminobutyric acid (GABA) and glutamate (GLU) were correlated with response to phenobarbital treatment in dogs with primary epilepsy. Eleven untreated dogs, 6 males and 5 females, with a median age of onset of seizures of 3 years (range: 0.5-5 years) were selected for therapy based on progressive or serious seizure patterns. The median interval between the first observed seizure and start of phenobarbital therapy was 485 days (range: 101-1,765 days). All dogs were purebred, with the exception of I male dog. Oral phenobarbital was started at 2.5 mg/kg every 12 hours. Trough serum phenobarbital concentrations were measured at 15, 45, 90, 180, 360, 540, and 720 days after the start of treatment. There was no difference in the mean trough serum concentration or in the mean number of seizures recorded between each time period of phenobarbital measurement over the 2-year evaluation. No correlation was found between CSF GLU, GABA, or GLU: GABA ratio and the total number of seizures recorded before or after initiation of phenobarbital therapy. Lower CSF GABA concentration, however, was correlated with a lower seizure frequency difference (the total number of seizures before phenobarbital therapy minus the total number of seizures after phenobarbital therapy for an identical time period of evaluation) and lower percentage reduction in seizures: ([total number of seizures before phenobarbital therapy minus the total number of seizures after phenobarbital therapy] divided by the total number of seizures before phenobarbital therapy) x 100. There was no correlation between CSF GLU and the seizure frequency difference and percentage reduction in seizures. A negative correlation between the CSF GLU:GABA ratio and seizure frequency difference was found. Thus, dogs with an initial lower CSF GABA concentration before phenobarbital therapy did not respond as well as did dogs with a higher CSF GABA concentration.     

Significance of surface epithelial cells in canine cerebrospinal fluid and relationship to central nervous system disease

Background: The term “surface epithelium” is used to describe cells, including meningeal, choroid plexus, ependymal, and endothelial cells, that are found in human cerebrospinal fluid (CSF) and are difficult to distinguish cytologically. We hypothesized that the presence of surface epithelial cells in canine CSF was associated with specific diseases of the central nervous system (CNS). Objectives: In this retrospective study the frequency of surface epithelial cells in CSF from dogs with neurologic disease was investigated along with the potential association with age, specific type of CNS disease, and CSF total nucleated cell count (TNCC) and protein concentration. Methods: The frequency of surface epithelial cells in 359 canine CSF samples was analyzed for 5 disease groups: CNS neoplasia, CNS compression, CNS inflammation, idiopathic epilepsy, and miscellaneous diseases. Groups were also combined into those with and without expected meningeal involvement. Association of the presence of surface epithelial cells in CSF with age, disease type, and CSF TNCC and protein concentration was investigated. Results: Surface epithelial cells were found in 27 of 359 (7.5%) CSF samples: CNS neoplasia 2/30 (6.7%), CNS compression 7/64 (10.9%), CNS inflammation 1/39 (2.6%), idiopathic epilepsy 8/124 (6.5%), and miscellaneous diseases 9/102 (8.8%). Significant associations between surface epithelial cell presence in CSF and age, disease type, CSF TNCC, and CSF protein concentration were not found. Conclusions: The presence of surface epithelial cells was not related to a specific disease group or CSF changes in the studied population. Thus, the presence of surface epithelial cells should be interpreted carefully, as it could represent an incidental finding in CSF specimens.     

Immunoglobulin M-capture enzyme-linked immunosorbent assay testing of cerebrospinal fluid and serum from horses exposed to west nile virus by vaccination or natural infection

The West Nile (WN) virus, present in the United States since 1999, is a cause of encephalomyelitis in birds, alligators, humans, and horses. No data exist regarding detection of anti-WN virus immunoglobins in equine cerebrospinal fluid (CSF). The aims of this study were to evaluate the blood-brain barrier (BBB) in WN virus-infected (WNE) horses, to compare diagnostic testing in serum and CSF, and to describe the immunoglobulin M (IgM) response in serum and CSF of vaccinated horses. CSF was collected from the lumbosacral (LS) space (n = 13) or the allanto-occipital (AO) space (n = 14) of WNE horses. The albumin quotient (AQ) and IgG index were calculated, and the IgM-capture-enzyme-linked immunosorbent assay (MAC-ELISA) was used to detect anti-WN virus IgM in serum and CSF. CSF collected from the LS site had a higher (P < .02) IgG index compared to the AO site (0.34 +/- 0.04 versus 0.22 +/- 0.04 [mean +/- SE], respectively). The mean AQ, irrespective of collection site, did not exceed reference values. There was 100% agreement between CSF and serum testing for IgM by MAC-ELISA testing. However, the positive to negative antigen ratios were higher (P < .001) in CSF (34.5) versus serum (8.5), indicating lower nonspecific reactivity in CSF samples. Horses vaccinated against WN virus did not develop an IgM response at 1:400 mg/dL in serum; however, a few horses developed a weak IgM response in serum but not in CSF. In conclusion, MAC-ELISA testing of serum and CSF were equivocal. Also, examination of CSF data from WNE horses suggests a normal BBB integrity and increased intrathecal production of antibodies.     

Detection of an autoantibody from Pug dogs with necrotizing encephalitis (Pug dog encephalitis).

An autoantibody against canine brain tissue was detected in the cerebrospinal fluid (CSF) and serum of two Pug dogs (Nos. 1 and 2) by indirect immunofluorescence assay (IFA). Dog No. 1, a 2-year-old male, exhibited severe depression, ataxia, and generalized seizures and died 2 months after the onset of symptoms. Dog No. 2, a 9-month-old male, exhibited severe generalized seizures and died 17 months after the onset of symptoms. Histopathologic examination revealed a moderate to severe multifocal accumulation of lymphocytes, plasma cells, and a few neutrophils in both the gray and white matter of the cerebrum in dog No. 1. In dog No. 2, the cellular infiltrates were mild, but there was a severe, diffuse, and multifocal necrosis in the cerebral cortex with prominent astrocytosis. With the aid of IFA using fluorescein isothiocyanate-labeled antidog IgG goat serum and a confocal imaging system, specific reactions for glial cells were detected in the CSF of these Pug dogs but not in six canine control CSF samples. Double-labeling IFA using CSF from these Pug dogs and a rabbit antiserum against glial fibrillary acidic protein (GFAP) revealed that the autoantibody recognized GFAP-positive astrocytes and their cytoplasmic projections. By immunoblot analysis, the autoantibody from CSF of these Pug dogs recognized two common positive bands at 58 and 54 kd, which corresponded to the molecular mass of human GFAP. The role of this autoantibody for astrocytes is not yet clear. However, if the presence of the autoantibody is a specific feature of Pug dog encephalitis, it will be a useful clinical diagnostic marker and a key to the pathogenesis of this unique canine neurologic disease.     

Cerebrospinal Fluid PCR and Antibody Concentrations against Anaplasma phagocytophilum and Borrelia burgdorferi sensu lato in Dogs with Neurological Signs

Background: The tick-borne bacteria Borrelia burgdorferi sensu lato (sl) and Anaplasma phagocytophilum have been suspected to cause neurological signs in dogs. Diagnosis often has been made based on positive antibody titers in serum of dogs with neurological signs, but a high seroprevalence in dogs in at-risk populations makes diagnosis difficult.
Objective: To determine if the neurological signs in dogs examined were caused by any of these bacteria.
Animals: Fifty-four dogs presented to a board-certified neurologist.
Methods: Prospective study. We divided dogs into 2 groups: those with inflammatory diseases of the central nervous system (CNS) and those with neurological signs from other diseases. Blood and cerebrospinal fluid (CSF) from all dogs were analyzed.
Results: Dogs with inflammatory CNS diseases showed no serum antibodies against any of the agents. Among dogs with neurological signs from other diseases, 10.3% had serum antibodies for B. burgdorferi sl and 20.5% for A. phagocytophilum . All blood samples analyzed for bacterial deoxyribonucleic acid (DNA) and all CSF analyzed for antibodies and bacterial DNA for the 2 agents were negative.
Conclusions and Clinical Importance: Based on this study, these bacteria are unlikely causes of neurologic disease in dogs and the presence of serum antibodies alone does not document or establish a definitive diagnosis of CNS disease caused by these organisms. Dogs that have neurologic disease and corresponding serum antibodies against these agents should have additional tests performed to assess for other potential etiologies of the signs.     

Cerebrospinal fluid constituents collected at the atlanto-occipital site of xylazine hydrochloride sedated, healthy 8-week-old Holstein calves.

Cerebrospinal fluid (CSF) collected at the atlanto-occipital site and serum were obtained from 10 male, 8-week-old, Holstein calves after sedation with xylazine hydrochloride. Glucose, creatine kinase, alkaline phosphatase, urea nitrogen, creatinine, sodium, potassium, chloride, calcium, phosphorus, total protein, and albumin were determined in serum and CSF. Optical characteristics, specific gravity, total red blood cell and nucleated cell counts and differentials were also evaluated in the CSF. Additionally, CSF protein electrophoresis and immunoglobulin concentrations were determined. Then, albumin quotients (AQ) were derived. Erythrocytes were observed in 9 of 10 CSF samples. Total nucleated cell counts ranged from 0-10 cells x 10(6)/L with a mean of 3 cells x 10(6)/L. Differential nucleated cell count in the CSF consisted primarily of lymphocytes/small mononuclear cells (57%), fewer monocytes/ large mononuclear cells (38%), and scant neutrophils (4%) and eosinophils (0.05%). The concentration of sodium (134 to 139 mEq/L) was similar to that of serum, but the concentration of potassium (2.8 to 3 mEq/L) was lower than that of serum. Creatine kinase activity (0 to 4 U/L) of CSF was markedly lower than serum activity. The CSF glucose concentration was approximately 80% of the serum value. Cerebrospinal fluid total protein concentration determined by electrophoresis ranged from 110 to 330 mg/L with a mean of 159 mg/L. Cerebrospinal fluid albumin ranged from 48 to 209 mg/L with a mean of 86 mg/L. In all CSF samples, radial immunodiffusion of unaltered CSF and concentrated CSF (four-fold concentration) revealed quantities undetectable by the present techniques in which the lowest standard values for IgG1, IgG, and IgM determinations was 70 mg/L and IgG2 was 30 mg/L. The albumin quotient ranged from 0.15 to 0.65 with a mean of 0.25. Based on the results of this study, CSF may be collected at the atlanto-occipital site safely and efficiently in calves, and reported values for CSF from adult cattle may not be suitable for evaluation of CSF collected from immature cattle.     

Detection of serum alpha-fetoprotein in dogs with hepatic tumors.

Serum alpha-fetoprotein (AFP) concentration was detected by use of 2 commercially available kits containing antibodies to human AFP--a radioimmunoassay and an enzymetric test. Using neonatal canine serum (a source high in AFP), it was determined that reagents from both kits were able to bind to canine AFP, but a significant difference was detected in AFP concentration. The enzymetric test was superior in detecting canine AFP. Sera from dogs were classified into 6 groups: from dogs with primary hepatic tumors only (group 1); from dogs with primary hepatic tumors and other tumors (group 2); from dogs with normal liver but with other types of neoplasia (group 3); from dogs with nonneoplastic hepatic disease and tumors originating in other organs (group 4); from dogs with nonneoplastic hepatic disease only (group 5); and from clinically normal dogs (group 6). Serum biochemical determinations (alkaline phosphatase, alanine transaminase, albumin, total protein, total bilirubin, and serum bile acids) and values from the 2 AFP assays were obtained for all dogs. Serum AFP concentration detected by the enzymetric test was significantly higher in dogs with hepatocellular carcinoma and cholangiocarcinoma. Values greater than 250 ng/ml were detected in 5 of 9 dogs with cholangiocarcinoma and in 3 of 4 dogs with hepatocellular carcinoma. High serum AFP concentration also was indicative of liver involvement in 2 of 3 dogs with primary hepatic lymphosarcoma; 2 dogs had values greater than 225 ng/ml. Serum AFP concentration in dogs with other types of hepatic tumors was less than 250 ng/ml, and serum AFP concentration could not be correlated with such tumors.(ABSTRACT TRUNCATED AT 250 WORDS)