Use of albumin quotient and IgG index to differentiate blood- vs brain-derived proteins in the cerebrospinal fluid of cats with feline infectious peritonitis

BACKGROUND: Inflammation of the central nervous system (CNS) is a frequent condition in cats but etiology often remains unsolved. Routine cerebrospinal fluid (CSF) analysis can be extended through the calculation of the albumin quotient (Q(alb)), a marker of the integrity of the blood-brain barrier (BBB), and IgG index, an estimate of intrathecal IgG synthesis. OBJECTIVES: The purpose of this study was to validate nephelometric methods for CSF protein analysis, and to use the Q(alb) and IgG index to discriminate blood- and brain-derived immunoglobulin fractions in cats with feline infectious peritonitis (FIP). METHODS: Cats presented to our clinic between 2001 and 2005 were included in the study based on clinical and laboratory data and histopathologic findings at necropsy. Cats were grouped as having nonneurologic disease (controls; n=37), brain tumors (n=8), FIP involving the CNS (n=12), and extraneural FIP (n=12). CSF-total protein (TP) was measured and albumin and IgG concentrations were measured in paired CSF/serum samples; Q(alb) and IgG index were calculated. Intraassay and interassay precision of the nephelometric assays were determined using pooled samples. RESULTS: Coefficients of variation for the nephelometric assays ranged from 2.7% to 7.2%. In control cats, CSF-TP concentration ranged from 0.06 to 0.36 g/L, Q(alb) ranged from 0.6 to 5.7 x 10(-3), and IgG index ranged from 0.3 to 0.6. Q(alb) and IgG index were significantly higher in cats with brain tumors and cats with CNS-FIP compared with other groups. Compared with control cats, pleocytosis was evident in 8 of 12 (67%) cats and CSF-TP was increased in 3 of 12 (25%) cats with CNS-FIP. CONCLUSION: Nephelometry is a reliable method for measurement of CSF protein, albumin, and IgG in cats. The Q(alb) and IgG index did not identify a CSF protein pattern specific for BBB dysfunction or intrathecal IgG synthesis in cats with CNS-FIP.     

Bartonella species antibodies and DNA in cerebral spinal fluid of cats with central nervous system disease

Bartonella species infection is associated with central nervous system (CNS) disease in some humans and cats but the diagnosis is difficult to confirm with blood or serum test results. In this retrospective study of 100 client-owned cats, serum and cerebral spinal fluid (CSF) were assayed for Bartonella species IgG antibodies and CSF was assayed for Bartonella species DNA. Bartonella species IgG antibodies were detected in serum of 36 cats, Bartonella species C-values>1 (suggesting antibody production by the CNS) were detected in CSF of 11 cats, and B henselae DNA was amplified from the CSF of 10 cats. While the clinical significance of these findings cannot be assessed without a control group, the development of neurological signs in some cats inoculated with B henselae and the results of this study warrant prospective evaluation of the association of Bartonella species with feline CNS disease.     

An immunoturbidimetric assay for rapid quantitative measurement of feline alpha-1-acid glycoprotein in serum and peritoneal fluid

BACKGROUND: Alpha-1-acid glycoprotein (AGP) is an acute phase protein that increases in concentration in infectious and inflammatory conditions. The serum and peritoneal fluid concentrations of AGP may be useful in the diagnosis of feline infectious peritonitis (FIP), a lethal disease of cats. Currently AGP can be measured by radioimmunodiffusion (RID) assays, which are time consuming and difficult. OBJECTIVES: The objectives of this study were to develop a rapid immunoturbidimetric assay for measurement of AGP in feline serum and peritoneal fluid and to compare the results with those obtained by RID. METHODS: AGP was purified by perchloric acid precipitation and ion-exchange chromatography from a pool of peritoneal fluid obtained from cats with FIP, as determined by a panel of laboratory tests, including serum AGP concentration, albumin: globulin ratio, and total protein concentration, anti-coronavirus antibody titers, and effusion analysis. The purified AGP in a complete Freund's adjuvant and Tween 20 mixture was injected into a sheep and blood was collected at monthly intervals. Anti-AGP antiserum, as confirmed by ELISA and Western blot techniques, and a pool of peritoneal fluid from cats with FIP were used to prepare standards. Clinical samples of feline peritoneal fluid (n=55) and serum (n=59) were assayed for AGP and results from the immunoturbidimetric and RID methods were compared. RESULTS: Significant correlation (P < .001) was obtained between methods for both peritoneal fluid (R2=.9259) and serum (R2=.9448) samples. Coefficients of variation for the immunoturbidimetric method were <5%. CONCLUSIONS: This rapid immunoturbidimetric assay for measurement of feline AGP in serum and peritoneal fluid may be of value in the diagnosis of FIP and possibly other inflammatory diseases in cats.     

Diagnostic Features of Clinical Neurologic Feline Infectious Peritonitis

Feline infectious peritonitis (FIP) is a fatal Arthus-type immune response of cats to infection with FIP virus, a mutant of the ubiquitous feline enteric coronavirus (FECV). The disease may occur systemically or in any single organ system, and primary neurologic disease is a common subset of such manifestations. We examined 16 domestic cats with clinical neurologic FIP and 8 control cats with nonneurologic FIP, with the intention of identifying the ante-and postmortem diagnostic tests that most contribute to accurate diagnosis. Of the 16 cats with neurologic FIP, 15 were less than 2 years of age and all 16 originated from large multiplecat households. The most useful antemortem indicators of disease were positive anti-coronavirus IgG titer in cerebrospinal fluid, high serum total protein concentration, and findings on magnetic resonance imaging suggesting periventricular contrast enhancement, ventricular dilatation, and hydrocephalus. Postmortem diagnosis was facilitated by FIP monoclonal antibody staining of affected tissue and coronavirus-specific polymerase chain reaction. Most cats with neurologic and ocular forms of FIP had patchy, focal lesions, suggesting that recently developed technologies described in this report may be useful for evaluation of cats with suspected FIP.     

Clinical, cerebrospinal fluid, and histological data from twenty-seven cats with primary inflammatory disease of the central nervous system.

The purpose of this report is to present the clinical, cerebrospinal fluid (CSF) and histological data from 27 cats with inflammatory disease of the central nervous system (CNS). The cats were part of a study of 61 cats admitted to two university clinics over an eight-year period because of signs of CNS disease. The most frequent diseases were feline infectious peritonitis (FIP) (12/27) and suspected viral disease other than FIP (10/27). Typical CSF findings in cats with FIP were a protein concentration of greater than 2 g/L (200 mg/dL) and a white cell count of over 100 cells/microL, which consisted predominantly of neutrophils. In contrast, the CSF of cats with suspected viral disease had a protein concentration of less than 1 g/L (100 mg/dL) and a total white cell count of less than 50 cells/microL. In general, cats with FIP or suspected viral disease were less than four years of age. Neurological signs were usually multifocal in cats with FIP, but focal in cats with suspected viral disease. The CSF findings were variable in five other inflammatory diseases represented. Two cats with protozoan infection had normal CSF total cell counts but abnormal differential counts. The CSF findings were invaluable in differentiating FIP from other causes of inflammatory CNS disease.     

An enzyme-linked immunosorbent assay using canine coronavirus-infected CRFK cells as antigen for detection of anti-coronavirus antibody in cat

From the reasons that canine coronavirus (CCV) grows more efficiently than feline coronavirus in a cell culture and they are mutually related in their antigenicities, an enzyme-linked immunosorbent assay (ELISA) using CCV-infected feline kidney (CRFK) cells as substrate antigens was developed for detection of anti-coronavirus antibodies in cats. It was indispensable for generating coronavirus-specific ELISA antibody activities that the sample was applied to the mock-infected, normal CRFK cells in parallel with the CCV-infected cells and then the optical density values given by the mock-infected cell antigen were subtracted from those given by the virus-infected cell antigen. On the basis of ELISA antibody titers obtained in sera from the cats experimentally infected with CCV and from the spontaneous feline infectious peritonitis (FIP) cases, the ELISA described in the present study was found to be applicable as a simple and easy serologic test which was able to detect anti-coronavirus antibodies as efficiently as the indirect immunofluorescence assay with homologous FIP virus.     

Changes in some acute phase protein and immunoglobulin concentrations in cats affected by feline infectious peritonitis or exposed to feline coronavirus infection

The possible role of some acute phase proteins (APPs) and immunoglobulins in both the pathogenesis and diagnosis of feline infectious peritonitis (FIP) has been investigated. Serum protein electrophoresis and the concentration of haptoglobin (Hp), serum amyloid A (SAA), alpha(1)-acid glycoprotein (AGP), IgG and IgM were evaluated in cats exposed to feline coronavirus (FCoV) and in cats with FIP. The highest concentration of APPs was detected in affected cats, confirming the role of these proteins in supporting a clinical diagnosis of FIP. Repeated samplings from both FIP affected and FCoV-exposed cats showed that when FIP appeared in the group, all the cats had increased APP levels. This increase persisted only in cats that developed FIP (in spite of a decrease in alpha(2)-globulins) but it was only transient in FCoV-exposed cats, in which a long lasting increase in alpha(2)-globulins was observed. These results suggest that changes in the electrophoretic motility of APPs or APPs other than Hp, SAA and AGP might be involved in the pathogenesis of FIP or in protecting cats from the disease.     

Toxoplasma gondii-induced abortion in dairy goats

Three of 8 goats on a Maryland farm aborted or had dystocia associated with toxoplasmosis during the winter of 1984. Doe 1 aborted a decomposed fetus 30 days before term. Modified agglutination test (MAT) antibody titers against Toxoplasma gondii were found in pleural fluid of the fetus (1:1,024) and in serum of doe (1:4,096 at 31 days after abortion). Doe 2 aborted a fetus 5 days before term; MAT antibody was found in the pleural fluid of the fetus (1:16,384) and in the doe's serum (1:4,096 on the day of abortion). Placenta from both does had foci of necrosis characteristic of toxoplasmosis, and T gondii was identified in lesions. Doe 3 had dystocia 7 days before term and a partially decomposed fetus was delivered by cesarian section; MAT was found in pleural fluid of the fetus (1:1,024) and in serum from the doe (1:4,096 on the day of abortion). Focal gliosis and calcification were seen in brain specimens from 2 of the 3 fetuses. None of the does produced milk after abortion. Two other does (No. 4 and 5) delivered apparently healthy kids transplacentally infected with T gondii; MAT in serum of both does was 1:4,096. Doe 4 delivered 3 kids; MAT titer in a serum from each kid 38 days after birth was 1:16,384. Doe 5 delivered 1 kid with a serum MAT titer of 1:1,024 at 38 days after birth. The 3 remaining does had MAT titers of 1:256, 1:16, and 1:16, and all delivered healthy kids. Epizootiologic evidence suggested that the does acquired T gondii infection from oocysts passed in feces of domestic cats on the farm. The MAT titers of 4 cats on the farm were 1:65,356; 1:1,024; 1:16; and 1:1,024.     

Prevalence of Toxoplasma gondii infection in cats in Georgia using enzyme-linked immunosorbent assays for IgM, IgG, and antigens

Serologic prevalence of Toxoplasma gondii infection was determined using enzyme-linked immunosorbent assays detecting immunoglobulin M (IgM), immunoglobulin G (IgG), and circulating T. gondii antigens (Ag) in 81 healthy cats and 107 cats with clinical signs referable to toxoplasmosis. A higher prevalence of infection was detected using the three assays together in healthy cats, clinically ill cats, and combined healthy and clinically ill cats than when IgG class antibody detection alone was used. IgM titers greater than or equal to 1:256 and IgG titers greater than or equal to 1:512 were present more frequently in cats with clinical signs of disease. Prevalence of present or prior infection as defined by these three assays combined increased with advancing age in both groups of cats.     

Flow cytometric detection of alpha-1-acid glycoprotein on feline circulating leucocytes

To assess whether alpha‐1‐acid glycoprotein (AGP) can be detected on the membrane of feline circulating leucocytes. Design The presence of AGP on circulating leucocytes was investigated in both clinically healthy cats and cats with different diseases. A group of feline coronavirus (FCoV)‐positive cats, comprising cats with feline infectious peritonitis (FIP) and cats not affected by FIP but seropositive for FCoV, were included in this study because the serum concentration of AGP increases during FCoV infection. Procedure Flow cytometry (using an anti‐feline AGP antibody), serum protein electrophoresis, routine haematology and measurement of the serum AGP concentration were performed using blood samples from 32 healthy cats (19 FCoV‐seropositive), 13 cats with FIP and 12 with other diseases (6 FCoV‐seropositive). The proportion of cats with AGP‐positive leucocytes in the different groups (e.g. controls vs sick; FIP vs other diseases, etc.) or in cats with different intensities of inflammatory response was compared using a Chi‐square test. Results AGP‐positive leucocytes were found in 23% of cats. Compared with controls, the proportion of patients with positive granulocytes and monocytes was higher among sick cats (especially cats with diseases other than FIP) and cats with high serum AGP concentration, but not in cats with leucocytosis or that were FCoV‐seropositive. Conclusion AGP‐positive leucocytes can be found in feline blood, especially during inflammation. Conversely, no association between AGP‐positive leucocytes and FIP was found. Further studies are needed to elucidate the mechanism responsible for this finding and its diagnostic role in cats with inflammation.     

Effects of blood contamination of cerebrospinal fluid on western blot analysis for detection of antibodies against Sarcocystis neurona and on albumin quotient and immunoglobulin G index in horses.

OBJECTIVE: To determine effects of blood contamination on western blot (WB) analysis of CSF samples for detection of anti-Sarcocystis neurona antibodies, and on CSF albumin and IgG concentrations, albumin quotient (AQ), and IgG index in horses. DESIGN: Prospective in vitro study. SAMPLES: Blood with various degrees of immunoreactivity against S neurona was collected from 12 healthy horses. Cerebrospinal fluid without immunoreactivity against S neurona was harvested from 4 recently euthanatized horses. PROCEDURE: Blood was serially diluted with pooled nonimmunoreactive CSF so that final dilutions corresponded to 10(-3) to 100 microliters of blood/ml CSF, and WB analysis was performed on contaminated CSF samples. Number of RBC, albumin and IgG concentrations, AQ, and IgG index were also determined. RESULTS: Antibodies against S neurona were detected in CSF contaminated with 10(-3) microliters of strongly immunoreactive blood/ml. In CSF samples contaminated with 10 microliters of blood/ml, AQ remained within reference range. Volume of blood required to increase IgG index varied among blood samples and was primarily influenced by serum IgG concentrations. Number of RBC in contaminated samples was correlated with volume of blood added, but not with degree of immunoreactivity detected in contaminated CSF samples. CONCLUSIONS AND CLINICAL RELEVANCE: During collection of CSF from horses, contamination with blood may introduce serum antibodies against S neurona at concentrations sufficient for detection by WB analysis, thus yielding false-positive results. When blood is moderately or strongly immunoreactive, the amount of contaminating albumin may be small enough as to not increase AQ above reference range. In these cases, AQ and IgG index should be interpreted with caution.     

Cerebrospinal fluid glutamine,tryptophan, and tryptophan metabolite concentrations in dogs with portosystemic shunts

OBJECTIVE: To determine whether glutamine (GLN), tryptophan (TRP), and tryptophan metabolite concentrations are higher in cerebralspinal fluid (CSF) dogs with naturally occurring portosystemic shunts (PSS), compared with control dogs. ANIMALS: 11 dogs with confirmed PSS and 12 control dogs fed low- and high-protein diets. PROCEDURE: Cerebrospinal fluid and blood samples were collected from all dogs. Serum and CSF concentrations of GLN, alanine, serine, TRP, 5-hydroxyindoleacetic acid (5-HIAA), and quinolinic acid (QUIN) were measured. RESULTS: Cerebrospinal fluid concentrations of GLN, TRP, and 5-HIAA were significantly higher in PSS dogs, compared with control dogs fed high- or low-protein diets. Cerebrospinal fluid QUIN concentration was significantly higher in PSS dogs, compared with control dogs fed the low-protein diet. Serum QUIN concentration was significantly lower in PSS dogs, compared with control dogs fed either high- or low-protein diets. CONCLUSIONS AND CLINICAL RELEVANCE: An increase in CNS GLN concentration is associated with high CSF concentrations of TRP and TRP metabolites in dogs with PSS. High CSF 5-HIAA concentrations indicate an increased flux of TRP through the CNS serotonin metabolic pathway, whereas high CSF QUIN concentrations indicate an increased metabolism of TRP through the indolamine-2,3-dioxygenase pathway. The high CSF QUIN concentrations in the face of low serum QUIN concentrations in dogs with PSS indicates that QUIN production from TRP is occurring in the CNS. High concentrations of QUIN and other TRP metabolites in the CNS may contribute to neurologic abnormalities found in dogs with PSS and hepatic encephalopathy.     

Virion polypeptide specificity of immune complexes and antibodies in cats inoculated with feline infectious peritonitis virus

Immune complexes purified from sera and ascitic fluids of cats after inoculation with feline infectious peritonitis (FIP) virus contained proteins and proteolytic fragments of the peplomer, nucleocapsid, and envelope polypeptides; in addition, host proteins were demonstrated in the immune complexes. Free (uncomplexed) antibodies against the 3 classes of virion polypeptides were detected and quantitated; the weakest and latest response was directed against the peplomer protein. Immunofluorescence titers showed the best correlation with the antibody response directed against the envelope polypeptides. Differences in reactivity were not found between sera and ascitic fluids from the same animals and between seropositive healthy cats and cats which had died of FIP. Humoral antibody and hypergammaglobulinemia showed a linear correlation, but the wide variation in antiviral titers at a given concentration of gamma-globulin indicated that additional (autoimmune) reactions occur during the pathogenesis of FIP.     

Antibody index and specific antibody quotient in horses after intragastric administration of Sarcocystis neurona sporocysts

OBJECTIVE: To investigate the use of a specific antibody index (AI) that relates Sarcocystis neurona-specific IgG quotient (Q(SN)) to total IgG quotient (Q(IgG)) for the detection of the anti-S neurona antibody fraction of CNS origin in CSF samples obtained from horses after intragastric administration of S neurona sporocysts. ANIMALS: 18 adult horses. PROCEDURES: 14 horses underwent intragastric inoculation (day 0) with S neurona sporocysts, and 4 horses remained unchallenged; blood and CSF samples were collected on days - 1 and 84. For purposes of another study, some challenged horses received intermittent administration of ponazuril (20 mg/kg, PO). Sarcocystis neurona-specific IgG concentrations in CSF (SN(CSF)) and plasma (SN(plasma)) were measured via a direct ELISA involving merozoite lysate antigen and reported as ELISA units (EUs; arbitrary units based on a nominal titer for undiluted immune plasma of 100,000 EUs/mL). Total IgG concentrations in CSF (IgG(CSF)) and plasma (IgG(plasma)) were quantified via a sandwich ELISA and a radial immunodiffusion assay, respectively; Q(SN), Q(IgG), and AI were calculated. RESULTS: Following sporocyst challenge, mean +/- SEM SN(CSF) and SN(plasma) increased significantly (from 8.8 +/- 1.0 EUs/mL to 270.0 +/- 112.7 EUs/mL and from 1,737 +/- 245 EUs/mL to 43,169 +/- 13,770 EUs/mL, respectively). Challenge did not affect total IgG concentration, Q(SN), Q(IgG), or AI. CONCLUSIONS AND CLINICAL RELEVANCE: S neurona-specific IgG detected in CSF samples from sporocyst-challenged horses appeared to be extraneural in origin; thus, this experimental challenge may not reliably result in CNS infection. Calculation of a specific AI may have application to the diagnosis of S neurona-associated myeloencephalitis in horses.     

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.     

Reference intervals for feline cerebrospinal fluid: biochemical and serologic variables, IgG concentration, and electrophoretic fractionation

Reference intervals are reported for feline CSF biochemical and serologic variables, IgG concentration, and electrophoretic fractionation, derived from 58 clinically normal adult cats that did not have histologic lesions of the CNS. There was no apparent effect of age on any variable. The CSF total protein concentration was significantly (P = 0.012) greater in males than in females, but all other variables were unaffected by gender. The only variable that had a statistically significant correlation with its corresponding blood concentration was IgG. Blood contamination of the CSF affected the following CSF variables: total protein concentration, activities of lactate dehydrogenase and creatine kinase, IgG ratio, and gamma-globulin percentage. The reference intervals proposed for feline CSF were derived from 33 cats with CSF RBC count less than 31 cells/microliters. Reference limits for CSF with 31 to 1,700 RBC/microliters also are reported.     

Beta-2-microglobulin levels in the cerebrospinal fluid of normal dogs and dogs with neurological disease

BACKGROUND: Cerebrospinal fluid (CSF) analysis is the basis for establishing a diagnosis of central nervous system (CNS) inflammation. However, the information provided by routine CSF analysis is limited. Determination of CSF beta-2-microglobulin (beta2m) concentration has been used diagnostically in humans to identify inflammatory CNS disease; we hypothesized that it may have similar value in dogs. OBJECTIVES: The objective of this study was to measure (beta2m concentration in the CSF of clinically healthy dogs and compare the values to those observed in dogs with inflammatory CNS disease and intervertebral disc disease (IVDD). METHODS: CSF was collected from 10 clinically healthy laboratory dogs and 11 dogs each with inflammatory CNS disease and IVDD. Routine CSF analysis was performed, and (beta2m concentration was measured by ELISA. CSF (beta2m concentration and CSF:serum (beta2m ratio were compared between groups by ANOVA. Linear relationships between CSF total nucleated cell count (TNCC), RBC count, total protein concentration, and (beta2m concentration were assessed by regression analysis. RESULTS: The mean (+/- SD) CSF (beta2m concentration in clinically healthy dogs was 0.36 (+/- 0.05 microg/mL (cisternal) and 0.40 (+/- 0.07 microg/mL (lumbar). Median CSF (beta2m concentration in dogs with IVDD (0.46 microg/mL) and inflammatory CNS disease (0.85 microg/mL) differed from that of controls (0.36 microg/mL; P=.002). The concentration also differed between the 2 disease groups (P=.01). Five dogs with inflammatory CNS disease had CSF:serum (beta2m ratios >1. A correlation was identified between TNCC and (beta2m concentration (r=0.69, P=.0003). CONCLUSIONS: CSF (beta2m concentration is higher in dogs with IVDD and inflammatory CNS disease, with highest values seen with inflammatory disease. This may be attributed in part to the correlation between CSF (beta2m concentration and TNCC, but also may reflect intrathecal immune activation.