der Tierärztlichen Hochschule Hannover
Concentrations of Acute-Phase Proteins in dogs with Steroid Responsive Meningitis-Arteritis
INAUGURAL – DISSERTATION zur Erlangung des Grades einer
Doktorin der Veterinärmedizin (Dr. med. vet.)
durch die Tierärztliche Hochschule Hannover
Vorgelegt von Andrea Bathen-Nöthen aus Bergisch Gladbach
Hannover 2008
Wissenschaftliche Betreuung: Univ. Prof. Dr. Andrea Tipold
und Univ. Prof. Dr. Reinhard Mischke
1. Gutachter/in: Univ. Prof. Dr. Andrea Tipold 2. Gutachter/in: Univ. Prof. Dr. Hewicker-Trautwein
Tag der mündlichen Prüfung: 28. Oktober 2008
Meinen Eltern und meinem Mann
Vorläufige Resultate der zugrunde liegenden Studie wurden als Vortragspräsentation auf dem Jahreskongress des European College of Veterinary Neurology im September 2006 in Barcelona unter dem Titel „ IS C-REACTIVE PROTEIN (CRP) A VALUABLE TOOL IN DIAGNOSIS AND TREATMENT CONTROL IN DOGS WITH STEROID-RESPONSIVE MENINGITIS-ARTERIITIS (SRMA)? “ (J Vet Intern Med 2007;21(5): 1141) und als Vortragspräsentation auf dem Jahreskongress des American College of Internal Medicine im Juni 2007 in Seattle, USA unter dem Titel “STEROID-RESPONSIVE MENINGITIS- ARTERITIS IN DOGS: CENTRAL NERVOUS SYSTEM OR SYSTEMIC DISEASE?”
vorgestellt.
Diese Dissertation basiert auf einer Veröffentlichung in einer international anerkannten und international erscheinenden Wissenschaftszeitschrift (Journal of Veterinary Internal
Medicine) mit Gutachtersystem.
INHALTSVERZEICHNIS:
Seite
I. Einleitung 9
II. Manuskript
Concentrations of Acute-Phase Proteins in dogs with Steroid Responsive
Meningitis-Arteritis 12
Introduction 13
Material and methods 15
Results 20
Discussion 31
Footnotes 35
References 36
III. Zusammenfassung / Abstract 41
IV. Anhang (tabellarische Darstellung der Patientendaten) 46 V. Bestätigung des Verlages / Online – Literaturstelle 56
VI. Danksagung 57
Abkürzungsverzeichnis:
AMG α2-Makroglobulin
(α2-macroglobulin)
APP Akute Phase Proteine
(acute-phase proteins)
CNS Zentrales Nervensystem (ZNS)
(central nervous system)
CRP C-reaktives Protein
(C-reactive protein)
CSF Cerebrospinalflüssigkeit
(cerebrospinal fluid)
IE Idiopathische Epilepsie
(idiopathic epilepsy)
IVDD/DLSS Intervertebral Disk Disease/Degenerative Lumbosakrale Stenose
(Intervertebral disk disease/degenerative lumbosacral stenosis)
ME Meningoenzephalitis unklarer Genese
(meningoencephalitis of unknown origin)
OD optische Dichte
(optical density)
pNA p-nitroaniline
SRMA Steroid Responsive Meningitis-Arteritis
TCNS primäre und sekundäre Tumoren des ZNS
(primary and secondary tumors affecting the CNS)
I. Einleitung
Akute-Phase-Proteine (APPs) sind Teil der Akute-Phase-Reaktion, eines phylogenetisch alten Anteils des unspezifischen Immunsystems. 1 Die Akute- Phase-Reaktion wird durch verschiedene systemische Erkrankungen ausgelöst, wie z.B. Entzündung, Neoplasie oder Trauma.1 Sie ist charakterisiert durch zahlreiche systemische Effekte, wie z.B. eine erhöhte Körpertemperatur, Anstieg der Leukozyten, Appetitlosigkeit, Depression und eine Erhöhung oder Senkung einer Vielzahl von Plasmaproteinen, sog. Akute-Phase-Proteinen (APP; Tizard
& Schubot, 2004). APP verhalten sich im Falle einer Erkrankung unterschiedlich. Die sog.
negativen APP zeigen bei Akuter-Phase-Reaktion eine Abnahme im Blut, die sog. positiven APP reagieren mit einer Zunahme. Zu den positiven APP gehören C-reaktives Protein (CRP), Serum Amyloid A (SAA), Haptoglobin (Hp), α1-saures Glycoprotein (AGP), Fibrinogen (Fb), Caeruloplasmin (Cp) und α2-Makroglobulin (AMG), zu den negativen zählen Transferrin und Albumin. Die Bedeutung von APP in der unspezifischen Immunantwort ist vielfältig. Einige APP zählen zu Komponenten des Komplement-Systems oder des Gerinnungssystems, andere gehören zu Protease-Inhibitoren oder metallbindenden Proteinen (Tizard & Schubot, 2004).
Die Aktivierung von APP erfolgt durch Makrophagen, die am Ort der entzündlichen Läsion Zytokine freisetzen, wie Interleukin-1, Interleukin-6 und Tumornekrosefaktor-α. Diese Zytokine stimulieren die Bildung von APP in Hepatozyten, außerdem auch in Leukozyten und in Zellen des Magen-Darm-Trakts.
Die labordiagnostische Bestimmung der APP im Blut wurde in den letzten fünf Jahrzenten in der Humanmedizin durchgeführt und findet auch in der Veterinärmedizin zunehmend
Verwendung1,3 . Die Bestimmung des APPs C-reaktives Protein (CRP) wird in der Humanmedizin dazu verwendet, zwischen viralen und bakteriellen Infektionen zu unterscheiden, z.B. bei Meningitiden oder Pneumonien.4,5
CRP ist sowohl beim Menschen als auch beim Hund ein sogenanntes Major-APP, d.h. es steigt bei einer Akute-Phase-Reaktion um ein Vielfaches an.4,6 So wurde bei infektiösen und immun-mediierten Entzündungen beim Hund eine massive Erhöhung des CRP im Serum festgestellt.7,8
Die Veränderung eines einzelnen APPs ist nicht spezifisch für eine bestimmte Erkrankung.
Daher wird empfohlen, ein sogenanntes APP-Profil zu erstellen. Dieses setzt sich zusammen aus APPs, die sich bei einer Akute-Phase-Reaktion um ein Vielfaches erhöhen (Major-APP), aus APPs, die nur moderat (Minor-APP) bzw. mit zeitlicher Verzögerung ansteigen und negativen APPs, also APPs, die bei einer Akute-Phase-Reaktion reduziert sind. So kann die Spezifität der Diagnostik nachweislich verbessert werden.3,9
Daher sollte in der vorliegenden Studie ein APP-Profil erstellt und auf seine diagnostische Aussagekraft geprüft werden. Dafür wurden neben CRP Alpha2 (α2)-Makroglobulin (AMG) und Albumin ausgewählt. AMG ist erhöht bei experimentell induzierter Entzündung bei Mäusen10 und bei neoplastischen Erkrankungen beim Menschen11, wogegen die AMG Konzentration bei Menschen mit Sepsis signifikant erniedrigt ist.12 Für den Hund konnte bisher nur in einer experimentellen Studie eine Abnahme von AMG gezeigt werden.13 Albumin ist als negatives APP aus der Literatur hinreichend bekannt.3,6,13
Die Steroid responsive Meningitis-Arteritis (SRMA, Steril-eitrige Meningitis-Arteritis) ist eine der meist diagnostizierten Meningitiden beim Hund.14 Die Erkrankung zeichnet sich durch eine Entzündung der Meningen und der meningealen Arterien aus.15,16 Die Diagnose wird erstellt auf der Grundlage typischer klinischer Symptome wie Halsbiegeschmerz, Fieber, Leukozytose und einer Zellzahlerhöhung im Liquor mit neutrophilen Granulozyten.
Darüberhinaus lassen sich in Liquor und Serum erhöhte IgA Werte nachweisen.17,18 Das Behandlungsmonitoring wird mittels Liquorkontrolluntersuchung alle 4-6 Wochen durchgeführt.17 Der Nachteil dieser Methode liegt in der erforderlichen Allgemeinanästhesie für die Liquorpunktion. Daher wäre es wünschenswert, eine weniger invasive Methode zum Therapiemonitoring bei Hunden mit SRMA zu finden.
Gegenstand der vorliegenden Studie war es, Konzentrationen von APP in Serum und Liquor von Hunden mit SRMA im Vergleich zu Hunden mit anderen neurologischen Erkrankungen zu bestimmen, und ihren Nutzen für die Diagnose und Therapiekontrolle der SRMA zu evaluieren.
Literatur zur Einleitung, die nicht im Manuskript erscheint:
Tizard, I.R. & Schubot, R.M. (2004) Veterinary Immunology: An Introduction, 7th edn., Saunders, Philadelphia
II. Manuskript
Eingereicht am 23.12.2007 und akzeptiert zur Veröffentlichung am 23.06.2008 (siehe S. 54 ).
Erschienen im „Journal of Veterinary Internal Medicine“, Impact Factor 1.776 (2008), J Vet Intern Med 2008; 22(5): 1149-1156
Concentrations of Acute-Phase Proteins in dogs with Steroid Responsive Meningitis-Arteritis
Andrea Bathen-Noethen1, 2, Regina Carlson1, Dirk Menzel1, Reinhard Mischke1, Andrea Tipold1
1Klinik für Kleintiere der Tierärztlichen Hochschule Hannover
2Tierarztpraxis Andrea Bathen-Noethen
Introduction
Acute phase proteins (APPs) are part of the acute phase reaction, a phylogenetically old component of the non-specific innate immune system.1 The acute phase response is generated by a variety of systemic diseases, such as inflammation, neoplasia or trauma.1 The measurement of these proteins has been used for diagnostic purposes during the last five decades in human and increasingly in veterinary medicine.1,3 For example, the determination of the APP C-reactive protein (CRP) concentration in serum is used to differentiate between viral and bacterial infections in humans, especially in patients with meningitis and pneumonia4,5. CRP is a major APP in humans and dogs, with a significant increase within an acute phase response.4,6 Indeed, a markedly elevated serum CRP concentration was found in infectious and immune-mediated diseases in dogs.7,8
The increase of one major APP is not disease-specific. Therefore, it is recommended to measure an APP-profile including positive and negative APPs, to improve the specificity of this diagnostic finding in respect of a special disease.3,9
Alpha2 (α2)-macroglobulin (AMG) is increased in experimental inflammation in mice10 and in neoplastic disease in humans11, whereas AMG concentration is significantly diminished in patients with sepsis.12 Experimentally induced inflammation in dogs causes a reduction of AMG.13 Albumin is known to decrease in acute phase response in dogs.3,6,13
Steroid responsive meningitis-arteritis (SRMA) is one of the most commonly diagnosed meningitides in dogs.14 The disease consists of an inflammation of the meninges and meningeal arteries.15,16 The diagnosis is confirmed by typical clinical signs such as cervical rigidity, fever, leucocytosis and pleocytosis with polymorphonuclear cells in the cerebrospinal fluid (CSF), and elevated IgA levels in serum and CSF.17,18 Treatment is monitored by repetitive CSF examinations.17 The disadvantage of this diagnostic procedure is the need for
general anaesthesia for CSF collection. Therefore, it would be useful to find a less invasive method for monitoring efficacy of treatment in dogs with SRMA.
The purpose of this study was to determine concentrations of APPs in serum and CSF of dogs with SRMA in comparison to dogs with other neurological diseases, and to evaluate their usefulness for diagnosis and during monitoring of treatment efficacy of SRMA.
Material and Methods Study Population
The study population was 133 dogs with varying CNS diseases, and six dogs with a diagnosis of sepsis, that were a positive control group, which were examined to the Department of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover, Germany, between September 2004 and August 2007. Additionally, eight healthy Beagles served as negative controls (animal experiment number 33.42502/05-12.05). The dogs were categorized as healthy on the basis of a complete physical examination, electrocardiographya, and blood biochemistry.
Of the dogs examined, 36 had SRMA, 14 had other meningoencephalitides (ME), 32 had intervertebral disk disease (IVDD) or degenerative lumbosacral stenosis (DLSS), 26 had primary or secondary tumors affecting the central nervous system (TCNS), and 25 had idiopathic epilepsy (IE). Thirty-one of the SRMA-dogs were also examined for monitoring of treatment efficacy. The treatment schedule used was as described.17 The monitoring period was 1 - 28 months (mean 5.4 months, 1 to 10 controls, respectively). Repeat examinations were performed four, eight, twelve and sixteen weeks after the first examination. Five dogs with SRMA suffered a relapse of SRMA during the period of study.
SRMA was diagnosed on the basis of characteristic clinical signs, CSF cell count (≥ 8/3/µl) with neutrophilic pleocytosis, and IgA concentrations ≥ 0.2 µg/ml in CSF and ≥ 100 µg/ml in serum.18 In dogs with other meningoencephalitides, intervertebral disk disease or degenerative lumbosacral stenosis, tumors affecting the CNS, and idiopathic epilepsy, diagnostic procedure consisted of physical and neurological examination, complete blood examinations, magnetic resonance imaging (MRI), and examination of CSF. In cases of canine distemper virus – encephalitis, histopathology and immunohistochemistry were performed. Furthermore,
diagnosis was confirmed by surgery in 23 cases with intervertebral disk disease and degenerative lumbosacral stenosis, and histopathology in 12 dogs with tumors affecting the CNS (2 meningiomas, 2 hemangiosarcomas and 1 nephroblastoma, malignant histiocytosis, ganglioneuroma, anaplastic ependymoma, anaplastic astrocytoma, malignant blastoma, osteosarcoma, or metastatic adenocarcinoma each). Dogs with sepsis were diagnosed on the basis of clinical signs, blood work and positive bacterial culture.
Sample Collection and Handling
Blood was collected by venipuncture. A complete blood cell count, blood biochemistry analysis (including serum-alkaline phosphatase, total protein, and albumin) was performed using routine methodsb, and in dogs with suspected SRMA, serum IgA concentration (enzyme linked immunosorbent assay, ELISA18) was also determined.
CSF was collected under general anaesthesia by suboccipital puncture with the dog in lateral recumbency and was analyzed for total nucleated cell count, cell differentiation, total protein concentration, IgA and CRP. CSF was considered normal with a total nucleated cell count <
8 cells/3/µl, a total red blood cell count < 12,000 cells/3/µl19, a total protein concentration <
25 mg/dl, and an IgA content < 0.2 µg/ml.
For measurement of CRP concentration, serum and CSF samples were separated into aliquots.
For measurement of AMG, citrated plasma (9 parts of blood are mixed with 1 part of 0.11 mol/l tri-sodium citrate solution, centrifuged for 20 minutes at 2000 x g) was used as sample material. All samples were stored frozen at -20 oC until analysis. The numbers of samples measured are described in Table 1.
Table 1
Number of samples measured for each variable in each disease; steroid responsive meningitis- arteritis: SRMA; other meningoencephalitides: ME; intervertebral disk disease/degenerative lumbosacral stenosis: IVDD/DLSS; tumors affecting the central nervous system: TCNS; idiopathic epilepsy: IE; C-reactive protein in the serum: s-CRP; C-reactive protein in the cerebrospinal fluid:
CSF CRP; α2-macroglobulin: AMG; albumin: Alb
Variable SRMA n = 36
ME n = 14
IVDD/DLSS n = 32
TCNS n = 26
IE n = 25
Normal n = 8
Sepsis n = 6
s-CRP 36 12 32 26 25 8 6
CSF CRP 35 13 32 26 25 8 3
AMG 17 11 32 21 25 8 6
Alb 31 14 30 23 24 8 6
C-reactive Protein
CRP was measured in serum and CSF samples with a commercially available ELISA kitc. As recommended by Cerón et al, a laboratory-specific reference range was created by measuring serum samples of the eight healthy Beagles.3 The detection limit was determined by making a serial dilution of serum of a known concentration. The lowest concentration with an optical density (O.D.) which was two-fold over the background O.D. was considered to be the detection limit of the assay (DL: 0.1 µg/ml).
α2-macroglobulin
The concentration of AMG was measured using a commercial chromogenic substrate assay kitd.
Diluted plasma is mixed with excess trypsin, and the AMG becomes complexed with trypsin.
The remaining, non-complexed trypsin is inhibited with soybean trypsin inhibitor. The trypsin complexed with the AMG is still able to cleave small substrates and releases p-nitroaniline (pNA) from a suitable chromogenic peptide substrate. The pNA concentration which is measured photometrically is proportional to the AMG concentration.20
The test was performed manually using a 96-well polystyrene microtitre plate. The test procedure followed the method description of the manufacturer with the following exceptions:
Test samples were diluted 1:80 instead of 1:160 due to the lower AMG activity in canine plasma when compared to human plasma. The lower sample dilution was also considered for the preparation of standards. Dilutions of a canine pooled plasma from equal aliquots from the citrated plasma of 100 healthy dogs were used as standards. Standard curves were prepared on the basis of the results of standards with the following activities: 200, 150, 125, 100, 75, 50, 25, and 0 %.
Statistical Analysis
Arithmetic means and medians were calculated using routine descriptive statistical procedures.
Statistical evaluation was performed by computer softwaree. Data were analyzed using the One-Way-ANOVA approach with grouping of dogs as classification factor and mean values (CRP, AMG, albumin) as dependent variables. The effect of age was tested as a covariate.
The Levene`s test was used to assess the equality of variance in different samples. The
P-value was highly significant (P < .001), therefore the null hypothesis (no difference between neurological diseases) of equal variances was rejected and the level of significance of the ANOVA was lowered to 1 %. R2 proved that the total variance of parameters can be explained by difference of groups of diseases; R2 indicates the goodness of model fit. Multiple comparisons between the groups of diseases were performed by tests following Scheffé for unbalanced models. An error probability of .01 (P) was used as the significance level for ANOVA and Scheffé-Test.
An unpaired t-test to compare concentrations of serum CRP and alkaline phosphatase between SRMA-dogs with and without glucocorticosteroid pre-treatment and between dogs with relapse and unremarkable recovery during monitoring of treatment efficacy of SRMA was performed.
Analyses of associations of blood and CSF values (serum CRP, CSF CRP, CSF total nucleated cells, CSF neutrophils, CSF erythrocytes, CSF total protein, blood leucocytes, serum total protein, serum albumin, serum alkaline phosphatase, CSF IgA, serum IgA) within the SRMA-group were performed by calculating Pearson correlation coefficients with a significance level of P < .05.
Boxplots were used to visualize minimal and maximal values, median of the values. The box contains the middle 50% of the sample values.
Results
Breeds, Age, Sex, and Weight
In the presented SRMA population Jack Russell Terriers were affected in a proportion similar to boxers and beagles (Table 2). 17,21
Table 2
Breed distribution of 133 dogs with neurological diseases; steroid responsive meningitis-arteritis:
SRMA; other meningoencephalitides: ME; intervertebral disk disease/degenerative lumbosacral stenosis: IVDD/DLSS; tumors affecting the central nervous system: TCNS; idiopathic epilepsy: IE
Breed SRMA
n=36
ME n=14
IVDD/DLSS n=32
TCNS n=26
IE n= 25
Beagle 5 -- 1 -- 1
Boxer 5 -- -- 1
Jack Russell Terrier
4 3 1 -- --
Dachshund -- -- 5 -- --
Westhighland White Terrier
-- 4 1 2
Golden Retriever
2 -- 2 -- 2
Labrador 2 -- -- 1 1
Cocker Spaniel
2 -- -- -- 1
Doberman -- -- 2 -- --
German Shepherd
Dog
1 -- 3 -- --
Mix 6 6 7 10 4
Further breeds (n = 1)
Bernese Mountain Dog, German Pinscher, Nova Scotia Duck Tolling
Airedale Terrier
Bernese
Mountain Dog, Russian
Terrier, Malinois, Bobtail, Small
Saluki, Irish Wolfhound, Barsoi, Rottweiler, Greyhound, Border Collie,
Pyrenean Mountain dog, Cairn Terrier, Eurasier, Great Dane, Scottish
Weimaraner, Brandlbracke, Miniature Pinscher, German Shorthair Pointer, German Wirehaired Pointer
Hovawart, Miniature Schnauzer, Briard, Fila Brasileiro
Small
Munsterlander, Mastino, Appenzeller Mountain Dog, Bullmastiff, Malinois
Shepherd, Groenendal, Hovawart, Swiss Mountain Dog, Gordon Setter,
Brandlbracke, Magyar Viszla, Yorkshire Terrier, Border Collie, French
Bulldog, German Wirehaired Pointer
Dogs with SRMA had a median age of ten months (25-75% quantile range, 8-14.5 months), and were younger than the dogs with other neurological diseases (other meningoencephalitides median 42.5 months, 21-74.3 months; intervertebral disk disease/
degenerative lumbosacral stenosis 87 months, 44.5-116 months; tumors affecting the CNS 96 months, 70-115 months; idiopathic epilepsy 39 months, 21-68.5 months, respectively). The SRMA group consisted of 13 females, one spayed female and 22 males. The median weight of the SRMA group was 19 kg (25-75% quantile range 10-27 kg; other meningoencephalitides 11.6 kg, 8.1-19 kg; intervertebral disk disease/degenerative lumbosacral stenosis 25.7 kg, 9-39.2 kg; tumors affecting the CNS 27.5 kg, 16.8-35.6 kg;
idiopathic epilepsy 28.5 kg, 12.8-37.5 kg, respectively).
Serum CRP Concentrations
The laboratory specific reference range for serum CRP was 0.3 to 0.9 µg/ml. Serum CRP was significantly higher in dogs with SRMA than in dogs with other neurological diseases and
negative controls (mean SRMA: 142 µg/ml ± 75, P < .001), but did not differ from values of dogs with sepsis (mean Sepsis: 114 µg/ml ± 67, P = .941; Fig 1). Within the group of dogs with SRMA, CRP was not significantly different between dogs pre-treated with glucocorticosteroids and dogs which were not treated with glucocorticosteroids before diagnosis (meanpre-treated = 163 µg/ml ± 98, meannon-pre-treated = 131 µg/ml ± 55, P = .242).
There was no effect of age on serum CRP values.
Fig 1 Serum C-reactive protein (CRP) concentrations in the groups of dogs at the time of diagnosis
(steroid responsive meningitis-arteritis: SRMA n = 36; other meningoencephalitides: ME n = 12;
intervertebral disk disease/degenerative lumbosacral stenosis: IVDD/DLSS n = 32; tumors affecting the central nervous system: TCNS n = 26; idiopathic epilepsy: IE n = 25; healthy controls: Normal n = 8, dogs with sepsis: Sepsis n = 6); Boxplots: median of values, minimal and maximal values, boxes contain the middle 50% of sample values. Groups SRMA, Sepsis differed significantly from other groups (P < .001; )
CSF CRP Concentrations
The healthy Beagles had CRP concentrations in CSF that were below the detection limit.
Therefore, a nondetectable CRP concentration (< 0.1 µg/ml) in the CSF was considered to be normal. All the dogs of the groups of intervertebral disk disease/degenerative lumbosacral stenosis and idiopathic epilepsy had nondetectable CRP values in the CSF. Six dogs with SRMA had undetectable CRP in CSF. In 29 dogs with SRMA, the values ranged from 0.19 up to 13.6 µg/ml. In SRMA, CRP values in the CSF were significantly higher than in the groups disk disease, CNS tumors, and idiopathic epilepsy (mean SRMA= 1.59 ± 0.48 µg/ml, meanIVDD/DLSS= 0 µg/ml, P < .01, meanTCNS= 0.07 µg/ml ± 0.04, P = .01, meanIE= 0 µg/ml, P < .01; Fig 2). Because of high intra-group differences (81 %), only 19 % of variance could be explained by disease grouping. In dogs with various CNS tumors (n=26), CRP in the CSF was non-detectable (n = 22), with the exception of two meningiomas and two hemangiosarcomas (1.03, 0.23 µg/ml, and 0.53, 0.1 µg/ml). It was remarkable, that in two of three dogs with sepsis, the concentration was also equal to or above the detection limit (0.1 and 0.14 µg/ml).
Fig 2 C-reactive protein (CRP) concentrations in the cerebrospinal fluid (CSF) in the groups of dogs at
the time of diagnosis (steroid responsive meningitis-arteritis: SRMA n = 35; other meningoencephalitides: ME n = 13; intervertebral disk disease/degenerative lumbosacral stenosis:
IVDD/DLSS n = 32; tumors affecting the central nervous system: TCNS n = 26; idiopathic epilepsy: IE n = 25; healthy controls: Normal n = 8, dogs with sepsis: Sepsis n = 3); Boxplots: median of values, minimal and maximal values, boxes contain the middle 50% of sample values. Groups with different letters (A, B) are significantly different at P ≤ .01.
Plasma AMG Concentrations
No significant differences were detected between the groups (Fig 3). Results for dogs with SRMA or sepsis did not differ from healthy controls (meanSRMA= 109 % ± 20, meanSepsis= 86
% ± 54, meanNormal= 91 % ± 17). The age of the dogs had no effect on AMG values.
Fig 3 Plasma α2-macroglobulin (AMG) concentrations in the groups of dogs at the time of diagnosis (steroid responsive meningitis-arteritis: SRMA n = 17; other meningoencephalitides: ME n = 11;
intervertebral disk disease/degenerative lumbosacral stenosis: IVDD/DLSS n = 32; tumors affecting the central nervous system: TCNS n = 21; idiopathic epilepsy: IE n = 25; healthy controls: Normal n = 8; dogs with sepsis: Sepsis n = 6); Boxplots: median of values, minimal and maximal values, boxes contain the middle 50% of sample values.
Serum Albumin Concentrations
Dogs with SRMA had albumin concentrations significantly lower than those in dogs with other meningoencephalitides, intervertebral disk disease/degenerative lumbosacral stenosis, and tumors of the CNS (meanSRMA = 3.20 g/dl ± 0.412 g/dl, P < .01; Fig 4). Septic dogs had lower albumin concentrations but without significance because of a high standard deviation (meanSepsis = 3.16 g/dl ± 0.899 , P ≥ .07; Fig 4). The age of the dogs did not influence these results.
Fig 4 Serum albumin concentrations in the groups of dogs at the time of diagnosis (steroid responsive
meningitis-arteritis: SRMA n = 31; other meningoencephalitides: ME n = 14; intervertebral disk disease/degenerative lumbosacral stenosis: IVDD/DLSS n = 30; tumors affecting the central nervous system: TCNS n = 23; idiopathic epilepsy: IE n = 24; healthy controls: Normal n = 8, dogs with sepsis:
Sepsis n = 6); Boxplots: median of values, minimal and maximal values, boxes contain the middle 50% of sample values. Groups with different letters (A, B) are significantly different at P < .01.
Results of Blood and CSF examinations in the SRMA Group
Dogs from the SRMA-group had leucocytosis (n = 34), increased alkaline phosphatase (n = 23; medianleucocytes = 26,620/µl, range 11,500-48,060, ref. 6,000-12,000/µl; medianalkaline
phosphatase = 231 U/l, range 82-553 , ref. < 150 U/l) activity, or both. The serum alkaline phosphatase activity in dogs pre-treated with glucocorticosteroids was not higher than in dogs not pre-treated with glucocorticosteroids (meanpre-treated = 245 U/l ± 119, meannon-pre-treated = 241 U/l ± 122, P = .925).
Dogs with SRMA had high numbers of nucleated cells with a neutrophilic pleocytosis within the CSF (mediantotal nucleated cells = 1,775/3/µl, range 6-9,720, ref. < 8/3/µl; medianneutrophils = 79
%, range 34-100). The median of total protein concentration of the CSF was 66 mg/dl ( range 7-1333, ref. < 25 mg/dl). In many cases, the CSF was xanthochromic, suggestive of subarachnoid bleeding (medianerythrocytes = 235/3/µl, range 0-76,800; ref. < 12,000/3/µl19). IgA levels in serum and CSF were high in this disease group (medianIgA serum=188 µg/ml, range 40-994, ref. < 100 µg/ml; medianIgA CSF =2 µg/ml, range 0.06-14.77, ref. < 0.2 µg/ml18).
Correlations in the SRMA group
There was only a weak, non-significant correlation between the concentration of CRP in serum and CSF (r = .301, P = .084) in dogs with SRMA. There was no association between concentration of CRP in serum and other characteristics of the CSF, such as CSF cell count, CSF neutrophils, the total protein content of the CSF, or IgA. There was also no correlation between CRP in serum and total protein content or albumin in the blood. However, there was an association between CRP in serum and serum alkaline phosphatase (r =.515, P = .003; Fig 5). The CRP concentration in the CSF was significantly correlated to the number of erythrocytes in the CSF (r = .382, P = .026) and a weak correlation without significance could be found between CRP in the CSF and the total protein content of the CSF (r = .308, P = .072).
Fig 5 Serum C-reactive protein (CRP) concentration and serum alkaline phosphatase activity
correlated highly significant in dogs suffering from steroid responsive meningitis-arteritis (SRMA).
Scatter plot of 31 paired measurements of serum alkaline phosphatase activity and serum CRP concentration, showing the regression line (solid) with 95% confidence intervals (dashed lines). The linear regression equation was y = 63.32 + 0.3409x, R² = 0.265, P = .003.
Monitoring of Treatment Efficacy in Dogs with SRMA
Serum CRP decreased markedly from the time of diagnosis until the first repeat examination in 30 of 31 dogs. The remaining dog had a relapse four weeks after diagnosis. The decrease of serum CRP of the clinically normal dogs was in the same relative dimension as the decline of total nucleated cell count in the CSF (Fig 6 a). During monitoring of treatment efficacy, the concentration of serum CRP and the total nucleated cell count remained within the reference range (0.3-0.9 µg/ml; < 8 cells/3/µl).
Fig 6 a Course of serum C-reactive protein (CRP) and CSF total nucleated cell count in dogs with
steroid responsive meningitis-arteritis (SRMA) at the time of diagnosis and at monitoring of treatment efficacy; O: diagnosis, I: 4 weeks after diagnosis, II: 8 weeks, III: 12 weeks, and IV: 16 weeks;
reference range CRP 0.3 – 0.9 µg/ml, total nucleated cell count < 8 cells/3/µl; values as means.
There was no difference in the concentration of serum CRP between the dogs which recovered and the dogs which showed a relapse at the time of diagnosis and the second control (P = .36, P = .49, respectively; Fig 6 b), though there was a tendency towards higher serum CRP levels in the group of dogs, which showed a relapse later on, at the time of the first control (mean recovery = 0.31 µg/ml ± 0.22, mean future relapse = 1.05 µg/ml ± 0.79, P = .063;
Fig 6 b). In four out of five cases a markedly increased serum CRP could be measured at the time of relapse (Fig 6 b).
Fig 6 b Course of serum C-reactive protein (CRP) in dogs with steroid responsive meningitis-arteritis
(SRMA) which made a full recovery and dogs which showed a relapse within the observation period . Serum CRP at the time of relapse . O: diagnosis, I: 4 weeks after diagnosis, II: 8 weeks, III: 12 weeks, and IV: 16 weeks, reference range CRP 0.3 – 0.9 µg/ml; values as means.
Discussion
In the present study a marked acute phase response in dogs with SRMA could be shown supporting the hypothesis that SRMA is a systemic disease, or part of the systemic inflammatory response syndrome (SIRS).
CRP is an indicative parameter for the acute phase response in dogs with SRMA, whereas AMG seems not to be a valuable APP in neurological disorders in the dog. Thus, there is still no evidence, that AMG is changed in acute phase response in naturally diseased dogs at all.
Albumin levels - as a negative APP – tally with the findings of CRP levels, but no significant difference between SRMA dogs and negative controls could be found. Therefore, a decreased concentration of serum albumin could only support but not lead to a diagnosis of systemic inflammation.
Though the elevation of the concentration of serum CRP in dogs suffering from SRMA was very impressive, other systemic inflammatory diseases such as sepsis cannot be differentiated using this test. This limitation was also found in other studies examining the diagnostic use of CRP in dogs.3
CRP in the CSF was high in the SRMA-group. In this group, arteritis is expected and can lead to an increased risk of subarachnoid bleeding resulting in xanthochromic CSF.16,21,22 In fact, a correlation between CRP concentration in the CSF and the number of erythrocytes in the CSF could be shown (r = 0.382; P = .026), even though the erythrocyte count was still within the reference range. Hence, it is possible, that the elevated concentration of CRP in the CSF originates from subarachnoid bleeding. This corresponds to the findings in two dogs with sepsis, where CRP was also elevated in the CSF. In sepsis, a secondary CNS-vasculitis with increased permeability or a deranged function of the blood brain barrier may occur and could explain an elevation of CRP in the CSF. 23
Another explanation for the increased CRP values in CSF samples could be an intrathecal production, e.g. by leucocytes in the meninges. A production of APPs by leucocytes was suspected in former studies. 24,25 Determining a CRP-index in accordance with the IgG- index26 would be useful in further studies for deciding whether the CRP in the CSF originates from the blood or from intrathecal production. In addition to dogs with SRMA, CRP in the CSF was markedly increased in four dogs with tumors affecting the CNS, two with meningioma, and two with hemangiosarcoma. These are two types of tumors, where an alteration of the blood-brain-barrier or hemorrhage might occur. 27,28
Hepatocytes are known to produce CRP.1,2 Therefore, it is not surprising, that serum CRP and alkaline phosphatase correlated highly significantly. Both parameters are supposed to be induced by proinflammatory mediators, especially interleukin-1, in liver cells.29
An alternative explanation for a high CRP and a high alkaline phosphatase is pre-treatment with glucocorticosteroids. It is well known, that in dogs alkaline phosphatase synthesis is induced by glucocorticosteroids. 30 Furthermore, glucocorticosteroids generally enhance the stimulatory effects of cytokines on the production of APPs. 31 Otherwise, production of cytokines is downregulated by glucocorticosteroids.Though Martínez-Subiela et al32 showed, that CRP concentration in the blood of dogs is not affected by glucocorticosteroids, others supposed an association, although in pigs33. In the underlying study population, no difference between dogs with pre-treatment with glucocorticosteroids and dogs without pre-treatment could be shown regarding the concentration of serum CRP and alkaline phosphatase (P = .242; P = .925). Thus, in the current study, glucocorticosteroids had no effect on CRP levels in the blood.
Besides its usefulness in diagnostics, measurement of CRP is used to monitor treatment success and as a predicting parameter for healing and treatment failure in human and
veterinary medicine.34-37 In all these studies, a marked decline in the CRP concentration could be observed between the time of diagnosis and treatment control. This corresponds to the findings of the present study in SRMA: A perceptible decrease in serum CRP parallels the decline of CSF cell count after initiation of treatment. Therefore, measurement of serum CRP can be considered to be a useful monitoring parameter in SRMA. Up to now, remission of clinical signs and number of nucleated cells in the CSF have been the only parameters for monitoring the treatment schedule. 17 As serum CRP concentrations decline in the same way as cells in the CSF during treatment and as both analytes increased again in cases of relapse in the dogs of this study, serum CRP concentrations could be considered as a valuable tool in diagnosing relapses, albeit other inflammatory diseases have to be ruled out.
For management of dogs with SRMA, it would be beneficial to have a laboratory parameter, which could predict a relapse before recurrence of clinical signs. At the first repeat examination for monitoring of treatment efficacy, serum CRP levels in dogs suffering from a relapse during the observation time showed a tendency towards higher CRP levels in comparison to dogs which made a full, unremarkable recovery. This difference was not observed anymore comparing these two groups four weeks later. CRP in the dog is known to increase significantly four hours after stimuli with a maximum peak 24 hours after surgical trauma38 and 48 hours after turpentine oil injection.6 In the latter study, the amount of serum CRP decreased markedly during the following days and reached nearly normal values after approximately three weeks. Therefore, measuring CRP values to monitor treatment of SRMA can only be an indicator for the current status of the disease, but clearly predicting relapses seems not to be feasible.
CRP was markedly elevated in serum and CSF of dogs with SRMA in contrast to dogs with other neurological diseases. Hence, CRP seems to be a valuable parameter for SRMA in
comparison to other neurological diseases. However, other systemic diseases of the SIRS complex have to be ruled out. Serum albumin values support these findings as they were significantly lower in dogs with SRMA than in dogs with other neurological diseases. Since albumin as a negative acute-phase protein was also lower in septic dogs, the measurement of a combination of CRP and albumin does not help distinguishing these two groups of diseases.
AMG-values did not differ between groups of dogs with neurological diseases. A combination of measuring CRP, albumin and AMG as an APP profile unfortunately could not improve the diagnostic workup of SRMA in comparison to the determination of serum CRP alone. In conclusion, measuring CRP as an acute phase protein supports the diagnosis of SRMA very well, but cannot be used as a single diagnostic tool.
Footnotes
a MAC 5000 marquette, GE Medical Systems, IT GmbH Freiburg, Germany
b Hitachi 912, Boehringer-Mannheim, Germany
c Tridelta development Ltd, Kildare, Ireland
d UnitestTM, Unicorn Diagnostics, Kent, UK
e SPSS, version 13.0 for Windows; SPSS Inc., Chicago, IL
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III. Zusammenfassung
Andrea Bathen-Nöthen
Konzentrationen von Akute-Phase-Proteinen bei Hunden mit Steroid Responsiver Meningitis- Arteritis
Akute-Phase-Proteine (APP) werden im Zusammenhang mit der Akute-Phase-Reaktion vom Körper synthetisiert. Die Bezeichnung Akute-Phase-Reaktion bezieht sich auf die unspezifische Immunabwehr eines Wirtes kurz nach einem Gewebstrauma im Rahmen einer Entzündungsreaktion. Sie ist charakterisiert durch zahlreiche systemische Effekte, wie z.B.
eine erhöhte Körpertemperatur, Anstieg der Leukozyten, Appetitlosigkeit, Depression und eine Erhöhung oder Senkung einer Vielzahl von Plasmaproteinen, sog. Akute-Phase- Proteinen.
Seit über 50 Jahren werden APP in der Humanmedizin in der Diagnostik eingesetzt, seit ungefähr 20 Jahren finden sie auch in der Veterinärmedizin zunehmend Verwendung in der Diagnostik und auch zur Therapiekontrolle. Besonders bei bakteriellen Entzündungen lassen sich hohe Konzentrationen von APP im Serum finden. Daher eignet sich z.B. die CRP- Bestimmung (C-reaktives Protein) zur Differenzierung zwischen viraler und bakterieller Genese von Meningitiden oder Pneumonien.
In der vorliegenden Studie sollte der diagnostische Nutzen verschiedener APP bei der Steroid Responsiven Meningitis-Arteritis (SRMA) im Vergleich zu anderen neurologischen Erkrankungen untersucht werden. Dazu wurden bei 133 Hunden mit
neurologischen Erkrankungen, sechs Hunden mit Sepsis und 8 gesunden Kontrolltieren CRP, α2-Makroglobulin (AMG) und Albumin im Serum/Plasma, und CRP darüber hinaus auch im Liquor cerebrospinalis bestimmt. 36 der neurologisch erkrankten Tiere hatten SRMA (bei 31 dieser Hunde wurden ebenfalls Therapiekontrollen durchgeführt), 14 Tiere hatten Meningitiden unklarer Genese, 32 Hunde litten an degenerativen Bandscheibenerkrankungen, 26 Hunde hatten primäre oder sekundäre Tumore des zentralen Nervensystems (ZNS) und 25 Hunde idiopathische Epilepsie.
Die Messung von CRP im Serum zeigte hochsignifikant höhere Werte bei den an SRMA (x= 142 µg/ml ± 75) und Sepsis (x= 114 µg/ml ± 67) erkrankten Tieren im Vergleich zu den Hunden mit anderen neurologischen Erkrankungen (x= 2.3 - 21 µg/ml; P < .001) und den gesunden Kontrolltieren. Die Serum CRP Konzentration bei SRMA Hunden korrelierte positiv mit Serumwerten der alkalischen Phosphatase (r = .515, P = .003).
Bei der Messung von CRP im Liquor wurde die Nachweisgrenze bei den gesunden Tieren nicht überschritten (<0,1 µg/ml). Liquor-CRP Werte waren bei SRMA Hunden am höchsten. Sie waren signifikant höher als bei Hunden mit Bandscheibenerkrankungen, ZNS Tumoren und idiopathischer Epilepsie (xSRMA= 1.59 ± 0.48 µg/ml, xIVDD/DLSS= 0 µg/ml, P < .01, xTCNS= 0.07 µg/ml ± 0.04, P = .01, xIE= 0 µg/ml, P < .01).
Bezüglich AMG im Plasma konnte kein signifikanter Unterschied zwischen den Gruppen gefunden werden. Die Albumin Konzentration im Serum war signifikant niedriger bei SRMA-Patienten (x= 3.2 g/dl ± 0.41) als bei Hunden mit anderen neurologischen Erkrankungen (x= 3.6 - 3.9 g/dl, P < .01).
Bei den SRMA-Hunden mit Kontrolluntersuchungen unter Therapie konnte ein dramatischer Abfall der Serum-CRP Werte zwischen dem Zeitpunkt der Diagnose und der ersten
Therapiekontrolle nach vier Wochen festgestellt werden. Bei Rückfällen stiegen die Werte wieder an.
In vorliegender Arbeit konnte daher gezeigt werden, dass CRP Konzentrationen in Serum und Liquor sinnvolle Parameter sowohl bei der Diagnosestellung als auch bei der
Therapiekontrolle der SRMA beim Hund sind.
Abstract (Manuskript)
Andrea Bathen-Nöthen
Concentrations of Acute-Phase Proteins in dogs with Steroid Responsive Meningitis- Arteritis
Measurement of concentrations of acute-phase proteins (APPs) is used as an aid in the diagnosis of a variety of diseases of animals.
To determine concentration of APPs in dogs with steroid responsive meningitis-arteritis (SRMA) and other neurological diseases.
133 dogs with neurological diseases, 6 dogs with sepsis, and 8 healthy dogs were included in the study. Thirty-six dogs had SRMA (31 of which had monitoring), 14 dogs had other meningoencephalitides (ME), 32 had degenerative disk disease (IVDD), 26 had tumors affecting the central nervous system (TCNS), and 25 had idiopathic epilepsy (IE).
Prospective, observational study. C-reactive protein (CRP), α2-macroglobulin (AMG) and albumin concentrations were determined in the serum or plasma. CRP was also measured in the cerebrospinal fluid.
Serum CRP was significantly higher in dogs with SRMA (x= 142 µg/ml ± 75) and sepsis (x= 114 µg/ml ± 67) in comparison to dogs with other neurological diseases (x= 2.3 - 21 µg/ml; P < .001). There was no significant difference detected in AMG between groups.
Serum albumin concentration was significantly lower (P < .01) in dogs with SRMA (x= 3.2 g/dl ± 0.41) than in other groups (x= 3.6 - 3.9 g/dl). Serum CRP concentration of SRMA- dogs correlated with alkaline phosphatase levels (r = .515, P = .003).
Conclusions and clinical importance: CRP concentrations in serum are useful in diagnosis of dogs with SRMA. Serum CRP could be used as a monitoring parameter in treatment management of these dogs.
IV. Anhang
Im Anhang befinden sich die Originaldaten aller 147 Hunde der Studie (Tabelle A-E).
Abkürzungsverzeichnis
AMG α2-Makroglobulin
CRP C-reaktives Protein
IE Idiopathische Epilepsie
IVDD/DLSS Intervertebral Disk Disease/Degenerative Lumbosakrale Stenose
m männlich
ME Meningoenzephalitis unklarer Genese
mk männlich kastriert
Negativ Kontrollgruppe mit gesunden Tieren
Nr. Probandennummer des Hundes
Sepsis Kontrollgruppe mit an Sepsis erkrankten Hunden
SRMA Steroid Responsive Meningitis-Arteritis
TZNS primäre und sekundäre Tumoren des ZNS
W weiblich
wk weiblich kastriert
ZNS Zentrales Nervensystem
Tabelle A
Nr. Rasse Geschlecht
Alter bei Diagnose
in Monaten Gewicht in kg SRMA
1 Mix m 13 51
2 Berner Sennenhund m 8 27
3 Beagle m 9 18
4 Mix w 5 20
5 Golden Retriever m 21 27
6 Mix m 12 28
7 Beagle m 9 18
8 Labrador m 8 27
9 Deutsch Drahthaar m 64 34
10 Golden Retriever w 19 35
11 Deutscher Pinscher m 11 19
12 Beagle m 11 21
13 Boxer w 11 26
14 Beagle m 7 19
15 Nova Retriever m 10 16
16 Cocker Spaniel m 8 10
17 Deutsch Kurzhaar m 10 28
18 Griffon m 12 14
19 Mix m 5 17
20 Jack Russell Terrier w 11 5
21 Labrador w 10 10
22 Jack Russell Terrier w 67 6
23 Cocker Spaniel m 15 11
24 Weimaraner w 7 23
25 Beagle m 6 9,2
26 Boxer m 8 34,5
27 Deutscher Schäferhund w 9 28
28 Mix w 10 8
29 Boxer m 16 37
30 Brandelbracke w 9 18,5
31 Mix w 15 19
32 Jack Russell Terrier m 18 10
33 Boxer w 8 15
34 Jack Russell Terrier wk 11 5
35 Zwergpinscher m 17 2,5
36 Boxer w 8 21
ME
37 Mix w 12 15,5
38 Mix w 27 18
39 Airedale Terrier m 24 22
40 West Highland White Terrier wk 89 8
41 Jack Russell Terrier m 10 7,4
42 Collie-Mix m 47 27,6
43 West Highland White Terrier m 129 10,7
44 Mix w 5 24
45 Jack Russell Terrier mk 34 9
46 West Highland White Terrier w 38 10
47 Jack Russell Terrier w 81 8,15
48 West Highland White Terrier mk 51 12,6
49 Mastiff-Mix m 48 2,2
Signalement der Hunde mit neurologischen Erkrankungen (SRMA: Steroid Responsive Meningitis Arteritis, ME:
Meningoenzephalitis unklarer Genese, IVDD/DLSS: intervertebral disk disease/ lumbosakrale Stenose, TZNS:
primäre und sekundäre Tumoren des ZNS, IE: Idiopathische Epilepsie), Negativ- (Normal) und Positivkontrollen (Sepsis)
Tabelle A - Fortsetzung
Nr. Rasse Geschlecht
Alter bei Diagnose
in Monaten Gewicht in kg IVDD/DLSS
51 Mix m 122 19
52 Russisch Terrier m 21 47
53 Dobermann mk 17 23,5
54 Malinois m 37 36,4
55 Bobtail wk 115 29,3
56 Deutscher Schäferhund wk 44 34
57 Kleiner Münsterländer mk 96 31,5
58 West Highland White Terrier m 127 9
59 Langhaarteckel mk 135 9,1
60 Dobermann m 89 50
61 Deutscher Schäferhund m 89 49
62 Cairn Terrier mk 114 9
63 Langhaarteckel m 109 7,3
64 Golden Retriever mk 133 33
65 Berner Sennenhund m 27 44
66 Kurzhaarteckel m 34 6,3
67 Mix m 116 30
68 Deutscher Schäferhund m 46 43
69 Golden Retriever mk 83 40,8
70 Mix mk 44 28
71 Rauhhaarteckel m 122 12,7
72 Hovawart w 104 33
73 Zwergschnauzer mk 123 10,75
74 Mix m 73 7
75 Briard m 44 40,15
76 Fila Brasileiro w 85 52,1
77 Mix wk 132 10,7
78 Mix w 96 7,3
79 Mix w 54 8,55
80 Jack Russell Terrier m 72 7,2
81 Beagle w 60 15
82 Rauhhaarteckel mk 74 8,2
TZNS
83 Saluki m 113 24
84 Irischer Wolfshund w 3 14,2
85 Deutscher Schäferhund-Mix wk 156 40,5
86 Labrador m 72 30,6
87 Barsoi m 78 35,5
88 Rottweiler m 108 50
89 Greyhound wk 120 20,3
90 Mix mk 120 9,1
91 Border Collie m 12 16,1
92 Mix wk 111 14,2
93 Riesenschnauzer w 86 36
94 Kleiner Münsterländer w 60 17
95 Mastino m 96 60
96 Appenzeller wk 108 26,5
97 Bullmastiff mk 96 47
98 West Highland White Terrier m 84 9,1
99 Mix wk 35 28
100 Mix wk 81 34,5
101 Mix w 33 28,5
102 Boxer m 74 32
103 Mix mk 96 35
104 Mix w 144 26
105 Mix wk 156 47,5
106 Deutscher Schäferhund-Mix wk 128 27
