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The collection and analysis of cerebrospinal fluid as an aid to diagnosis in ruminant neurological disease
137"vet..[. (1995). 151,603
REVIEW T H E COIJ,F, C T I O N A N D ANALYSIS OF CEREBROSPINAL FLUID AS A N AID T O D I A G N O S I S IN R U M I N A N T N E U R O L O G I C A L DISEASE
P. R. SCOTT Department of Veterina U Clinical Studies, Royal (Dick) School of Veterinary Studies, Veterina U FieM Station, Easter Bush, Roslin, Midlothian EH25 9RG, Scotland
SUMMARY
In ruminant species cerebrospinal fluid (CSF) collection and analysis prorides rapid and, in some situations, instant information to the veterinary clinician investigating a disease problem in the living animal. CSF analysis is particularly useful with respect to confirming the presence of an inflammatory lesion involving the leptomeninges such as bacterial meningoencephalitis. When correctly performed under local anaesthesia, lumbosacral CSF collection in ruminants is a safe procedure and there are no harmful sequelae. There are few indications for cisternal CSF collection in food animal practice. IZ~WORDS:Cerebrospinal fluid; nervous disease; cattle; sheep.
INTRODUCTION
Altered mental state, particularly depression, is a common feature of many disease processes. The investigation of possible central nervous system (CNS) disease involves a detailed assessment of the history and a complete physical and neurological examination of the animal (Mayhew, 1989; George, 1990). Despite such investigations, the data collected in neurological disease cases may not be pathognomic. In many situations the response to treatment is used as an adjunct to diagnosis (Little & Sorenson, 1969) but commonly a variety of drugs are administered simultaneously (Power el al., 1985) which limits the interpretation of the outcome. A specific diagnosis of the cause of the neurological signs is often only reached in fatal cases which are examined in detail at necropsy. Histopathological examination of the CNS is time-consuming, expensive and requires expert interpretation. In the absence of information to guide the pathologist to potentially affected area(s) of the brain, multiple tissue sections are required, which usually renders this investigative procedure prohibitive on the basis of cost. 0007-1935/95/060603-12/$12.00/0
© 1995 Bailli6reTindall
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Man), neurological diseases of cattle and sheep occur as outbreaks. It is, therefore, essential to reach an accurate diagnosis as soon as possible in order to expedite preventive and control measures. Cerebrospinal fluid (CSF) can be obtained from li~6ng animals with analysis results available within 1 h of submission to the laboratory. ~qaile there are many references to the principles of CSF collection and analysis in ruminant neurological disease (see, for example, Tvedten, 1987; George, 1990; Holbrook & White, 1992), there are few studies which report actual results (Binkhorst, 1982; Rebhun & de Lahunta, 1982; Scott et al., 1990; Scott, 1992; Green & Smith, 1992; Scott & Penny, 1993). Tiffs article reviews the recent literature on CSF collection techniques, analytical procedures and intepretation of results. The usefulness of CSF analysis in providing the veterinarian in general practice with diagnostic and prognostic information is discussed.
Formation of CSF CSF is formed largely from the choroid plexuses of the lateral ventricles by the ultrafiltration of plasma and the active transport of selected substances across the blood-brain barrier (Cserr, 1971; Milhorat, 1975). The CSF in the ventricular system flows caudally and diffuses out of the lateral aperture in the fourth ventricle to circulate around the brain and spinal cord. The presence of CSF in the subarachnoid space separates the brain and spinal cord fi'om the bony craniuna and vertebral column which reduces traulna to the underlying delicate nervous tissue. CSF also has excretol T functions with the removal of products of cerebral metabolism.
Collection of CSF in ruminant species In contrast to companion animals, lumbosacral CSF can be collected from ruminant species under local anaesthesia (Scott et al., 1990; Scott, 1992). The risks associated with general anaesthesia, particularly in an alfimal with possible CNS dysfunction, are thereby avoided. Adequate restraint and identification of bony landmarks are essential during the sampling procedure. In order to appreciate the relevant anatomical structures it is recommended that the technique is first attempted on cadavers. For CSF collection and examination it is necessary to puncture the subarachnoid space in the cerebellomedullary cistern (cisternal sample) or at the lumbosacral site (lumbosacral sample). In the absence of a focal spinal cord compressive lesion there is usually no substantial difference between the composition of cisternal and lumbosacral CSF samples (Scott & Will, 1991). While theoretically it may be desirable to collect CSF fi'om the site nearer the suspected location of the lesion, this is not possible in the field. In those runfinant neurological diseases studied by the author, a lumbosacral CSF sample was representative. Thus the risk of needle penetration of the brainstem associated with cisternal puncture is difficult to justify. Collection of lumbosacral CSF is facilitated when the animal is positioned in sternal recumbency with the hips flexed and the pelvic limbs extended alongside the abdomen. Aversion of the animal's head against the flank may assist in maintaining sternal recumbency during the collection procedure. Alternatively, lambs
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and kids can be restrained in lateral recumbency. In adult cattle that are still ambulatory, the lumbosacrai CSF sample is collected in the standing animal which must be suitably restrained in cattle stocks. T h e site for lumbosacral CSF collection is the m i d p o i n t o f the lumbosacral space which can be identified as the midline depression between the last palpable dorsal l u m b a r spine (L6) and the first palpable sacral dorsal spine ($2). T h e site must be clipped, surgically p r e p a r e d and between 1-2 ml local anaesthetic injected subcutaneously. Sterile surgical gloves should be worn for the collection p r o c e d u r e . In sheep and y o u n g cattle an internal styler is not r e q u i r e d (Table I). T h e n e e d l e is slowly advanced at a right angle to the plane o f the vertebral c o l u m n or with the h u b directed 5-10 ° caudally. It is essential to appreciate the changes in tissue resistance as the n e e d l e p o i n t passes sequentially t h r o u g h the subcutaneous tissue, interarcuate ligament then the s u d d e n ' p o p ' due to the loss o f resistance as the n e e d l e point finally p e n e t r a t e s the l i g a m e n t u m flavum into the epidural space. O n c e the n e e d l e point has p e n e t r a t e d the dorsal s u b a r a c h n o i d space CSF will well up in the n e e d l e h u b within 2-3 s. Failure to appreciate the c h a n g e in resistance to n e e d l e travel may result in n e e d l e p u n c t u r e o f the conus medullaris. This may elicit an i m m e d i a t e pain response and cause unnecessary discomfort to the animal. M o v e m e n t o f the pelvic limbs may dislodge the n e e d l e point with the risk o f causing local t r a u m a and h a e m o r r h a g e in the l e p t o m e n i n g e s and may affect the CSF sample which is ultimately collected. If restraint o f the animal proyes difficult, for e x a m p l e as a c o n s e q u e n c e o f hyperaesthesia to tactile stimuli c o m m o n l y observed in scrapie cases, the collection p r o c e d u r e should be abandoned. Between 2-3 ml CSF is sufficient for laboratory analysis and, while the sample can be collected by free flow, it is lnore c o n v e n i e n t to e m p l o y gentle syringe aspiration over a 10-30 s period. Care must be taken not to dislodge the n e e d l e point from the dorsal s u b a r a c h n o i d space when the syringe is attached to the n e e d l e hub. Stabilizing the position o f the n e e d l e can be assisted by firmly resting the forearms and wrists on the animal's back. T h e seal on the syringe should be b r o k e n before it is c o n n e c t e d to the n e e d l e h u b which must be a n c h o r e d firmly between the t h u m b and index finger. Selection o f the correct n e e d l e length (Table I) ensures that the n e e d l e h u b is close to the skin thereby assisting stabilization. Failure to obtain CSF is most c o m m o n l y caused by i n c o r r e c t direction o f the
Table 1 Guide to needle length and gauge for lumbar cerebrospinal fluid sampling in ruminants Lambs Ewes Rams Calves Calves Cattle
<30 kg 40-80 kg >80 kg <100 kg 100-200 kg >200 kg
0.8x25 mm 0.9x40 mm 0.9x50 mm 1.1x40 mm 1.1x50 mm 1.2x100 mm
(1 in) (1.5 in) (2 in) (1.5 in) (2 in) (4 in)
21 gauge 20 gauge 20 gauge 19 gauge 19 gauge 18 gauge+ internal stylet
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needle, in which case the reference bony landmarks must be rechecked and the needle correctly aligned. A new needle must be used on each occasion but no more than two attempts should be made to collect CSF. Sedation of the animal is not usually necessary but intravenous xylazine (0.05-0.1 mg kg -~ bodDveight) or diazepam (0.04 mg kg -~ bodyweight) can facilitate positioning of the animal. In the author's experience compression of the jugular veins to aid CSF collection (Jamison & Prescott, 1987) has not been necessary. No adverse sequelae, such as epidural abscess formation or focal spinal meningitis, have been recognized by the author following lumbosacral CSF sampling. In animals with CSF hypertension the potential problems which may result from rapid reduction of inu'acranial pressure, due to withdrawal of CSF and continued leakage from the subarachnoid space, can be minimized by using the correct gauge needle and removing the minimum amount of CSF necessary for relevant analyses.
Measurement of CSF pressure While CSF pressure can be determined with a simple water manometer connected via the needle hub to the needle point in the subarachnoid space, this measurement is not routinely performed in ruminant species because of problems associated with restraint and the large variation which occurs in healthy animals (Binkhorst, 1982). In addition, small changes in CSF pressure may be difficult to interpret as CSF pressure is directly affected by venous pressure, posture and dehydration (Jamison & Prescott, 1987). In ruminant species normal CSF pressure is within the range of 60-150 mm H 2 0 , and for an animal in sternal recumbency this represents a steady flow of one drop of CSF per 1-2 s from the needle hub. In normal adult cattle standing in stocks, CSF may simply appear at the hub and not flow freely from a 10 cm (4 in) spinal needle.
ANALYSIS O F CSF
Specific gravity The specific gravity value for normal ruminant CSF is <1.010. While there is highly significant positive correlation between CSF protein concentration and specific gravity, the specific gravity results are not sufficiently precise to be useful in the investigation of ruminant neurological disease (unpublished data).
Protein concentration Due to low protein content, many CSF samples must first be concentrated (Macrosolute concentrators, Danvers, USA) and the CSF protein concentration then determined using the pyrogallol method (Randox Laboratories Ltd). CSF protein concentration can be quoted in mg dl -~ or g 1-~ units (multiply concentration in g 1-1 by 10 for concentration in mg dl-~). The normal range for CSF protein concentration quoted for cattle is <0.4 g 1-~ (Binkhorst, 1982; Oliver & Lorenz, 1983; Scott et al., 1990; Scott & Penny, 1993) and for sheep <0.4 g l -~ (Scott & Will, 1990; Scott, 1992) and <0.7 g 1-~ (Fankhauser, 1963).
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A rapid approximation of CSF globulin concentration can be made using the Pandy test whereby one drop of CSF is added to 1 ml saturated phenol solution. A blue/white turbidity indicates a significant increase in the CSF globulin concentration. A urinary protein dipstick test is not sufficiently sensitive to determine low CSF protein concentrations (Jacobs et al., 1990) and should be relied upon only to estimate CSF protein concentrations >2.0 g 1-1. In food animal practice, protein fractionation of CSF by agarose gel electrophoresis, to detect changes in the globulin fractions, is not routinely performed. In animal species intrathecal immunoglobulin production results from many chronic viral infections of the CNS but there are relatively few common viral encephalopathies in ruminant species. Visna is the most important chronic CNS viral infection of sheep but this disease has only recently been identified in the United Kingdom (Watt et al., 1990). Protein etectrophoresis provides useful information for the investigation of CNS disease in the dog (Sorjonen, 1987), a species in which viral encephalitides such as distemper are relatively common.
White cell concentration White cell concentration in CSF can be determined using a haemocytometer. Cytological examination of CSF is performed within 2 h of collection and is greatly facilitated if the sample is first concentrated by cytospin [LSI (UK) Ltd]. The sample is then air-dried and stained with Leishman stain. The differential white cell count should be based on a minimum of 20 cells. Alternatively, a sedimentation chamber can be used to examine CSF cellular morphology (Jamison & Prescott, 1987). Presence of bacteria in CSF Samples of CSF can be examined microscopically for the presence of bacteria following preparation with Gram's stain. This technique is most useful for the confirmation of neonatal suppurative meningitis because of the common finding of large numbers of bacteria within the CSF. Baaeriology of CSF Positive bacteriological culture of CSF has only been reported in cases of neonatal bacterial meningitis (Binkhorst, 1982; Green & Smith, 1992; Scott & Penny, 1993; Scott et al., 1994). There are conflicting reports as to whether Listeria monocytogenes can be cultured fi-om CSF (Holbrook & White, 1992) or not (Low & Donachie, 1991). Cerebrospinal fluid bacteriology results would not assist the clinician in the immediate diagnosis and choice of treatments due to the time taken for such results and antibiotic sensitivity testing to be reported. A provisional result of bacterial meningitis could be provided by a direct Gram's stain. Neonatal bacterial meningitis in calves probably results from either the umbilical route or entero-invasion by opportunistic bacteria, particularly Eschelqchia coll. The development of a bacteraemia, with subsequent invasion of the meninges, is predisposed by the failure of adequate passive antibody transfer. Other CSF constituents Concentrations of glucose, creatine kinase, lactate dehydrogenase and other
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CSF constituents have been reported for clinically normal adult cattle (Welles et al., 1992). It is unlikely that such tests, would be routinely performed on CSF samples as they provide little additional information in the diagnosis of the common ruminant CNS diseases.
INTERPRETATION OF RESULTS
Red blood cells may be present in CSF following pathological haemorrhage into the subarachnoid space but this is u n c o m m o n in ruminant neurological disease. More frequendy, the presence of erythrocytes in the CSF sample results from needle puncture of blood vessels on the dura, and particularly the leptomeninges, during the sampling procedure. Accidental u'auma resulting in intrathecal haemorrhage is more common following repeated attempts to obtain CSF. Haemorrhage caused by the sampling technique will appear as streaking of blood in clear fluid and gradually clear as more CSF is allowed to flow freely fi-om the needle hub. Turbidity caused by recent haemorrhage into the CSF will clear after centrifugation to leave a clear supernatant. Alternatively, if the sample is left to stand for approximately 2 h the red blood cells gravitate and form a small plug at the bottom of the collection tube. Pathological haemorrhage within the CSF is most confidendy diagnosed by the presence of phagocytosed red blood cells within macrophages (Tvedten, 1987). A yellow discolouration of the CSF, referred to as xanthochromia, appears within a few hours after subarachnoid haemorrhage and may persist for 2-4 weeks (Mayhew & Beal, 1980). Xanthochromia is caused by release of pigment following lysis of red blood cells resulting from membrane fragility as a consequence of the low CSF protein concentration. Xanthochromia caused by icterus or carotenoids has not been recognized by the author.
Protein fractionation Protein fractionation studies of CSF from bovine spongiform encephalopathy (BSE) and scrapie cases have revealed no evidence of intrathecal immunoglobulin production. Within the range of common ovine neurological diseases investigated, protein fractionation of CSF did not aid in the further diagnosis of ruminant CNS disease and the expense of such analysis is not justified in general practice (Scott, 1993b). Normal CSF contains <0.012x 10u cells 1-1 which are predominantly lymphocytes with occasional neutrophils. As a general rule, a predominantly polymorphonuclear intrathecal inflammatory response is found in acute CNS bacterial infections (Scott, 1992) whereas a mononuclear response is seen in viral CNS infections (Tvedten, 1987). Macrophages are seen following destruction of cerebral tissue or following haemorrhage and are variably seen in polioencephalomalacia (PEM) (Tvedten, 1987). There are few reports in the veterinary literature of a consistent association between an increased CSF eosinophil concentration and parasitic infection of the CNS (Doherty et al., 1989; Lunn & Hinchcliff, 1989). Glucose concentration of CSF is reported to be low in cases of diffuse generalized meningeal disorders particularly bacterial meningo-encephalitis. How-
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ever, the CSF glucose concentration is too variable for specific diagnostic purposes and does not add to the data more reliably collected from CSF protein, cell count and cytological examinations (Jamison & Prescott, 1987). CSF electrolyte and enzyme concentrations are not usually determined in ruminant neurological disease as little additional information can be gained with the exception of CSF magnesium concentration. Under experimental conditions, a significant correlation has been demonstrated between serum and CSF magnesium which correlated well with the clinical appearance of hypomagnesaemic tetany (Allsop & Pauli, 1985). In the field investigation of sudden death in cattle, the determination of CSF magnesium concentration is reliable up to 24 h after death and quantitites of CSF are more readily collected than aqueous humour for magnesium analysis (unpublished data).
CSF protein concentration Changes in the CSF protein composition have been classified into three broad categories (Sorjonen, 1987): (i) blood-brain disturbance (increased albumin quotient or increased CSF albumin concentration) ; (ii) intrathecal immunoglobin production (decreased albumin percentage); (iii) categories (i) and (ii) together. Such classification depends upon the fact that an increased CSF albumin concentration can arise only from serum following disruption of the blood-brain barrier whereas immunolgobulins can also be produced by lymphocytes which have invaded the CNS. The albumin percentage in CSF remains within a narrow range and a significant reduction is suggestive of intrathecal immunoglobulin production. The albumin quotient is a more precise indicator of blood-brain barrier disruption: albumin quotient=CSF albumin concentration/serum albumin concentration In previously healthy animals, serum albumin is maintained within a fairly narrow range from 28-34 g 1-~ compared to a two- to 10-fold increase in CSF albumin concentration that occurs following bacterial infection of the CNS. There was no evidence of intrathecal immunoglobulin production in the range of bacterial CNS infections investigated previously (Scott, 1993b), therefore, it is valid that the CSF albumin concentration can be used as an approximate guide to the degree of disruption of the blood-brain barrier. The degree of disruption of the blood-brain barrier is an important concept with reference to the passage of penicillin G into the CSF (Scott & Sutton, 1992).
Bacterial meningo-encephalitis In cattle, a moderate increase in CSF protein concentration in the range 0 . 8 - 2 . 0 g l -l is observed in meningo-encephalitis caused by L. monocytogenes (Rebhun & de Lahunta, 1982; Scott & Sutton, 1992). In ovine listeriosis a wide range of CSF protein concentration has been reported; 0.4-4.0 g 1-1 (Scott, 1992;
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Scott, 1993a). Sufficient data are not yet available to provide a reliable prognostic index of the CSF protein concenu'ation in ovine listeriosis cases. As a general rule, those sheep with listeriosis which have a CSF protein concenta'ation <1.0 g l -I warrant aggressive antibiotic therapy and supportive care because they are more likely to SUl~,ive and are therefore worth treating. Penicillin G is the antibiotic of choice to u-i~at listeriosis with the first dose given intravenously (Scott, 1993a). Calves with bacterial meningo-encephalitis frequently have a CSF protein concentration >1.0 g 1-~ (de Lahunta, 1983) and >2.0 g 1-~ (Green & Smith, 1992; Scott & Penny, 1993). There is also a marked increase in CSF total white cell count of the order of 100fold and a change in the predominant white cell type from lymphocyte to neutrophil, commonly referred to as a neutrophil pleocytosis. Similar CSF changes have been reported in lambs with melaingo-encephalitis (Scott, 1992; Scott et al., 1994). Even with pronounced intrathecal inflammatory changes immature neutrophils are rarely seen in CSF.
Metabolic diseases Metabolic diseases which restllt in CNS dysfunction such as ovine pregnancy toxaemia and bovine ketosis do not result in alterations in CSF composition (Scott, 1992). In PEM CSF changes are variable (Scott, 1992) and may include a moderate increase in protein concentration and mononnclear pleocytosis. 7)ansmissible spongiform encephalopathies The transmissible spongiform encephalopatlaic diseases, scrapie and BSE, cause no significant increase in CSF protein or white cell concentrations (Strain et al., 1987; Scott et al., 1990).
DISCUSSION
Lnlnbosacral CSF can be safety collected tinder local anaesthesia from runlinants of all ages with no harmful effecLs. There are few indications for cisternal sampling in food animal practice because of die inherent risk of needle puncture of the brainstem and the need for general anaesthesia to ensure adequate restraint of the animal. Gross visual examination of CSF is only useful for detecting the presence of xanthochromia or marked turbidity caused by high concentrations of white cells observed in some cases of suppurative meningitis. The degree of foaming of the CSF sample after vigorous shaking is only an approximate guide to CSF protein concentration. Laboratory analysis of CSF is necessary for meaningful restllts in almost all neurological diseases. When detailed neurological examination indicates that dysfunction is restricted to the pelvic a n d / o r thoracic limbs, then demonstration of an increased lumbosacral CSF protein concentration indicates effusion of protein from a spinal lesion. Obstruction to the cranial flow of CSF may restllt from spinal cord swelling, as reported in sarcocystis encephalomyelitis (Scott et al., 1993), or from vertebral body or epidural abscessation causing spinal cord compression (Scott & Will, 1991). Such analysis can be employed as a practical ancillary aid in the differential
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diagnosis of pelvic limb paresis or tetraparesis (Scott, 1994). For example: in a 3month-old lamb with pelvic limb paresis, demyelination of the spinal cord resulting from copper deficiency (normal CSF values) can be differentiated from a thoracolumbar lesion causing cord compression such as a vertebral body abscess (high CSF protein concentration) as possible aetiologies. An increase in CSF pressure has been observed in some cases of neonatal bacterial meningitis (Scott & Penny, 1993) whereby penetration of the lumbosacral subarachnoid space released CSF as a continuous flow from the needle hub compared to the normal flow rate of one drop of CSF every 1-2 s. A more precise measure of CSF pressure was not performed as it was not possible to attach a manometer to the needle hub and guarantee that the needle point remained within the lumbosacral dorsal subarachnoid space in a conscious animal. Metabolic diseases such as ketosis, metabolic acidosis, ovine pregnancy toxaemia and the slow viral encephalopathies scrapie and BSE do not alter CSF composition. The major application of CSF analysis in these diseases is that a normal result would allow the clinician to be reasonably confident that there was no inflammatory lesion involving the leptomeninges and perhaps deeper tissues. For example, when a veterinary surgeon is presented with a recumbent, stuporous, 5day-old calf with no suck reflex, two conditions that would feature high on the list of differential diagnoses would be metabolic acidosis and bacterial meningoencephalitis. Collection of lumbosacral CSF and gross inspection for turbidity and foaming (caused by a high white cell concentration and increased protein concentration, respectively) would go some way to confirm or refute a diagnosis of bacterial meningo-encephalitis. The gross interpretation of CSF must be supported by same day laboratory analysis. This initial calf-side gross examination of CSF would avoid any delay in the initiation of appropriate treatment and may be used routinely in the investigation of stupor in young calves. Insufficient clinical results are available to form a database for the prognosis of specific neurological diseases in ruminants based on CSF parameters. CSF is unchanged in metabolic and slow viral CNS diseases and, therefore, a prognostic guide could not be developed for these diseases. In the case of bacterial CNS infections, duration of clinical signs and causal organism are important considerations. /ha attempt has been made to formulate a prognostic guide for ovine listeriosis (Scott, 1993a) based on CSF protein concentration but further data are required to improve the specificity of this test. Few broad-spectrum bacteriocidal antibiotics are capable of penetrating the intact blood-brain barrier although it is commonly assumed that the disruption of the blood-brain barrier, which occurs in bacterial CNS diseases, increases the degree of antibiotic penetration (Barlow, 1983). This increased membrane permeability may allow sufficient passage of antibiotics to achieve minimum bactericidal concentrations (MBC) within the CSF. A peak CSF antibiotic concentration 10-30 times the effective MBC may be more important than the maintenance of CSF antibiotic MBC (Prescott & Baggot, 1988). These data would suggest that intensive intravenous antibiotic therapy is indicated as soon as possible after the onset of clinical signs of bacterial CNS infection. CSF penicillin concentrations were increased in cases of bacterial meningitis where the blood-brain barrier was disturbed compared to control healthy calves (Scott & Sutton, 1992). The
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response to therapy was disappointing (Scott & Sutton, 1992), presumably because the most c o m m o n isolate in bacterial neonatal meningitis is Esche~{chia coli ( G r e e n & Smith, 1992). More recently, a g o o d survival rate for calves with clinical signs o f bacterial meningo-encephalitis and m a r k e d CSF changes including positive bacterial cultures has b e e n r e p o r t e d for calves treated with c h l o r a m p h e n i c o l (Scott & Penny, 1993). O n c e again, detailed work i n v o M n g m o r e calves is r e q u i r e d b e f o r e firm t r e a t m e n t r e c o m m e n d a t i o n s can be offered to veterinary surgeons in general practice. A g o o d t r e a t m e n t response to high dose penicillin G (44 000 iu kg -~ body weight twice daily) has b e e n r e p o r t e d in box4ne listeriosis cases that are still ambulatory when t r e a t m e n t c o m m e n c e s ( R e b h u n & de Lahunta, 1982). A similar t r e a t m e n t response rate was r e c o r d e d for cattle but a survival rate o f only 23% was r e p o r t e d in oxdne listeriosis using this t r e a t m e n t r e g i m e n (Scott, 1993b). Lumbosacral CSF can be safely collected from r u m i n a n t neurological disease cases and laboratory analysis provides the veterinary clinician with m u c h useful i n f o r m a t i o n o f the living animal. Rapid CSF collection and analysis greatly assists diagnosis thus permitting accurate u ' e a t m e n t and the a d o p t i o n o f a p p r o p r i a t e control and p r e v e n t i o n measures for o t h e r animals in the group.
REFERENCES At.IASOP,T. F. & P.-\t'H,J. v. (1985). Magnesimn concentrations in the ventricular and lumbar cerebrospinal fluid of hypomagnesaemic cows. Research in Vete~qnan,Science 38, 61-4. B..xRCOW, R. M. (1983). In Diseases of Sheep, 1st edn., p. 81, ed. W. t3. Martin, Oxford: Blackwell Scientific Puhlications. BRV:WER,B. D. (1987). In Bovine Neurological Diseases, p. 22, ed.J.C. Baker, Â:etelT~naO, Clinics ~ North Ame~4ca 3, Philadelphia: W. B. Saunders Co. BINKIIORST, G. J. (1982). Cereb,-ospinal fluid as an aid in the differential diagnosis of nervous diseases. In Proceedin~ XII WorM Congress on Diseases of Cattle, The Netherlands, Vol. II, pp. 964-8. CSERR,H. F. (1971). Physiology of the choroid plexus. Physiolo~, Review51, 273-310. DOHERT~',M. L., BASSETr,H. F., BRI~ATHNACH,R., MONA(;HAN,M. L. & Mc:ERL~\N,B. A. (1989). Outbreak of acute coenuriasis in aclult sheep in Ireland. Veterinarl, Record 125, 185. F.~.X}U-I.-Xt'SER,R. (1963). The cerehrospinal fluid. In Comparative Neuropatholo~,, p. 21, ed.J. R. M. hines, New York: Acade,nic Press. GEORC;E, L. W. (1990). Localisation and differentiation of neurological diseases. In Large Animal hT.ternal Medicine, ed. B. P. Smith, p. 145. St Louis: CV Mosby. GREEN, S. L. & S,XUTH,L. L. (1992). Meningitis in neonatal cah,es: 32 cases (1983-1990). Journal Ame14can Veterinm3, Medical Association 201,125-8. HOLBROOK,T. C. &: WHrrE, S. L. (1992). In PhysicalExamination, p. 312, e d . J . H . Wilson, Vete~4narl, Clinics of North America 8. Philadelphia: W. B. Saunde,'s Co. JacoBs, t(. M., COCHRANE,S. M., Lt~..x4sDE.x,J. H. & NORRIS,A. M. (1990). Relationship of cerebrospinal fluid protein concentration determined by dye-binding and urinary dipstick methodologies. Canadian VeterinmyJourna131,587-8. JaMlSO,X,J. M. & PREscovr,J. F. (1987). Bacterial meningitis in large animals. Compendium of Continuing Education 9, F399-F406. DE LAHUNT.~,A. (1983). VeterinaT), Neuroanatomy and Clinical Neurology, 2nd edn. Philadelphia: W. B. Saunders Co. LI'FFLE, P. B. & SORENSON, D. K. (1969). Bovine polioencephalo,nalacia, infectious embolic
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meningo-encephalitis, and acute lead poisoning in feedlot cattle. Journal American Veterinary Medical Association 155, 1892-903. Low, J. C. & DONACHJE,W. (1991). Listeriosis. In Diseases of Sheep, 2nd edn., p. 177, eds W. B. Martin & I. D. Aitken. Oxford: Blackwell Scientific Publications. LUNN, D. P. & HINf;I-ICt.IFI:,K. W. (1989). Cerebrospinal fluid eosinophilia and ataxia in five llamas. Vete~naTyRecord 124, 302-5. MAw-tKw, I. G. (1989). Large Animal Neurology: A Handbook for Vete~Jnary Clinicians, p. 15 Philadelphia: Lea & Febiger. MA~I-IEW,I. G. & BZAL,C. R. (1980). Techniques of analysis of cerebrospinal fluid. VeterinaTy Clinics of North America 10, 155. Mit.Hor~w', T. H. (1975). The third circulation revisited. Journal Neurosurge~y42, 628--45. McGvmK, S. M. (1987). In Bovine Neurologic Diseases, p. 112, ed. J. c. Baker, Veterinary Clinics of North America 3, Philadelphia: W. B. Saunders Co. OIJvER, J. E. & LORENZ, M. D. (1983). Handbook of Veterinary NeacrologicDiagnosis. Philadelphia: W. B. Saunders Co. POWER, E. P., CROWLEV,J.J., B',~yE,J. F., O'I~Ew, M. P. & W~WZRS, E. D. (1985). Cerebrocortical necrosis in dairy cows. lrish Vete~TnaryJourna139, 81-3. PRFSCO'I-r,J. F. & BA~;C;o'I',J. D. (1988). In Antimicrobial Therapy in Veterinary Medicine, p. 78. London: Blackwell Scientific Publications. Rt:.~Huy, W. C. & DE L-`'HUNTA,A. (1982). Diagnosis and treatment of bovine listeriosis. Journal Ame~qcan Veterinaly Medical Association 180, 395-8. ScoTt, P. R. (1992). Analysis of cerebrospinal fluid from field cases of some c o m m o n ovine neurological diseases. B~itish VeterinaryJournal 148, 15-22. Scoa r, P. R. (1994). Cerebrospinal fluid analysis in the differential diagnosis of spinal cord lesions in ruminants. In Practice (in press). ScoTt, P. R. (1993a). A field study of listerial meningo-encephalitis with particular reference to cerebrospinal fluid analysis as an aid to diagnosis and prognosis. British Veterinary Journal 149, 165-70. Scolq', P. R. (1993b). Total protein and electrophoretic pattern of cerebrospinal fluid in sheep with some c o m m o n neurological disorders. Cornell Vete74na~ian83, 199-204. Sc:orl", P. R., At.DRmC;E,B. M., CL-xRKE,M. & Wmt, R. G. (1990). Cerebrospinal fluid studies in normal cows and cases of bovine spongiform encephalopathy. British Vete~Tna~yJournal 146, 88-90. Scorr, P. R. & Wu.L, R. G. (1991). A report of Froin's syndrome in four ovine thoracolumbar epidural abscess cases. British I/eterinaryJou~za1147, 582-4. Sc:oI-r, P. R. & Stq-ro~, D. (1992). A study of some factors which may influence the concentrations of penicillin G in cerebrospinal fluid in some c o m m o n bovine neurological diseases. Proceedings of XVIl World Buiatffcs Congress, St Paul, USA 2, 63-8. S~:ol-l, P. R. & PENNV,C. D. (1993). A field study of meningo-encephalitis in calves with particular reference to cerebrospinal fluid analysis. Vete~ina~),Record 133, 119-21. Sco-ra', P. R., WAaq, N. J., WOOOMA~, M. P., McGoRuM, B. M. & McDL~HD, A. (1993). Protozoan enceplaalomyelitis causing pelvic limb paresis in a yearling sheep. New Zealand Vete~7;na~yJournal 41, 139-41. S¢:orT, P. R., SARC,lSON, N. D., PENN',, C. D. & PIRIE, R. S. (1994). A field study of naturallyoccurring ovine bacterial meningo-encephalitis. Vete~71na~yRecord (in press). SORJONEN, D. C. (1987). Total protein, albumin quota and electrophoretic patterns in cerebrospinal fluid of dogs with central nervous system disorders. American Journal of Veterina~. Research48, 301-5. STILMN,G. M., Ct.~xroN, M. S., TURNQUIST,S. E. & KREEGER,J. M. (1987). Evoked potential and electro-encephalographic assessment of central blindness in a steer. Co~7zellVeterinarian 77, 374--82. T v ~ x ~ , H. W. (1987). In Bovine Neurologic Diseases, pp. 25-44, ed. J. C. Baker. Veterinary Clinics of North America. Food Aaaimal Practice 3. Philadelphia: W. B. Saunders. WATt, N.J., Roy, D. J., McCo~nzta., I. & KJNG, T.J. (1990). A case of visna in the United Kingdom. VeterinaryRecord 126, 600-1. Wzt.~zs, E. G., T~zR, J. W., So~jo,~Fy, D. C. & Wrtaxuzv, E. M. (1992). Composition and analy-
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sis of cerebrospinal fluid in clinically normal adult cattle. American Journal of Veterina O, Research 53, 2050-7.
(Acceptedfor publication 13 September 1994)
REVIEW T H E COIJ,F, C T I O N A N D ANALYSIS OF CEREBROSPINAL FLUID AS A N AID T O D I A G N O S I S IN R U M I N A N T N E U R O L O G I C A L DISEASE
P. R. SCOTT Department of Veterina U Clinical Studies, Royal (Dick) School of Veterinary Studies, Veterina U FieM Station, Easter Bush, Roslin, Midlothian EH25 9RG, Scotland
SUMMARY
In ruminant species cerebrospinal fluid (CSF) collection and analysis prorides rapid and, in some situations, instant information to the veterinary clinician investigating a disease problem in the living animal. CSF analysis is particularly useful with respect to confirming the presence of an inflammatory lesion involving the leptomeninges such as bacterial meningoencephalitis. When correctly performed under local anaesthesia, lumbosacral CSF collection in ruminants is a safe procedure and there are no harmful sequelae. There are few indications for cisternal CSF collection in food animal practice. IZ~WORDS:Cerebrospinal fluid; nervous disease; cattle; sheep.
INTRODUCTION
Altered mental state, particularly depression, is a common feature of many disease processes. The investigation of possible central nervous system (CNS) disease involves a detailed assessment of the history and a complete physical and neurological examination of the animal (Mayhew, 1989; George, 1990). Despite such investigations, the data collected in neurological disease cases may not be pathognomic. In many situations the response to treatment is used as an adjunct to diagnosis (Little & Sorenson, 1969) but commonly a variety of drugs are administered simultaneously (Power el al., 1985) which limits the interpretation of the outcome. A specific diagnosis of the cause of the neurological signs is often only reached in fatal cases which are examined in detail at necropsy. Histopathological examination of the CNS is time-consuming, expensive and requires expert interpretation. In the absence of information to guide the pathologist to potentially affected area(s) of the brain, multiple tissue sections are required, which usually renders this investigative procedure prohibitive on the basis of cost. 0007-1935/95/060603-12/$12.00/0
© 1995 Bailli6reTindall
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Man), neurological diseases of cattle and sheep occur as outbreaks. It is, therefore, essential to reach an accurate diagnosis as soon as possible in order to expedite preventive and control measures. Cerebrospinal fluid (CSF) can be obtained from li~6ng animals with analysis results available within 1 h of submission to the laboratory. ~qaile there are many references to the principles of CSF collection and analysis in ruminant neurological disease (see, for example, Tvedten, 1987; George, 1990; Holbrook & White, 1992), there are few studies which report actual results (Binkhorst, 1982; Rebhun & de Lahunta, 1982; Scott et al., 1990; Scott, 1992; Green & Smith, 1992; Scott & Penny, 1993). Tiffs article reviews the recent literature on CSF collection techniques, analytical procedures and intepretation of results. The usefulness of CSF analysis in providing the veterinarian in general practice with diagnostic and prognostic information is discussed.
Formation of CSF CSF is formed largely from the choroid plexuses of the lateral ventricles by the ultrafiltration of plasma and the active transport of selected substances across the blood-brain barrier (Cserr, 1971; Milhorat, 1975). The CSF in the ventricular system flows caudally and diffuses out of the lateral aperture in the fourth ventricle to circulate around the brain and spinal cord. The presence of CSF in the subarachnoid space separates the brain and spinal cord fi'om the bony craniuna and vertebral column which reduces traulna to the underlying delicate nervous tissue. CSF also has excretol T functions with the removal of products of cerebral metabolism.
Collection of CSF in ruminant species In contrast to companion animals, lumbosacral CSF can be collected from ruminant species under local anaesthesia (Scott et al., 1990; Scott, 1992). The risks associated with general anaesthesia, particularly in an alfimal with possible CNS dysfunction, are thereby avoided. Adequate restraint and identification of bony landmarks are essential during the sampling procedure. In order to appreciate the relevant anatomical structures it is recommended that the technique is first attempted on cadavers. For CSF collection and examination it is necessary to puncture the subarachnoid space in the cerebellomedullary cistern (cisternal sample) or at the lumbosacral site (lumbosacral sample). In the absence of a focal spinal cord compressive lesion there is usually no substantial difference between the composition of cisternal and lumbosacral CSF samples (Scott & Will, 1991). While theoretically it may be desirable to collect CSF fi'om the site nearer the suspected location of the lesion, this is not possible in the field. In those runfinant neurological diseases studied by the author, a lumbosacral CSF sample was representative. Thus the risk of needle penetration of the brainstem associated with cisternal puncture is difficult to justify. Collection of lumbosacral CSF is facilitated when the animal is positioned in sternal recumbency with the hips flexed and the pelvic limbs extended alongside the abdomen. Aversion of the animal's head against the flank may assist in maintaining sternal recumbency during the collection procedure. Alternatively, lambs
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and kids can be restrained in lateral recumbency. In adult cattle that are still ambulatory, the lumbosacrai CSF sample is collected in the standing animal which must be suitably restrained in cattle stocks. T h e site for lumbosacral CSF collection is the m i d p o i n t o f the lumbosacral space which can be identified as the midline depression between the last palpable dorsal l u m b a r spine (L6) and the first palpable sacral dorsal spine ($2). T h e site must be clipped, surgically p r e p a r e d and between 1-2 ml local anaesthetic injected subcutaneously. Sterile surgical gloves should be worn for the collection p r o c e d u r e . In sheep and y o u n g cattle an internal styler is not r e q u i r e d (Table I). T h e n e e d l e is slowly advanced at a right angle to the plane o f the vertebral c o l u m n or with the h u b directed 5-10 ° caudally. It is essential to appreciate the changes in tissue resistance as the n e e d l e p o i n t passes sequentially t h r o u g h the subcutaneous tissue, interarcuate ligament then the s u d d e n ' p o p ' due to the loss o f resistance as the n e e d l e point finally p e n e t r a t e s the l i g a m e n t u m flavum into the epidural space. O n c e the n e e d l e point has p e n e t r a t e d the dorsal s u b a r a c h n o i d space CSF will well up in the n e e d l e h u b within 2-3 s. Failure to appreciate the c h a n g e in resistance to n e e d l e travel may result in n e e d l e p u n c t u r e o f the conus medullaris. This may elicit an i m m e d i a t e pain response and cause unnecessary discomfort to the animal. M o v e m e n t o f the pelvic limbs may dislodge the n e e d l e point with the risk o f causing local t r a u m a and h a e m o r r h a g e in the l e p t o m e n i n g e s and may affect the CSF sample which is ultimately collected. If restraint o f the animal proyes difficult, for e x a m p l e as a c o n s e q u e n c e o f hyperaesthesia to tactile stimuli c o m m o n l y observed in scrapie cases, the collection p r o c e d u r e should be abandoned. Between 2-3 ml CSF is sufficient for laboratory analysis and, while the sample can be collected by free flow, it is lnore c o n v e n i e n t to e m p l o y gentle syringe aspiration over a 10-30 s period. Care must be taken not to dislodge the n e e d l e point from the dorsal s u b a r a c h n o i d space when the syringe is attached to the n e e d l e hub. Stabilizing the position o f the n e e d l e can be assisted by firmly resting the forearms and wrists on the animal's back. T h e seal on the syringe should be b r o k e n before it is c o n n e c t e d to the n e e d l e h u b which must be a n c h o r e d firmly between the t h u m b and index finger. Selection o f the correct n e e d l e length (Table I) ensures that the n e e d l e h u b is close to the skin thereby assisting stabilization. Failure to obtain CSF is most c o m m o n l y caused by i n c o r r e c t direction o f the
Table 1 Guide to needle length and gauge for lumbar cerebrospinal fluid sampling in ruminants Lambs Ewes Rams Calves Calves Cattle
<30 kg 40-80 kg >80 kg <100 kg 100-200 kg >200 kg
0.8x25 mm 0.9x40 mm 0.9x50 mm 1.1x40 mm 1.1x50 mm 1.2x100 mm
(1 in) (1.5 in) (2 in) (1.5 in) (2 in) (4 in)
21 gauge 20 gauge 20 gauge 19 gauge 19 gauge 18 gauge+ internal stylet
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needle, in which case the reference bony landmarks must be rechecked and the needle correctly aligned. A new needle must be used on each occasion but no more than two attempts should be made to collect CSF. Sedation of the animal is not usually necessary but intravenous xylazine (0.05-0.1 mg kg -~ bodDveight) or diazepam (0.04 mg kg -~ bodyweight) can facilitate positioning of the animal. In the author's experience compression of the jugular veins to aid CSF collection (Jamison & Prescott, 1987) has not been necessary. No adverse sequelae, such as epidural abscess formation or focal spinal meningitis, have been recognized by the author following lumbosacral CSF sampling. In animals with CSF hypertension the potential problems which may result from rapid reduction of inu'acranial pressure, due to withdrawal of CSF and continued leakage from the subarachnoid space, can be minimized by using the correct gauge needle and removing the minimum amount of CSF necessary for relevant analyses.
Measurement of CSF pressure While CSF pressure can be determined with a simple water manometer connected via the needle hub to the needle point in the subarachnoid space, this measurement is not routinely performed in ruminant species because of problems associated with restraint and the large variation which occurs in healthy animals (Binkhorst, 1982). In addition, small changes in CSF pressure may be difficult to interpret as CSF pressure is directly affected by venous pressure, posture and dehydration (Jamison & Prescott, 1987). In ruminant species normal CSF pressure is within the range of 60-150 mm H 2 0 , and for an animal in sternal recumbency this represents a steady flow of one drop of CSF per 1-2 s from the needle hub. In normal adult cattle standing in stocks, CSF may simply appear at the hub and not flow freely from a 10 cm (4 in) spinal needle.
ANALYSIS O F CSF
Specific gravity The specific gravity value for normal ruminant CSF is <1.010. While there is highly significant positive correlation between CSF protein concentration and specific gravity, the specific gravity results are not sufficiently precise to be useful in the investigation of ruminant neurological disease (unpublished data).
Protein concentration Due to low protein content, many CSF samples must first be concentrated (Macrosolute concentrators, Danvers, USA) and the CSF protein concentration then determined using the pyrogallol method (Randox Laboratories Ltd). CSF protein concentration can be quoted in mg dl -~ or g 1-~ units (multiply concentration in g 1-1 by 10 for concentration in mg dl-~). The normal range for CSF protein concentration quoted for cattle is <0.4 g 1-~ (Binkhorst, 1982; Oliver & Lorenz, 1983; Scott et al., 1990; Scott & Penny, 1993) and for sheep <0.4 g l -~ (Scott & Will, 1990; Scott, 1992) and <0.7 g 1-~ (Fankhauser, 1963).
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A rapid approximation of CSF globulin concentration can be made using the Pandy test whereby one drop of CSF is added to 1 ml saturated phenol solution. A blue/white turbidity indicates a significant increase in the CSF globulin concentration. A urinary protein dipstick test is not sufficiently sensitive to determine low CSF protein concentrations (Jacobs et al., 1990) and should be relied upon only to estimate CSF protein concentrations >2.0 g 1-1. In food animal practice, protein fractionation of CSF by agarose gel electrophoresis, to detect changes in the globulin fractions, is not routinely performed. In animal species intrathecal immunoglobulin production results from many chronic viral infections of the CNS but there are relatively few common viral encephalopathies in ruminant species. Visna is the most important chronic CNS viral infection of sheep but this disease has only recently been identified in the United Kingdom (Watt et al., 1990). Protein etectrophoresis provides useful information for the investigation of CNS disease in the dog (Sorjonen, 1987), a species in which viral encephalitides such as distemper are relatively common.
White cell concentration White cell concentration in CSF can be determined using a haemocytometer. Cytological examination of CSF is performed within 2 h of collection and is greatly facilitated if the sample is first concentrated by cytospin [LSI (UK) Ltd]. The sample is then air-dried and stained with Leishman stain. The differential white cell count should be based on a minimum of 20 cells. Alternatively, a sedimentation chamber can be used to examine CSF cellular morphology (Jamison & Prescott, 1987). Presence of bacteria in CSF Samples of CSF can be examined microscopically for the presence of bacteria following preparation with Gram's stain. This technique is most useful for the confirmation of neonatal suppurative meningitis because of the common finding of large numbers of bacteria within the CSF. Baaeriology of CSF Positive bacteriological culture of CSF has only been reported in cases of neonatal bacterial meningitis (Binkhorst, 1982; Green & Smith, 1992; Scott & Penny, 1993; Scott et al., 1994). There are conflicting reports as to whether Listeria monocytogenes can be cultured fi-om CSF (Holbrook & White, 1992) or not (Low & Donachie, 1991). Cerebrospinal fluid bacteriology results would not assist the clinician in the immediate diagnosis and choice of treatments due to the time taken for such results and antibiotic sensitivity testing to be reported. A provisional result of bacterial meningitis could be provided by a direct Gram's stain. Neonatal bacterial meningitis in calves probably results from either the umbilical route or entero-invasion by opportunistic bacteria, particularly Eschelqchia coll. The development of a bacteraemia, with subsequent invasion of the meninges, is predisposed by the failure of adequate passive antibody transfer. Other CSF constituents Concentrations of glucose, creatine kinase, lactate dehydrogenase and other
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CSF constituents have been reported for clinically normal adult cattle (Welles et al., 1992). It is unlikely that such tests, would be routinely performed on CSF samples as they provide little additional information in the diagnosis of the common ruminant CNS diseases.
INTERPRETATION OF RESULTS
Red blood cells may be present in CSF following pathological haemorrhage into the subarachnoid space but this is u n c o m m o n in ruminant neurological disease. More frequendy, the presence of erythrocytes in the CSF sample results from needle puncture of blood vessels on the dura, and particularly the leptomeninges, during the sampling procedure. Accidental u'auma resulting in intrathecal haemorrhage is more common following repeated attempts to obtain CSF. Haemorrhage caused by the sampling technique will appear as streaking of blood in clear fluid and gradually clear as more CSF is allowed to flow freely fi-om the needle hub. Turbidity caused by recent haemorrhage into the CSF will clear after centrifugation to leave a clear supernatant. Alternatively, if the sample is left to stand for approximately 2 h the red blood cells gravitate and form a small plug at the bottom of the collection tube. Pathological haemorrhage within the CSF is most confidendy diagnosed by the presence of phagocytosed red blood cells within macrophages (Tvedten, 1987). A yellow discolouration of the CSF, referred to as xanthochromia, appears within a few hours after subarachnoid haemorrhage and may persist for 2-4 weeks (Mayhew & Beal, 1980). Xanthochromia is caused by release of pigment following lysis of red blood cells resulting from membrane fragility as a consequence of the low CSF protein concentration. Xanthochromia caused by icterus or carotenoids has not been recognized by the author.
Protein fractionation Protein fractionation studies of CSF from bovine spongiform encephalopathy (BSE) and scrapie cases have revealed no evidence of intrathecal immunoglobulin production. Within the range of common ovine neurological diseases investigated, protein fractionation of CSF did not aid in the further diagnosis of ruminant CNS disease and the expense of such analysis is not justified in general practice (Scott, 1993b). Normal CSF contains <0.012x 10u cells 1-1 which are predominantly lymphocytes with occasional neutrophils. As a general rule, a predominantly polymorphonuclear intrathecal inflammatory response is found in acute CNS bacterial infections (Scott, 1992) whereas a mononuclear response is seen in viral CNS infections (Tvedten, 1987). Macrophages are seen following destruction of cerebral tissue or following haemorrhage and are variably seen in polioencephalomalacia (PEM) (Tvedten, 1987). There are few reports in the veterinary literature of a consistent association between an increased CSF eosinophil concentration and parasitic infection of the CNS (Doherty et al., 1989; Lunn & Hinchcliff, 1989). Glucose concentration of CSF is reported to be low in cases of diffuse generalized meningeal disorders particularly bacterial meningo-encephalitis. How-
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ever, the CSF glucose concentration is too variable for specific diagnostic purposes and does not add to the data more reliably collected from CSF protein, cell count and cytological examinations (Jamison & Prescott, 1987). CSF electrolyte and enzyme concentrations are not usually determined in ruminant neurological disease as little additional information can be gained with the exception of CSF magnesium concentration. Under experimental conditions, a significant correlation has been demonstrated between serum and CSF magnesium which correlated well with the clinical appearance of hypomagnesaemic tetany (Allsop & Pauli, 1985). In the field investigation of sudden death in cattle, the determination of CSF magnesium concentration is reliable up to 24 h after death and quantitites of CSF are more readily collected than aqueous humour for magnesium analysis (unpublished data).
CSF protein concentration Changes in the CSF protein composition have been classified into three broad categories (Sorjonen, 1987): (i) blood-brain disturbance (increased albumin quotient or increased CSF albumin concentration) ; (ii) intrathecal immunoglobin production (decreased albumin percentage); (iii) categories (i) and (ii) together. Such classification depends upon the fact that an increased CSF albumin concentration can arise only from serum following disruption of the blood-brain barrier whereas immunolgobulins can also be produced by lymphocytes which have invaded the CNS. The albumin percentage in CSF remains within a narrow range and a significant reduction is suggestive of intrathecal immunoglobulin production. The albumin quotient is a more precise indicator of blood-brain barrier disruption: albumin quotient=CSF albumin concentration/serum albumin concentration In previously healthy animals, serum albumin is maintained within a fairly narrow range from 28-34 g 1-~ compared to a two- to 10-fold increase in CSF albumin concentration that occurs following bacterial infection of the CNS. There was no evidence of intrathecal immunoglobulin production in the range of bacterial CNS infections investigated previously (Scott, 1993b), therefore, it is valid that the CSF albumin concentration can be used as an approximate guide to the degree of disruption of the blood-brain barrier. The degree of disruption of the blood-brain barrier is an important concept with reference to the passage of penicillin G into the CSF (Scott & Sutton, 1992).
Bacterial meningo-encephalitis In cattle, a moderate increase in CSF protein concentration in the range 0 . 8 - 2 . 0 g l -l is observed in meningo-encephalitis caused by L. monocytogenes (Rebhun & de Lahunta, 1982; Scott & Sutton, 1992). In ovine listeriosis a wide range of CSF protein concentration has been reported; 0.4-4.0 g 1-1 (Scott, 1992;
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Scott, 1993a). Sufficient data are not yet available to provide a reliable prognostic index of the CSF protein concenu'ation in ovine listeriosis cases. As a general rule, those sheep with listeriosis which have a CSF protein concenta'ation <1.0 g l -I warrant aggressive antibiotic therapy and supportive care because they are more likely to SUl~,ive and are therefore worth treating. Penicillin G is the antibiotic of choice to u-i~at listeriosis with the first dose given intravenously (Scott, 1993a). Calves with bacterial meningo-encephalitis frequently have a CSF protein concentration >1.0 g 1-~ (de Lahunta, 1983) and >2.0 g 1-~ (Green & Smith, 1992; Scott & Penny, 1993). There is also a marked increase in CSF total white cell count of the order of 100fold and a change in the predominant white cell type from lymphocyte to neutrophil, commonly referred to as a neutrophil pleocytosis. Similar CSF changes have been reported in lambs with melaingo-encephalitis (Scott, 1992; Scott et al., 1994). Even with pronounced intrathecal inflammatory changes immature neutrophils are rarely seen in CSF.
Metabolic diseases Metabolic diseases which restllt in CNS dysfunction such as ovine pregnancy toxaemia and bovine ketosis do not result in alterations in CSF composition (Scott, 1992). In PEM CSF changes are variable (Scott, 1992) and may include a moderate increase in protein concentration and mononnclear pleocytosis. 7)ansmissible spongiform encephalopathies The transmissible spongiform encephalopatlaic diseases, scrapie and BSE, cause no significant increase in CSF protein or white cell concentrations (Strain et al., 1987; Scott et al., 1990).
DISCUSSION
Lnlnbosacral CSF can be safety collected tinder local anaesthesia from runlinants of all ages with no harmful effecLs. There are few indications for cisternal sampling in food animal practice because of die inherent risk of needle puncture of the brainstem and the need for general anaesthesia to ensure adequate restraint of the animal. Gross visual examination of CSF is only useful for detecting the presence of xanthochromia or marked turbidity caused by high concentrations of white cells observed in some cases of suppurative meningitis. The degree of foaming of the CSF sample after vigorous shaking is only an approximate guide to CSF protein concentration. Laboratory analysis of CSF is necessary for meaningful restllts in almost all neurological diseases. When detailed neurological examination indicates that dysfunction is restricted to the pelvic a n d / o r thoracic limbs, then demonstration of an increased lumbosacral CSF protein concentration indicates effusion of protein from a spinal lesion. Obstruction to the cranial flow of CSF may restllt from spinal cord swelling, as reported in sarcocystis encephalomyelitis (Scott et al., 1993), or from vertebral body or epidural abscessation causing spinal cord compression (Scott & Will, 1991). Such analysis can be employed as a practical ancillary aid in the differential
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diagnosis of pelvic limb paresis or tetraparesis (Scott, 1994). For example: in a 3month-old lamb with pelvic limb paresis, demyelination of the spinal cord resulting from copper deficiency (normal CSF values) can be differentiated from a thoracolumbar lesion causing cord compression such as a vertebral body abscess (high CSF protein concentration) as possible aetiologies. An increase in CSF pressure has been observed in some cases of neonatal bacterial meningitis (Scott & Penny, 1993) whereby penetration of the lumbosacral subarachnoid space released CSF as a continuous flow from the needle hub compared to the normal flow rate of one drop of CSF every 1-2 s. A more precise measure of CSF pressure was not performed as it was not possible to attach a manometer to the needle hub and guarantee that the needle point remained within the lumbosacral dorsal subarachnoid space in a conscious animal. Metabolic diseases such as ketosis, metabolic acidosis, ovine pregnancy toxaemia and the slow viral encephalopathies scrapie and BSE do not alter CSF composition. The major application of CSF analysis in these diseases is that a normal result would allow the clinician to be reasonably confident that there was no inflammatory lesion involving the leptomeninges and perhaps deeper tissues. For example, when a veterinary surgeon is presented with a recumbent, stuporous, 5day-old calf with no suck reflex, two conditions that would feature high on the list of differential diagnoses would be metabolic acidosis and bacterial meningoencephalitis. Collection of lumbosacral CSF and gross inspection for turbidity and foaming (caused by a high white cell concentration and increased protein concentration, respectively) would go some way to confirm or refute a diagnosis of bacterial meningo-encephalitis. The gross interpretation of CSF must be supported by same day laboratory analysis. This initial calf-side gross examination of CSF would avoid any delay in the initiation of appropriate treatment and may be used routinely in the investigation of stupor in young calves. Insufficient clinical results are available to form a database for the prognosis of specific neurological diseases in ruminants based on CSF parameters. CSF is unchanged in metabolic and slow viral CNS diseases and, therefore, a prognostic guide could not be developed for these diseases. In the case of bacterial CNS infections, duration of clinical signs and causal organism are important considerations. /ha attempt has been made to formulate a prognostic guide for ovine listeriosis (Scott, 1993a) based on CSF protein concentration but further data are required to improve the specificity of this test. Few broad-spectrum bacteriocidal antibiotics are capable of penetrating the intact blood-brain barrier although it is commonly assumed that the disruption of the blood-brain barrier, which occurs in bacterial CNS diseases, increases the degree of antibiotic penetration (Barlow, 1983). This increased membrane permeability may allow sufficient passage of antibiotics to achieve minimum bactericidal concentrations (MBC) within the CSF. A peak CSF antibiotic concentration 10-30 times the effective MBC may be more important than the maintenance of CSF antibiotic MBC (Prescott & Baggot, 1988). These data would suggest that intensive intravenous antibiotic therapy is indicated as soon as possible after the onset of clinical signs of bacterial CNS infection. CSF penicillin concentrations were increased in cases of bacterial meningitis where the blood-brain barrier was disturbed compared to control healthy calves (Scott & Sutton, 1992). The
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response to therapy was disappointing (Scott & Sutton, 1992), presumably because the most c o m m o n isolate in bacterial neonatal meningitis is Esche~{chia coli ( G r e e n & Smith, 1992). More recently, a g o o d survival rate for calves with clinical signs o f bacterial meningo-encephalitis and m a r k e d CSF changes including positive bacterial cultures has b e e n r e p o r t e d for calves treated with c h l o r a m p h e n i c o l (Scott & Penny, 1993). O n c e again, detailed work i n v o M n g m o r e calves is r e q u i r e d b e f o r e firm t r e a t m e n t r e c o m m e n d a t i o n s can be offered to veterinary surgeons in general practice. A g o o d t r e a t m e n t response to high dose penicillin G (44 000 iu kg -~ body weight twice daily) has b e e n r e p o r t e d in box4ne listeriosis cases that are still ambulatory when t r e a t m e n t c o m m e n c e s ( R e b h u n & de Lahunta, 1982). A similar t r e a t m e n t response rate was r e c o r d e d for cattle but a survival rate o f only 23% was r e p o r t e d in oxdne listeriosis using this t r e a t m e n t r e g i m e n (Scott, 1993b). Lumbosacral CSF can be safely collected from r u m i n a n t neurological disease cases and laboratory analysis provides the veterinary clinician with m u c h useful i n f o r m a t i o n o f the living animal. Rapid CSF collection and analysis greatly assists diagnosis thus permitting accurate u ' e a t m e n t and the a d o p t i o n o f a p p r o p r i a t e control and p r e v e n t i o n measures for o t h e r animals in the group.
REFERENCES At.IASOP,T. F. & P.-\t'H,J. v. (1985). Magnesimn concentrations in the ventricular and lumbar cerebrospinal fluid of hypomagnesaemic cows. Research in Vete~qnan,Science 38, 61-4. B..xRCOW, R. M. (1983). In Diseases of Sheep, 1st edn., p. 81, ed. W. t3. Martin, Oxford: Blackwell Scientific Puhlications. BRV:WER,B. D. (1987). In Bovine Neurological Diseases, p. 22, ed.J.C. Baker, Â:etelT~naO, Clinics ~ North Ame~4ca 3, Philadelphia: W. B. Saunders Co. BINKIIORST, G. J. (1982). Cereb,-ospinal fluid as an aid in the differential diagnosis of nervous diseases. In Proceedin~ XII WorM Congress on Diseases of Cattle, The Netherlands, Vol. II, pp. 964-8. CSERR,H. F. (1971). Physiology of the choroid plexus. Physiolo~, Review51, 273-310. DOHERT~',M. L., BASSETr,H. F., BRI~ATHNACH,R., MONA(;HAN,M. L. & Mc:ERL~\N,B. A. (1989). Outbreak of acute coenuriasis in aclult sheep in Ireland. Veterinarl, Record 125, 185. F.~.X}U-I.-Xt'SER,R. (1963). The cerehrospinal fluid. In Comparative Neuropatholo~,, p. 21, ed.J. R. M. hines, New York: Acade,nic Press. GEORC;E, L. W. (1990). Localisation and differentiation of neurological diseases. In Large Animal hT.ternal Medicine, ed. B. P. Smith, p. 145. St Louis: CV Mosby. GREEN, S. L. & S,XUTH,L. L. (1992). Meningitis in neonatal cah,es: 32 cases (1983-1990). Journal Ame14can Veterinm3, Medical Association 201,125-8. HOLBROOK,T. C. &: WHrrE, S. L. (1992). In PhysicalExamination, p. 312, e d . J . H . Wilson, Vete~4narl, Clinics of North America 8. Philadelphia: W. B. Saunde,'s Co. JacoBs, t(. M., COCHRANE,S. M., Lt~..x4sDE.x,J. H. & NORRIS,A. M. (1990). Relationship of cerebrospinal fluid protein concentration determined by dye-binding and urinary dipstick methodologies. Canadian VeterinmyJourna131,587-8. JaMlSO,X,J. M. & PREscovr,J. F. (1987). Bacterial meningitis in large animals. Compendium of Continuing Education 9, F399-F406. DE LAHUNT.~,A. (1983). VeterinaT), Neuroanatomy and Clinical Neurology, 2nd edn. Philadelphia: W. B. Saunders Co. LI'FFLE, P. B. & SORENSON, D. K. (1969). Bovine polioencephalo,nalacia, infectious embolic
CEREBROSPINAL FLUID ANALYSIS
613
meningo-encephalitis, and acute lead poisoning in feedlot cattle. Journal American Veterinary Medical Association 155, 1892-903. Low, J. C. & DONACHJE,W. (1991). Listeriosis. In Diseases of Sheep, 2nd edn., p. 177, eds W. B. Martin & I. D. Aitken. Oxford: Blackwell Scientific Publications. LUNN, D. P. & HINf;I-ICt.IFI:,K. W. (1989). Cerebrospinal fluid eosinophilia and ataxia in five llamas. Vete~naTyRecord 124, 302-5. MAw-tKw, I. G. (1989). Large Animal Neurology: A Handbook for Vete~Jnary Clinicians, p. 15 Philadelphia: Lea & Febiger. MA~I-IEW,I. G. & BZAL,C. R. (1980). Techniques of analysis of cerebrospinal fluid. VeterinaTy Clinics of North America 10, 155. Mit.Hor~w', T. H. (1975). The third circulation revisited. Journal Neurosurge~y42, 628--45. McGvmK, S. M. (1987). In Bovine Neurologic Diseases, p. 112, ed. J. c. Baker, Veterinary Clinics of North America 3, Philadelphia: W. B. Saunders Co. OIJvER, J. E. & LORENZ, M. D. (1983). Handbook of Veterinary NeacrologicDiagnosis. Philadelphia: W. B. Saunders Co. POWER, E. P., CROWLEV,J.J., B',~yE,J. F., O'I~Ew, M. P. & W~WZRS, E. D. (1985). Cerebrocortical necrosis in dairy cows. lrish Vete~TnaryJourna139, 81-3. PRFSCO'I-r,J. F. & BA~;C;o'I',J. D. (1988). In Antimicrobial Therapy in Veterinary Medicine, p. 78. London: Blackwell Scientific Publications. Rt:.~Huy, W. C. & DE L-`'HUNTA,A. (1982). Diagnosis and treatment of bovine listeriosis. Journal Ame~qcan Veterinaly Medical Association 180, 395-8. ScoTt, P. R. (1992). Analysis of cerebrospinal fluid from field cases of some c o m m o n ovine neurological diseases. B~itish VeterinaryJournal 148, 15-22. Scoa r, P. R. (1994). Cerebrospinal fluid analysis in the differential diagnosis of spinal cord lesions in ruminants. In Practice (in press). ScoTt, P. R. (1993a). A field study of listerial meningo-encephalitis with particular reference to cerebrospinal fluid analysis as an aid to diagnosis and prognosis. British Veterinary Journal 149, 165-70. Scolq', P. R. (1993b). Total protein and electrophoretic pattern of cerebrospinal fluid in sheep with some c o m m o n neurological disorders. Cornell Vete74na~ian83, 199-204. Sc:orl", P. R., At.DRmC;E,B. M., CL-xRKE,M. & Wmt, R. G. (1990). Cerebrospinal fluid studies in normal cows and cases of bovine spongiform encephalopathy. British Vete~Tna~yJournal 146, 88-90. Scorr, P. R. & Wu.L, R. G. (1991). A report of Froin's syndrome in four ovine thoracolumbar epidural abscess cases. British I/eterinaryJou~za1147, 582-4. Sc:oI-r, P. R. & Stq-ro~, D. (1992). A study of some factors which may influence the concentrations of penicillin G in cerebrospinal fluid in some c o m m o n bovine neurological diseases. Proceedings of XVIl World Buiatffcs Congress, St Paul, USA 2, 63-8. S~:ol-l, P. R. & PENNV,C. D. (1993). A field study of meningo-encephalitis in calves with particular reference to cerebrospinal fluid analysis. Vete~ina~),Record 133, 119-21. Sco-ra', P. R., WAaq, N. J., WOOOMA~, M. P., McGoRuM, B. M. & McDL~HD, A. (1993). Protozoan enceplaalomyelitis causing pelvic limb paresis in a yearling sheep. New Zealand Vete~7;na~yJournal 41, 139-41. S¢:orT, P. R., SARC,lSON, N. D., PENN',, C. D. & PIRIE, R. S. (1994). A field study of naturallyoccurring ovine bacterial meningo-encephalitis. Vete~71na~yRecord (in press). SORJONEN, D. C. (1987). Total protein, albumin quota and electrophoretic patterns in cerebrospinal fluid of dogs with central nervous system disorders. American Journal of Veterina~. Research48, 301-5. STILMN,G. M., Ct.~xroN, M. S., TURNQUIST,S. E. & KREEGER,J. M. (1987). Evoked potential and electro-encephalographic assessment of central blindness in a steer. Co~7zellVeterinarian 77, 374--82. T v ~ x ~ , H. W. (1987). In Bovine Neurologic Diseases, pp. 25-44, ed. J. C. Baker. Veterinary Clinics of North America. Food Aaaimal Practice 3. Philadelphia: W. B. Saunders. WATt, N.J., Roy, D. J., McCo~nzta., I. & KJNG, T.J. (1990). A case of visna in the United Kingdom. VeterinaryRecord 126, 600-1. Wzt.~zs, E. G., T~zR, J. W., So~jo,~Fy, D. C. & Wrtaxuzv, E. M. (1992). Composition and analy-
614
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sis of cerebrospinal fluid in clinically normal adult cattle. American Journal of Veterina O, Research 53, 2050-7.
(Acceptedfor publication 13 September 1994)