Albumin leakage into cerebrospinal fluid of dogs lethally infected with R252 canine distemper virus

Journal of Neuroimmunologv, 14 (1987) 61-74 Elsevier

61

JNI 00448

Albumin leakage into cerebrospinal fluid of dogs lethally infected with R252 canine distemper virus G.C. Johnson, S. Krakowka and M.K. Axthelm Department of Veterinary Pathobiology, The Ohio State Universi(v, Columbus, OH 43210, U.S.A. (Received 8 November 1985) (Revised, received 28 July 1986) (Accepted 28 July 1986)

Key words." Cerebrospinal fluid; Blood-brain barrier; Canine distemper; Morbillivirus; Albumin

Summary Cerebrospinal fluid (CSF) form nine lethally infected and three convalescent gnotobiotic dogs infected with the R252 strain of canine distemper virus (CDV) was evaluated prior to and following infection. Lethally infected dogs had a mean seven-fold increase in CSF albumin concentration compared to the preinoculation value, not present in dogs destined to survive. Immunochemical examination of tissue from these dogs revealed prominent perivascular localization of albumin. Examination of CSF cells demonstrated mild leukocytosis in both groups at the time when encephalopathic deaths occurred, with decreased lymphocyte percentages, particularly Thy-l-bearing lymphocytes, in lethally infected dogs. These dogs also had more extensive expression of viral antigens in CSF and peripheral blood leukocytes at the time of death than did surviving dogs, and failed to make antibody to viral antigens. The findings link terminal breakdown of the blood-brain barrier and extensive viral antigen expression in CSF leukocytes with experimental CDV infection resulting in death.

Address for correspondence: G.C. Johnson, Department of Veterinary Pathobiology, The Ohio State University, Columbus, OH 43210, U.S.A. 0165-5728/87/$03.50 ~:~1987 Elsevier Science Publishers B.V. (Biomedical Division)

62 Introduction

Acute encephalopathy is a very common manifestation of overwhelming canine distemper virus (CDV) infection of young animals, particularly under experimental circumstances (McCullough et al. 1974a; Krakowka and Koestner 1976; Krakowka et al. 1978c), and is analogous to measles encephalopathy in rodents (Griffin et al. 1974; Van et al. 1979), measles encephalopathy in immunosuppressed human beings (Roos et al. 1981; Chadwick et al. 1982), and neurotropic mumps encephalopathy in rodents (Kristensson et al. 1984). The short clinical course and fulminant nature of the disease is discordant with the absence of dramatic CNS lesions (Higgins et al. 1982a), which are limited to neuronal degeneration, with minimal inflammation. Viral antigen expression is limited largely to neurons and to perivascular macrophages. The genesis of clinical signs in CDV encephalopathy is unknown; neuronal changes have been attributed either to the direct effects of virus infection or to convulsion-induced hypoxia (Braund et al. 1982), despite the fact that neuronal degeneration occurs even if anticonvulsant therapy is given. We report that the terminal stages of CDV encephalopathy are characterized by leakage of albumin across the blood-brain barrier into the brain, a phenomenon which did not occur in dogs destined to recover from infection. Furthermore, dogs which died of encephalopathy had more extensive viral antigen expression in CSF leukocytes as well as a reduction in CSF lymphocytes, particularly Thy-l-bearing cells, relative to the surviving dogs. We speculate that altered blood-brain barrier permeability may have a role in determining the outcome of infection and that CSF albumin concentrations may predict the outcome of acute CDV infection in virus-infected dogs.

Material and Methods Animals

Twelve gnotobiotic beagle dogs from three litters were derived by cesarean section, as previously described (Greisemer 1963; Krakowka et al. 1978b), and housed with littermates. The dams of the litters were selected for low titers of canine distemper virus-neutralizing antibody, to ensure that maternal immunity derived in utero (Krakowka et al. 1978a) did not interfere with the course of postnatal infection. CSF samples were obtained from anesthetized dogs by puncture of the cisterna magna 7 days prior to infection with canine distemper virus, at 3 weeks of age. At 4 weeks of age, all puppies were inoculated intraperitoneally using 0.2 ml spleen suspension from a puppy infected with R252-CDV (10 4.5 TCIDs0/ml; McCullough et al. 1974b; Confer et al. 1975), which was two puppy passages removed from one brain passage of the original isolate (McCullough et al. 1974a). Paired serum and cisternal CSF samples were then taken at weekly intervals, commencing 4 weeks following infection. Dogs became clinically ill between 4 and 5 weeks after infection and died or were killed in a moribund state. Terminal CSF and serum samples were collected immediately after sacrifice or within 5 days prior to

63 death. Only CSF samples containing fewer than 1000 rbc//~l were used in data analysis.

Cerebrospinal fluid CSF samples were chilled (4 ° C) prior to evaluation. Cells were first gently mixed and leukocyte counts done using a hemocytometer. Samples were then centrifuged for 10 min at 800 × g to separate cells from liquid. The cell-free supernatant was aliquoted and frozen ( - 7 0 ° C ) for subsequent immunological determinants. Cells were resuspended in phosphate-buffered saline (0.01 M NaHzPO 4, pH 7.2 and 0.15 M NaCI) containing 5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO) and cytocentrifuged onto microscope slides (Shandon Southern, Piscataway, N J) for immunofluorescence (IF) studies. Peripheral blood leukocytes were separated from heparinized blood by Ficoll-Hypaque density gradient centrifugation (Ringler and Krakowka 1985) prior to cytocentrifugation of 1 × 105 leukocytes. Differential counts were done on Wright-Giemsa stained preparations, using standard methods. Examination of CSF sediment for canine distemper viral antigens utilized direct IF with canine hyperimmune serum to R252-CDV which had been treated with octanoic acid prior to conjugation with fluorescein isothiocyanate (Winters et al. 1983/1984). Cells expressing surface immunoglobulin were evaluated by treating cytospin preparations with rabbit anti-dog IgG F(ab)" (Cappel Laboratories, Cochranville, PA), while the procedure for demonstrating dog T cells involved indirect IF using mouse monoclonal antibody F3-20-7 (Serotec, Biester, Oxfordshire, U.K.) to dog Thy-1 antigen (Dalchau and Fabre 1979; McKenzke and Fabre 1981), followed by rabbit anti-mouse IgG-FITC conjugate (Miles Scientific, Naperville, IL). Hydrated unfixed specimens were used in each instance. Dog peripheral blood leukocytes and CDV-infected Vero cells were used as positive controls. After incubation with primary or secondary reagent, the slides were washed 3 times with PBS, and coverslips mounted in 90% glycerol (pH 8.2) containing 5% n-propyl gallate to reduce fading of fluorescence (Giloh and Sidat 1982). Differential counts of 100 cells were done to obtain results. Tissue evaluation Following death, tissues were fixed by immersion in 10% neutral buffered formalin, or immersed in embedding medium (O.C.T. Medium, LabTek, Naperville, IL) before freezing in liquid nitrogen. Multiple sections of brain, spinal cord and visceral organs were removed and processed by standard histologic methods. Localization of albumin in tissue sections was accomplished by cutting 8-10 /zm-thick sections of frozen brain, and staining them with fluorescein-conjugated rabbit anti-dog albumin, or fluorescein-conjugated rabbit anti-dog IgG F(ab)~ antiserum fragments (Traugott et al. 1985). Radial immunodiffusion Radial immunodiffusion was utilized to determine the albumin and IgG concentrations of serum and CSF samples, using previously published methods (Feyhey and McKelvey 1965; Johnson et al. 1985) and commercially available reagents

64 (Miles L a b o r a t o r i e s a n d C a p p e l L a b o r a t o r i e s ) . A l l d e t e r m i n a t i o n s were m a d e in q u a d r u p l i c a t e a n d averaged. C o o m a s s i e b r i l l i a n t blue staining of p r e c i p i t i n cylinders was used to increase the sensitivity of the assay for C S F d e t e r m i n a t i o n s . Each p l a t e h a d a series of s t a n d a r d s e r u m dilutions for d e t e r m i n i n g a s t a n d a r d curve, E n z y m e - l i n k e d i m m u n o s o r b e n t assays were p e r f o r m e d using lysates of C D V - i n fected Vero cells a t t a c h e d to m i c r o t i t e r plates. A f t e r i n c u b a t i o n of the plates with serum at a 1 : 10 dilution, b o u n d a n t i b o d y was d e t e r m i n e d b y the degree of b i n d i n g of h o r s e r a d i s h p e r o x i d a s e - c o n j u g a t e d r a b b i t a n t i - d o g I g G or IgM, as d e v e l o p e d b y 5 - a m i n o s a l i c y l i c a c i d c h r o m a g e n ( N o o n et al. 1980; B e r n a r d et al. 1982). Titers were expressed as ng s e c o n d a r y c o n j u g a t e b o u n d to the p l a t e as c o m p a r e d to the a m o u n t of c o l o r e d p r o d u c t d e v e l o p e d w h e n k n o w n a m o u n t s of c o n j u g a t e were a d d e d to the wells. T h e a b i l i t y of 10-fold serum dilutions to limit the r e p l i c a t i o n of 100 TCIDs0 R 2 5 2 - C D V was tested using m i c r o t i t r a t i o n m e t h o d s ( A p p e l a n d R o b s o n 1983). A f t e r 7 days, wells positive a n d negative for viral infection were d e t e r m i n e d by direct I F staining with a 1 : 1 0 d i l u t i o n of d o g a n t i - C D V - F I T C (Pursell a n d Cole 1976), a n d the titer d e t e r m i n e d using the R e e d - M u e n c h m e t h o d (Bichsel et al. 1984).

Results C o m p a r i s o n of s a m p l e s t a k e n p r i o r to i n o c u l a t i o n with those t a k e n at the time of d e a t h or convalescence ( T a b l e 1) d e m o n s t r a t e s that dogs which d i e d of C D V e n c e p h a l o p a t h y h a d significantly greater C S F a l b u m i n c o n c e n t r a t i o n s t h a n dogs which recovered ( m e a n 395.6 + 231.6 ~ g / m l c o m p a r e d to 56.9 + 17.3 btg/ml). C S F a l b u m i n c o n c e n t r a t i o n s of lethally infected dogs also were significantly increased f r o m m e a n p r e i n o c u l a t i o n c o n c e n t r a t i o n s (51.7 + 7 . 7 / ~ g / m l ) , whereas there was no difference b e t w e e n p r e i n o c u l a t i o n a n d p o s t i n o c u l a t i o n (p.i.) values from those dogs which recovered f r o m infection. S a m p l e s taken 6 - 1 0 weeks p.i. f r o m surviving dogs also failed to show i n c r e a s e d C S F a l b u m i n (overall m e a n 60.0 + 10 / ~ g / m l for 16 samples; d a t a not shown), so that a l b u m i n leakage into the C S F was n o t o b s e r v e d in these dogs at any time following infection. TABLE 1 CEREBROSPINAL FLUID ALBUMIN CONCENTRATIONS OF GNOTOBIOTIC DOGS INFECTED WITH R252 CANINE DISTEMPER VIRUS Sample

CSF albumin ~

Serum albumin

CSF : serum albumin ratio

Preinoculation ( n = 11) b Lethally infected ( n ~ 6) Recovered ( n = 3)

51.7 + 7.7 395.6 __231.6 * 56.9 + 17.3

23.1 + 12.1 46.4 + 0.1 41.5 + 9.2

0.0026 _4_0.001 0.0092 + 0.004 * 0.0013 _4-0.002

a Each data point represents the mean of quadruplicate determinations by single radial immunodiffusion. Mean + standard deviation. CSF in /~g/ml, serum in mg/ml. b Variation in dog numbers resulted from eliminating CSF samples contaminated by blood. * P < 0.05, one-way analysis of variance from both preinoculation and postinoculation treatment groups, pre- and postinoculation samples compared by repeated-measures analysis of variance.

65

Fig. 1. Frozen sections of cerebral cortex of a lethally infected dog stained by IF to localize canine albumin (a) or IgG (b). Bar = 25/zm.

Changes in CSF albumin were unaccompanied by differences in serum albumin concentrations between the lethally infected and surviving dogs (mean 46.4 + 0.1 m g / m l versus 41.5 ___9.2 m g / m l , respectively), although the serum albumin concentrations of the dogs increased significantly in both groups between preinoculation (mean 23.1 ___12.1 m g / m l ) and postinoculation (45.3 + 5.4 m g / m l at 4 weeks p.i.; P < 0.05). The C S F : s e r u m albumin ratio was not increased in dogs which survived infection (Table 1), indicating that the relative contribution of serum albumin to CSF had not increased during infection in survivors, while an increase in this ratio was present in terminally infected dogs. Thus, differences in CSF albumin between the groups could not be attributed to differences in the dogs at the time of inoculation, or to differences in serum albumin concentrations. To confirm that blood-brain barrier leakage of albumin had occurred, we examined frozen sections of affected brains by immunofluorescence for the presence of perivascular albumin in the neuropil. Numerous parenchymal vessels were outlined by localized deposits of albumin which diffused into the surrounding parenchyma. Capillaries and venules were affected, but cuffs were especially prominent around the latter (Fig. la). Sections from the same tissue stained with rabbit anti-dog I g G - F I T C conjugate failed to give a similar staining, although faint intravascular IgG could be seen in some sections (data not shown). Differences in CSF I g G were not detected in the two groups, perhaps due to the insensitivity of the assay. Gnotobiotic dogs in both groups had low (2.12 + 0.81 m g / m l ) serum IgG concentrations. Brain tissues collected from uninfected gnotobiotic dogs or from

66

Fig. 2. Histologic appearance of hippocampal tissue from dog in Fig. 1. Neuronal degeneration (N), mild spongiform change (S) and perivascular mononuclear cuffs (arrowheads) (C) are designated. Bar- 50 tLm.

dogs surviving infection and killed 11 months postinoculation did not stain with either conjugate. Histologically, lethally infected dogs had lesions typical of C D V encephalopathy (Fig. 2). Major nervous system lesions consisted of neuronal degeneration and necrosis, rare intracytoplasmic and intranuclear viral inclusions, and small loci of

67 TABLE 2 CEREBROSPINAL FLUID LEUKOCYTE COUNTS OF GNOTOBIOTIC DOGS LETHALLY AND NONLETHALLY INFECTED BY CANINE DISTEMPER VIRUS Preinoculation Postinoculation week 4-5 Postinoculation week 6-14

Lethally infected

Survived

3.8± 2.5(6) 20.9 ± 12.1 (7) ** ND

5.0± 0 (3) 22.8 ± 20.3 (6) **'* 3.0± 2.3 (16)

** Significantly greater than preinoculation values by Wilcoxon sign rank test (P < 0.005), one-tailed test. * Greater than preinoculation and 6-14 week values to P > 0.05.

p o l y m i c r o c a v i t a t i o n . I n f l a m m a t i o n was limited to mild n o n s u p p u r a t i v e meningitis a n d slight per±vascular cuffing. Mild, statistically significant C S F leukocytosis occurred in b o t h lethally a n d n o n l e t h a l l y infected dogs d u r i n g weeks 4 a n d 5 p o s t i n o c u l a t i o n , c o m p a r e d to p r e i n o c u l a t i o n s a m p l e s for the dogs ( T a b l e 2). However, there was no difference b e t w e e n dogs which d i e d of infection a n d those which survived. There was n o c o r r e l a t i o n b e t w e e n the C S F a l b u m i n c o n c e n t r a t i o n a n d the n u m b e r of cells in C S F ( r = 0.39, d a t a n o t shown). In the convalescing dogs, C S F cells r a p i d l y r e t u r n e d to p r e i n o c u l a t i o n n u m b e r s , a n d r e m a i n e d so thereafter. However, the t y p e of C S F leukocytes differed b e t w e e n the lethally infected a n d surviving dogs, as shown in T a b l e 3. Thus, lethally infected dogs h a d a p r e d o m i n a n c e of histiocytes in their C S F (Fig. 3a), while dogs d e s t i n e d to survive h a d significantly m o r e n u m e r o u s l y m p h o c y t e s (Fig. 3b). W h e n I F testing was used to distinguish B a n d T l y m p h o c y t e s p r e s e n t in C S F , lethally infected dogs t e n d e d to have r e d u c e d n u m b e r s of T h y - l - b e a r i n g cells (Table 3). F e w ( 0 - 1 % ) I a - b e a r i n g cells were f o u n d in either g r o u p so that q u a n t i t a t i o n of surface I g - b e a r i n g cells c o u l d not b e done. W h e n C S F cytological p r e p a r a t i o n s were e v a l u a t e d for viral antigen b y direct I F there was a striking difference in viral a n t i g e n expression b e t w e e n the lethally a n d n o n l e t h a l l y infected g r o u p s of dogs ( T a b l e 3). W h e r e a s dogs which d i e d of infection i n v a r i a b l y expressed C D V antigen in a high p e r c e n t a g e of their C S F leukocytes ( m e a n 35.9%; range 14-28%), dogs recovering from infection h a d viral antigen in

TABLE 3 CHARACTERISTICS OF CSF LEUKOCYTES OF SURVIVING AND LETHALLY INFECTED DOGS (4-5 WEEKS POSTINOCULATION) Outcome

Lymphocytes

T cells

CDV antigen

Positive PBL

Lethal Survived Significance b

33.6 ± 19.4 a 71.7± 16.5 _<0.005

14.4 + 8.7 25.4± 8.2 < 0.10

35.9 ± 11.0 3.3 ± 3.5 < 0.005

100% 0% --

a % cells + SD, determined by a manual count of 100 cells. b Determined by the Wilcoxon two-sample test.

68

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Fig. 3. Cytocentrifuge preparation of CSF cells from a lethally infected ( a ) and convalescent (b) dog at 4 weeks postinoculation. Bar = 50 t~m.

Fig. 4. Expression of canine distemper viral antigens in CSF cytocentrifuge preparation from a lethally infected dog, 4 weeks postinoculation. Bar = 20 btm.

69

IgG 6¸

~42-

ol o! o; oZ • ,

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•Surviving

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Weeks Post Infection

Fig. 5. Nanogramshorseradishperoxidase-conjugatedanti-dogIgG or IgM bound to CDV antigen-coated plates by serum of lethally infected(closed circles) and surviving(open circles) dogs followinginfection with R252-CDV.

few cells (mean 3.3%; range 0-7%). Both lymphocytes and macrophages demonstrated punctate cytoplasmic fluorescence (Fig. 4). Both groups of dogs also had antigen-positive peripheral blood leukocytes for the first 3 weeks following infection, although expression was more extensive in dogs which were lethally infected (data not shown). However, 4-week p.i. samples from all lethally infected dogs had peripheral blood leukocytes which were viral antigen-positive, while recovering dogs did not express viral antigens. Both CSF and peripheral blood leukocytes of convalescent dogs remained negative in succeeding weeks. Lethally infected dogs produced neither IgG or IgM anti-CDV antibodies, as demonstrated by ELISA testing (Fig. 5). Although lethally infected dogs displayed trace amounts of antiviral IgM one week following infection, they failed to sustain the response or produce detectable amounts of antiviral IgG. In contrast, dogs destined to recover from infection produced transient antiviral IgM by 4 weeks p.i., followed by a sustained antiviral IgG beginning 4-5 weeks p.i., at the time of greatest mortality in lethally infected dogs. Such increases were roughly coincident with increased serum IgM and IgG concentrations noted in surviving dogs. Further-

70 more, lethally infected dogs failed to produce viral neutralizing antibody, while low titers were evident in recovering dogs by week 8 (data not shown).

Discussion

Taken together, the data demonstrate the characteristics of cerebrospinal fluid following CDV infection, and denote differences in CSF of dogs destined to die of CDV encephalopathy compared to those destined to recover from acute stages of infection. Lethally infected dogs have increased CSF albumin concentrations in the terminal stages of disease. The suspicion that blood-brain barrier (BBB) leakage had occurred was substantiated by morphological demonstration of albumin localized around parenchymal vessels in the brain. Although both groups of dogs had mild CSF leukocytosis, lethally infected dogs had a lower percentage of lymphocytes and T cells in their CSF. More virus-positive cells were present in preparations of CSF sediment from lethally infected than convalescent dogs. Lethally infected dogs failed to make antibody to virus, either as detected by ELISA or virus neutralization. Measurement of CSF albumin concentrations in gnotobiotic dogs infected with R252-CDV indicated that dogs which die with acute encephalopathy 4 5 weeks following infection have a mean seven-fold increase (range 5.3-11.9) in terminal CSF albumin concentrations not present in dogs destined to recover from infection. These differences from control dogs could not be explained by differences in serum albumin concentrations, as indicated by the significantly increased C S F : s e r u m albumin ratio in the former groups. Although stasis of CSF protein resorption could conceivably contribute to the increase, the most probable explanation is increased permeability of the blood-brain barrier (BBB) to small prot~eins, including albumin. This hypothesis is supported by the presence of perivascular albumin in the brains of lethally infected dogs. A lack of detectable concomitant IgG leakage into the CSF probably reflects the low serum IgG concentration typical of gnotobiotic dogs; as increased CSF IgG concentrations typically accompany elevated albumin in instances of BBB disruption (Link and Tibbling 1977; Tourtellotte et al. 1980; Griffin 1981; Johnson 1984), although discrepancies between albumin and IgG concentration may occur (Livrea et al. 1984), particularly in severe meningitis (Prosiegel et al. 1983). Our findings in acute experimental CDV infection are similar to those of others in acute spontaneous distemper but differ from the increased CSF IgG in the absence of albumin leakage in CSF of dogs affected by more chronic forms of CDV encephalitis (Bichsel et al. 1984; Vandevelde et al. 1986; unpublished observations) and are similar to transient BBB leakage observed prior to inflammation in Sinbis viral encephalitis (Griffin 1981) and experimental allergic encephalomyelitis (Traugott et al. 1985). Furthermore, blood-brain barrier leakage appears to be a relatively terminal event. CSF samples with increased albumin concentrations were obtained 1-2 days prior to death (except for a single 5-day sample); in all instances these samples were obtained during the presence of convulsions or other clinical signs referable to CNS

71 disease. Additional samples obtained from lethally infected dogs earlier in infection (in some instances after the onset of convulsions) had a mean albumin concentration of 85.1 + 38.5 /~g/ml (unpublished observation), so that there was not a correlation between the presence of clinical signs and elevation of CSF albumin. However, experimental seizures can induce blood-brain barrier albumin leakage (Petito et al. 1977), indicating the clinical signs may have contributed to this phenomenon. Recent immunocytochemical evidence indicates that endothelial cells may become infected by CDV as early as 6 days following experimental infection (Axthelm 1985) providing a second possible mechanism of BBB derangement. Postmortem examination of the brains of lethally infected dogs revealed encephalopathic lesions similar to those previously described of widespread neuronal degeneration, with minimal inflammation and moderate focal vacuolar change (Higgins et al. 1982a) and are similar to those of acute measles virus and mumps virus infection (Van et al. 1979; Kristensson et al. 1984). When frozen sections of brain were examined, widespread areas of albumin leakage were revealed. Albumin was seen in and around vascular walls, in the Virchow-Robbin space, and in the adjacent neuropil and occurred more diffusely than the diencephalic distribution of seizure-induced leakage (Petito et al. 1977). Moreover, the morphological distribution of perivascular CNS albumin in these dogs closely approximates that produced by BBB injury rather than that resulting from acute oxygen deprivation (Brightman et al. 1970). The ultrastructural appearance of this lesion after antemortem administration of tracers is currently under investigation. Cytologic examination of cerebrospinal fluid indicated that mild transient leptomeningitis was present late in lethal infections and occurred during the same postinoculation week in dogs destined to recover. These changes were similar to those found by numerous other investigators (Cutler and Averill 1969; Krakowka and Koestner 1976; Vandevelde and Spano 1977; Higgins et al. 1982b) in both experimental and spontaneous disease. However, lethally infected dogs had a preponderance of monocyte-macrophage-like cells in their CSF, and fewer CSF Thy-l-bearing cells. This finding parallels reduction in peripheral blood lymphocytes in distemper, following viral infection (Krakowka et al. 1975): it is not evident whether these differences represent differential virocidal killing of lymphocytes (particularly Thy-l-bearing lymphocytes) within the CNS or whether there are merely fewer T cells to enter the CNS in lethally infected dogs. Alternatively, increased T cells in the CSF of convalescent dogs may indicate an increased immune traffic useful in stemming the viral infection, although increased CSF immunoglobulin was not apparent. The CSF sediment of lethally infected dogs was characterized by the presence of numerous virus-infected leukocytes, also an observation which has been made by others (Long et al. 1973; Higgins et al. 1982b). However, the reduced percentage of infected CSF leukocytes in dogs destined to recover from infection has not been reported. Moreover, although convalescent dogs did not express viral antigens in the PBL by week 4, their peripheral leukocytes had been antigen-positive in the preceding 3 weeks. Thus, although the present observations need to be extended to CSF samples taken earlier in infection, it appears that a high percentage of viral

72 antigen-positive C S F leukocytes m a y be a reliable indicator of poor prognosis in C D V infection. Following 5 weeks, virus antigen-positive leukocytes were not seen in the C S F or peripheral b l o o d of surviving dogs. The presence of low n u m b e r s of IF-positive cells in C S F of some convalescent dogs indicates that C N S infection occurs in m a n y dogs b u t that i m m u n e or n o n i m m u n e (Tsai et al. 1982) m e c h a n i s m s limit replication of C D V virus a n d t e r m i n a t e infection at this site in convalescent animals. I n conclusion, increased C S F a l b u m i n c o n c e n t r a t i o n s occur regularly in the t e r m i n a l stages of acutely lethal C D V infection of gnotobiotic dogs, following increased BBB p e r m e a b i l i t y a n d implicating vasogenic e d e m a as a c o n t r i b u t i n g factor to death. This p h e n o m e n o n occurs c o n c u r r e n t l y with r a m p a n t viral a n t i g e n expression i n C S F macrophages a n d with the r e d u c t i o n of T h y - l - b e a r i n g cells, at that site. These factors, c o n c o m i t a n t with the observations of A x t h e l m (1985) that endothelial infection occurs early in C D V infection suggest that this may be a c o n t r i b u t o r y factor to the d e v e l o p m e n t of encephalopathy.

Acknowledgements The authors t h a n k Sue Ringler, N a n c y A u s t i n a n d Judy D u b e n a for their excellent technical assistance, a n d Virginia S t u m p a n d Roslyn R y a n for their typing of the manuscript.

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