l "eterina(v Microbiology 27 ( 1991 ) 205-219 Elsevier Science Publishers B.V., A m s t e r d a m
205
Studies on the role of feline calicivirus in chronic stomatitis in cats J.O. Knowles a'b, F. McArdle b, S. Dawson a'b, S.D. Carter% C.J. Gaskell a and R.M. G a s k e l l b Departments of a Veterinary Clinical Science and b Veterinary Pathology, University ~/Livet7)ool, Liverpool, UK (Accepted 10 January 1991)
,ABSTRACT Knowles, J.O., McArdle, F., Dawson, S., Carter, S.D., Gaskell, C.J. and Gaskell, R.M., 1991. Studies on the role of feline calicivirus in chronic stomatitis in cats. Vet. Microbiol., 27:205-219. Two groups of cats were inoculated oro-nasally with one of two isolates of feline calicivirus ( FCV ) from clinical cases of chronic stomatitis. All cats developed signs typical of acute FCV infection: namely, ocular and nasal discharge, conjunctivitis, and marked oral ulceration. None of the cats shed virus beyond 28 days. Seronegative control cats were then infected with a lower dose of one isolate, but again only acute signs were seen and no carriers produced. The original cats were then re-infected with lhe heterologous isolate. As before, only signs of acute disease were seen, but the range of clinical signs and severity was reduced. Virus shedding patterns in one group were similar to those seen originally, but in the other the duration was reduced. No chronic stomatitis developed over the 10 months of the study. Serum virus neutralising and serum and salivary class specific immunoglobulin responses were investigated. Although long-term carriers were not induced, no relationship between cessation of virus shedding in an individual animal and systemic and local antibody responses was seen.
INTRODUCTION
Feline calicivirus is a common cause of upper respiratory disease and acute oral ulceration in cats (Gaskell, 1985 ). It has also been implicated in chronic stomatitis. Recent studies have shown that approximately 85% of cats with chronic stomatitis in the U K are carriers of feline calicivirus, compared to 20% controls (Knowles et al., 1989 ), confirming an earlier smaller Australian survey by Thompson et al. (1984). An association of feline immunodeficiency virus (FIV) infection with chronic stomatitis in cats has also been found (Yamamoto et al., 1989; Hosie et al., 1989; Knowles et al., 1989). However, the role of these agents either individually or in combination in inducing the disease has not been established. In both field and experimental infections, the carrier state appears to be a
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c o m m o n sequel to feline calicivirus ( F C V ) infection (Wardley cl al., 1974: Wardley and Povey, 1977: Orr el al., 1980: Gaskell ctal., 1982 ). Wardlcy and Povey (1977) have shown that although the duration of excretion is highl~ variable between individuals, in a group of infected cats the infective-animal half-life appeared to be approximately 75 days. Thus at 30 days after infection, the majority of cats were still shedding virus whereas at 75 days, 51)% had ceased. The possible immunological events involved in this apparenll) sudden cessation of virus shedding tbr an individual cat have not been examined. The purpose of this study was, firstly, to examine the role of feline calicivirus in chronic stomatitis in cats. There are a number of feline calicivirus isolates which differ slightly in their antigenicity and pathogenicity ( Kalunda et al., 1975; Povey and Hale, 1974 ). However, pathogenesis studies have been done with isolates of feline calicivirus from a variety of clinical syndromes, rather than isolates originating from cases of chronic stomatitis. The second aim of the study was to relate local and systemic humoral immune responses to the course of infection and to the cessation of virus shedding in carriers. MATERIALS AND METHODS
('ell culture A feline embryo-derived cell line (FEA; Jarrett et al., 1973 ) grown in B H K media (Wellcome) with antibiotics, sodium bicarbonate and Hepes buffers, and 4% (growth) or 1% (maintenance) foetal calf serum was used. [ ~II'IISCS Two isolates, from cats LS015 and LS027 with chronic stomatitis, and persistently infected with FCV, were used. Isolates were selected on the basis of their differing in vitro characteristics. Isolate LS027 was significantly neutralised by antiserum to FCV F9, and LS015 was not: F9 is a widely used FCV vaccine strain (Kahn and Hoover, 1976). LS015 formed large plaques (4.8 ram. diameter) under agar overlay, whereas LS027 produced small plaques (0.8 mm. ) (Ormerod and Jarrett, 1978; Knowles, 1988 ). Viral stocks were prepared and titrated in FEA cells as described previously (Wardley, 1976).
Virus neutralisation (VN) tests VN tests were performed using 100 TCID~o of homologous virus and serial two-fold dilutions of serum each contained in 50 microlitres in 96 well plates. Virus-serum mixtures were incubated at 37°C for one hour prior to the addition of FEA cells. The results were recorded after 72 hours.
FELINE C~L1CIVIRUS IN CHRONIC STOMATITIS IN CATS
207
Class-specific enzyme linked immunosorbent assay (ELISA) A single dilution ELISA assay was used with homologous virus precipitated with 7% polyethylene glycol (PEG) 6000 and then spun for two hours at 10 000 g, and resuspended in 1/ 10th of the original tissue culture volume in PBSA. ELISA plates (Flow) were coated at 10 #g/ml at 37°C for two hours. After washing the plates, the test cat sera ( 1 : 200 in PBSA+ 0.05% Tween 20) or test saliva ( 1:20 in PBSA+0.05% Tween 20) were incubated in duplicate at 37°C for one hour. Bound antibodies were detected with mouse monoclonal antibodies to IgM and IgG cat immunoglobulins and a monoclonal to human IgA which cross-reacts with cat IgA (Oxoid). The products were then developed with goat anti-mouse IgG (alkaline phosphatase-conjugated) and the substrate, p-nitrophenyl phosphate (Sigma). The colour conversion was read at 405 nm. Sera and saliva were also tested against ELISA plates previously coated with the PEG precipitate from a non-infected culture and any resulting Optical density (OD) subtracted from the antigen specific data. The data was expressed as relative ODs (i.e. values were expressed relative to the highest result within an individual immunoglobulin class for each virus group). CaIs Eight 16 week old SPF cats of mixed sexes were used. They were housed in three groups of two or three in barrier maintained rooms with positive pressure ventilation.
Experimental design Experiment 1. Two groups of three cats were inoculated with 10ss TCIDs0 in 2 ml of either LS015 or LS027, half given intranasally and half orally. Two control cats were similarly inoculated with 2 ml of uninfected cell culture fluid. The cats were monitored daily for the first nine days and thereafter at least twice weekly until 63 days after inoculation. Clinical signs were scored on a 0-5 scale, where 0 indicated a clinical sign was absent and 5 indicated the cat was severely affected. However oral ulcers were scored by their diameter in mm. Nasal and oropharyngeal swabs were placed in viral transport medium and stored at - 7 0 ° C prior to attempted virus isolation (Wardley et al., 1974; Cocker et al., 1984). Oropharyngeal swabs for class specific anti-FCV immunoglobulin estimation were taken into 1 ml PBSA containing 0.05% Tween 20 and stored at - 20 ° C. Serum samples were also obtained for VN tests and for class specific antibody estimations.
Experiment 2. Sixty-five days after the start of Experiment l, the two control cats were inoculated as above with a lower dose of LS015 (104 TCIDs0 in
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2 ml). These cats were monitored for clinical signs, virus shedding, and serology as in experiment 1, although sampling was slightly less frequent.
k\vperimenl 3. At the same time as Experiment 2, each of the two FCV-infected groups were then re-inoculated with the heterologous isolate, using an identical dose and route of infection as in Experiment 1. These cats were monitored for clinical signs, virus shedding, and serology as in Experiment 1. Clinical observation was carried out on all cats in these studies for a total of 10 months after the start of Experiment 1. RESULTS
E:vperirnenl 1 Clinical signs After an incubation period of one to four days, both isolates produced an acute disease of approximately two to three weeks duration (Table 1 ). This I.\BLtl I Q u a n t i t a l i x c scores o f c o m m o n clinical signs in cats i n f e c t e d oronasally with either F C V l.S015 or t_S027 ( Expert mcnl I ) ( l i n i c a l signs
Days post infection 0
I
l_S015 l_S027
0"* 0
1 0
Rcduccdappetite
LS015 l_S027
0 0
(l (1
Nasal d i s c h a r g e
LS015 LS027
0 0
()cular d i s c h a r g e
LS015 LS027
('onjunctivitis
()ral ulcers
Letharg 3
t ) t h e r signs +
2
3
Fotal* 4
6
7
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6 4
5 2
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16 9
t) I
1 1
2 1
3 1
1.5 1
1.5 1
0.5 1
0.5 0
0 ()
0 0
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1.5 1
1
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14.5 7.5
kS015 LS027
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1.5 2
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3.5 3.5
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3.5 3
3.5 2
2.5 2
2.5 1.5
1.5 I
0.5 I
0 I)
26 22.5
LSOI5 LS027
0 0
15
LSOI5 LS027
0 0
0 0
7 20 25
38 38
37 51
37 50
37 48
39 49
38 46
31 20
4 0
4 0
5 3
2 5
2 5
7 5
~ 3
6 0
17 8 . I 0
I0 ~ ' 7~+ 0 (I
301 345 43 21
* ( ' u m u l a t i v c total w i t h 13 o b s e r v a t i o n d a y s o v e r an 18 day period. P a r a m e t e r s were scored on a 0 5 basis w h e r e 0=paramelcrabsentand5 severe. **Total clinical scores lot all three cats per g r o u p per day. Oral ulcer sizes w e r e r e p r e s e n t e d by total d i a m e t e r o f all ulcers in ram. +See text. + + R e s o l v e d between days 24 a n d 28.
FELINECALICIVIRUSIN CHRONICSTOMATITISIN CATS
209
was characterised predominantly by oral ulceration which was generally most severe between days four and fourteen post-infection and persisted in three cats until days 24 to 28 (Table 1; Figs. la and lb - Experiment 1 ). Lingual ulceration was present in all six cats and one cat from each group also developed labial ulcers. Other relatively common, but generally milder signs which occurred in most cats were transient pyrexia (up to 41 ° C between days one and six ), lethargy, reduced appetite, nasal and ocular discharges and
20+ o u~
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p..__~c~-,--=~ t ,
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0
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I
10
20
Days Post-Infection
Fig. la. Mean ulcer score in cats infected with FCV LS015. Expt. l, three seronegative cats infected oronasally with 108-8 LS015 on day 0. Expt. 2, two seronegative cats infected oronasally with 104 LS015 on day 0 (see text). Expt. 3, three cats previously infected with LS027, and reinfected with 108.5 LS015 on day 0 (see text). *Ulcers present in two cats until days 24-28. + O r a l ulcer sizes were represented by total diameter of all ulcers in ram.
20-
(b)
+
-0- Expt.1 --e.- Expt.3
o o
,8 .=
0
,
10
•
i
20
Days Post Infection
Fig. lb. Mean ulcer score in cats infected with FCV LS027. Expt. 1, three seronegative cats infected oronasally with 108.8 LS027 on day 0. Expt. 3, three cats previously infected with LS015, and reinfected with 108.8 LS027 on day 0 (see text). *Ulcers present in one cat until days 2428. + O r a l ulcer sizes were represented by total diameter of all ulcers in ram.
.
ill
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conjunctivitis (Table I 1 days posl-infection ation ( LSOI 5, one cat (LS015. one cat) and
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I ). Less frequent clinical signs which devclopcd up lo included sneezing, b l e p h a r o s p a s m and ear pinna ulccr), sneezing, lameness and joint swelling and diarrhoea ulceration of the skin of lhe right tarsus ( 1.Si)27, one
,5
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20
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Fig. 2a. Mean titres F C V in o r o p h a r y n g c a l swabs f l o m cats infected wilh one o r lyre strains o f F('\,' (Expt. 1). [~ • kSO15, t h r c c c a t s i n f c c l c d oronasall.\, day 0 ~ i l h 10'~'~ LS()IS. 0 " LS027, three cats infected oronasally da~ 0 with 1()~ s 1_S027.
5 5 m o
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ta,3
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t
0
f
I
i
10
20
30
days post infection Fig. 2b. M e a n titres FCV in nasal swabs from cats infected with one o f two strains o f FCV (Expl. 1). [] : LS015, three cats infected oronasally day 0 with l0 ss LS015. 0 : 1,S027, three cats infected oronasally da} 0 with 10 a~ LS027.
FELINE CALICIVIRUS IN CHRONIC STOMATITIS IN CATS
21 [
cat). Overall there was no significant difference in the clinical scores between the two groups (total cumulative score, LS015 = 424.5; LS027 = 419 ). Beyond day 28 no clinical signs were seen except for mild transient inflammation of the gingival margins in association with eruption of some of the permanent teeth. This was also present in the control cats. No other clinical signs were seen at any time in the control cats.
Virus shedding For the two isolates, and for both oropharyngeal and nasal swabs, the level and duration of shedding was broadly similar (Figs. 2a and 2b and Table 2). Maximal shedding occurred between days one and nine with generally slightly higher levels being shed by cats infected with LS015. Detectable shedding in all six cats ceased between days 16 and 28 (Table 2). No virus was isolated from any of the cats on ten occasions between days 28 and 65. FCV was also isolated from the two ulcerated skin lesions which developed in two of the cats. No virus was isolated at any time from either of the control cats. TABLE 2
Virus shedding in cats infected oronasally with F C V L S 0 1 5 o r L S 0 2 7 Days postinfection
Virus group LS015
LS027
Cal. 1
Cat. 2
Cat. 3
Cat. 4
Cat. 5
Cat. 6
O* N
ON
ON
ON
ON
ON
O
--
--
1
+
+
2 3 4 6 7 8 9
11
+ + + + + + + +
NT + NT + NT + NT +
++ +NT ++ +NT ++ +NT ++ +NT ++
++ +NT ++ +NT ++ +NT ++ +NT ++
++ +NT ++ +NT ++ +NT ++ +NT ++
++ +NT ++ +NT ++ +NT ++ +NT ++
++ +NT ++ +NT ++ +NT ++ +NT ++
14 16
+ +
NT +
+ NT +-
+ NT --+
+ NT +--
+ NT +-
+ NT ++
18
+
NT
+NT
-NT
+NT
-NT
21 24 28-65
+ -
NT
+ . -NT
.
+ + _ _
-NT .
. . -NT
. --
* + / - F C V isolated/not-isolated from oropharyngeal (O) and nasal ( N ) swabs. N T = Not tested.
//#l/#ltlllo/oLTi('d[ l'd57]o/l.vCs Despite minor differences seen between cats in virus shedding patterns, there were no significant differences seen between their individual immunological responses which could be related to this. Mean data is therefore presented for each group. IW antibody re,v~onses. In both groups the serum VN antibody was first detected between days four and eight (Fig. 3a). Peak levels were attained in group LS027 by day eleven in contrast to group LS015 where there was a slower response and maximal levels were not reached until days 20-24.
108 -'=I-
4
--e- LS015 "~- LS027
2
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0
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8
I
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11
I
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14
I
•
18
I
'
21
I
'
24
I
'
28
Days Post-Infection
Fig. 3a. Homologous serum virus neutralising antibody response in cats infected with F ( V ( Expl. I ). LS015. seronegative cats int'ccted with LS015 on day 0. LS027, scronegative cats intO'trod with LS027 on day 0.
10
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8
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8
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11
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14
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18
!
21
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1
24
'
!
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28
Days Post Infection
Fig. 3b. Homologous serum virus neutralising response to reinfecting isolate in cats in Expt. 3. LS015, cats previously infected with LS027, and reinfected with LS015 on day 0 (see text }. LS027, cats previously infected with LS015, and reinfected with LS027 on day 0 (see text ).
FELINE CALICIVIRUSIN CHRONIC STOMATITIS IN CATS
2 13
Class specific anti-FCV immunoglobulin responses. In both groups the serum IgM response increased sharply by day eight, peaked at day twelve and returned to lower levels by day twenty-five (Figs. 4a and 4b). Salivary IgM showed similar response but was delayed by two or three days relative to serum (Figs. 4a and 4b). The IgG responses (Figs. 5a and 5b) were earlier in the LS027 group than in the LS015 group; serum IgG rose sharply by day 12 in the LS027 group compared to a slower rise in the LS015 group up to day 60 and beyond. The salivary IgG response to LS015 also took longer to develop fully, peaking around day 55. There is a good correlation in both groups (r> 0.75 ) between the IgG response and the VN response, the slower rise in LS015 VN antibody being mirrored by the slower rise in LS015 serum IgG. IgA responses to LS015 were very variable (Fig. 6a). In LS027 (Fig. 6b) serum and saliva responses were similar to each other, although maximal lev12
(a)
1.0 0
.,J
"o- Ser.um IgM
0.8-
ary IgM
0.6-
0.4IP
m
0.20.0
I
!
I
20
40
60
I
80
Days Post Infection
Fig. 4a. Mean salivary a n d serum IgM levels in LS015 infected cats (Expt. 1 ).
1.2
(b)
~
1.00
0.8" Serum IgM Salivary IgM
0.6IXl
0.4 0.2 0.0
I
0
I
0
20
i
I
30
40
I
•
50
i
60
•
|
70
•
i
80
Days Post Infection
Fig. 4b. M e a n salivary a n d serum IgM levels in LS027 infected cats (Expt. 1 ).
12 10
¸
08l/) .m
•-e- Serum IgG Salivary IgG
o6-
W
0.4O.20.0
I
i
I
I
20
40
60
80
day post infection
Fig. 5a. Mean salixary and serum lgG le',cls in LS015 infected cats (Expt. I ). 1.2 1.0
8
08 -~.-*-
0.6-
Serum IgG Salivary IgG
,-1
Lid
I1}
0.4 02
t.r 0002
i
~
10
20
I
I
30
T
i
40
•
i
50
[
I
60
70
•
i
80
Days Post Infection
Fig. 5b. Mean salix,ary and serum lgG le\els in LS027 infected cats (Expl. I ).
cls were reached more quickly in saliva (approx. 20 days ) compared to serum (approx.
4 0 d a y s ).
E.q~erimen! 2. Further attempts m induce FCl'carricrs Since no cat shed detectable virus beyond 28 days post int~ction, two further attempts to induce carriers were made ( Experiments 2 and 3 ). In Experiment 2, cats were infected by the same routes but with a significantly lower dose of LS015. The onset, severity, duration and range of clinical signs seen were generally similar to the previous experiment except that ulcers were less extensive and generally resolved earlier (Fig. la). In addition, no nasal discharge or any of the more infrequent clinical signs were seen. The pattern of oropharyngeal virus shedding was similar to that in cats infected with a high dose of virus with neither cat shedding virus beyond day 28, although one of
FELINE CALICIVIRUS IN CHRONIC STOMATITIS IN ('ATS
2 15 (Q)
1.2 1.0 O
u~
0.8
.J
0.6
14J
.~ '6
~-
Serum IgA
•- ~
Salivary IgA
0.4 0.2 0.0
1
i
i
i
20
40
60
80
Days Post-Infection.
Fig. 6a. Mean salivary and serum IgA levels in LS015 infected cats (Expt. 1 ). 1.2
==
(b)
1.0
u~
0,8
,,~
0.6
-.e.- Salivary IgA Serum IgA
0.4 0.2 0.0
i
0
10
•
i
i
I
20
30
40
•
I
i
50
60
•
i
70
•
i
80
Days Post Infection
Fig. 6b. Mean salivary and serum IgA levels in LS027 infected cats (Expt. 1 ).
the cats shed relatively higher levels of virus up until day 28. The serum V N response was also similar to that of cats in the previous experiment.
Experiment 3. Reinfection studies Both groups of cats reinfected with the heterologous isolate showed a more limited range of clinical signs than the n o n - i m m u n e cats, consisting of ocular discharge, conjunctivitis and oral ulceration. The severity and duration of ocular discharge and conjunctivitis was similar between Experiments 1 and 2 ( p > 0.05 for both groups, M a n n - W h i t n e y test), but mean ulcer size was significantly less ( p < 0 . 0 1 for both groups) and the duration slightly reduced (Figs. 1a and 1b). For LS015, both the overall level and duration oforopharyngeal virus shedding was similar to the non-immune cats and no cat shed virus beyond day
-~](}
it~
b. N I t \ \ I I ~ , l
I \1
28. For LS027, initial shedding patterns were similar, although shedding ceased earlier, by day 16. The serum neutralising response to the reinfection isolate was similar to the response in non-immune cats (Figs. 3a and 3b), with no evidence of prior cross-reactivity and no anamnestic response. There was a slight increase in neutralisation titres to the original infecting isolate (approximately 2 ( - l o g 2 ) ) in at least two of the three cats in each group (data not shown). No signs of chronic stomatitis were seen in any of the cats throughout the 10 months of the study. DISCUSSION
Neither of the two isolates from clinical cases of chronic stomatitis induced chronic stomatitis in experimental cats observed for the 10 months of the study. Both isolates produced clinical signs characteristic of acute FCV infection following experimental oronasal inoculation, namely mild malaise, nasal and ocular discharge and conjunctivitis, and severe oral ulceration, similar to those seen in previous studies (Povey and Hale, 1974; Ormerod et al., 1979; Gaskell et al., 1982). It is possible that the syndrome may have developed if the cats had been monitored for longer. Alternatively, the isolates chosen may have been unrepresentative of chronic stomatitis isolates, or, more likely, other cofactors may be involved, such as FIV (Knowles et al., 1989), or bacterial flora possibly not present in SPF cats (Mallonee et al., 1988). Other host factors may also have been involved. Another consideration is that virus shedding ceased by day 28 and no long term carriers were produced. As approximately 85% of cats with chronic stomatitis appear to be long term carriers, this state may be a prerequisite for the induction of chronic stomatitis. The reason why no carriers were produced in this study is not clear. In other work with experimental FCV infections, a significant proportion of cats excreted virus beyond 30 days (Wardley and Povey, 1977; Orr et al., 1980; Gaskell et al., 1982), although in an earlier study Povey and Hale (1974) found virus shedding normally ceased by three weeks post-infection. It is possible that some cats in the present study may have been very low level shedders only detectable by continuous swabbing (Wardley, 1976 ). However the sampling regime used would normally be sufficiently sensitive to detect carriers; although there might be some variation in sensitivity of different cell cultures. It is also possible that different strains have a different propensity to induce a carrier state. However, the two isolates used in the present study were from persistently infected cats, and in subsequent studies using one of these isolates and cats of the same age and source with a similar infection protocol, carriers have been produced (Dawson et al., 1991, unpublished data). In order to determine whether a lower infecting dose might influence the
FELINE CALICIV1RUS IN CHRONIC STOMATITIS IN CATS
217
outcome of infection with respect to chronic stomatitis or the development of carriers, the two control cats were inoculated with a significantly lower dose (4.8 ( - log~o) ) of LS015. However the level and duration of virus shedding was similar, and virus shedding again ceased by day 28. Despite similar amounts of virus being shed, clinical signs were milder in this group and the range of signs seen was reduced. In a previous study Povey and Hale (1974) reported reduced severity of clinical signs, but shorter duration of virus shedding with a lower infecting dose. In addition, heterologous reinfection of cats produced neither carriers nor chronic stomatitis, but resulted in signs typical of acute FCV infection. A more limited spectrum of signs was seen than in the non-immune cats, and signs were generally less severe. In some previous cross-infection studies with FCV, greater clinical cross-protection has been seen (Kahn et al., 1975; Povey and Ingersoll, 1975). However in the present study isolates were specifically selected for their divergent in vitro characteristics, and this may be why crossprotection was less evident. More recently, other workers have also noted an apparent lack of cross-protection with some isolates of FCV (Pedersen et al., 1983 ) and this may be of importance in terms of vaccination. In the heterologous reinfection study, virus shedding patterns were similar to those seen in non-immune cats, except for a slight reduction in duration of shedding in LS027. This is broadly in agreement with previous challenge studies, where some workers have found the duration of shedding remains unchanged (Gaskell et al., 1982), and some have found the duration to be reduced (Povey and Ingersoll, 1975 ). As no long term virus carriers were produced it was not possible to relate the cessation of the carrier state in an individual cat to immunological events. Even though minor differences in virus shedding patterns were seen up to 28 days post-infection no differences were seen in the immunological responses of individual animals to account for this. Overall, virus neutralising antibodies rose more rapidly in the LS027 group compared to the LS015. Previous investigators have also found that the VN response can vary considerably between different isolates (Wardley, 1974) and in some instances between cats infected with the same isolate (Kahn and Gillespie, 1971; Povey and Ingersoll, 1975 ). Class specific anti-FCV i m m u n e responses in both virus groups demonstrated a characteristic response to antigenic stimulation, with IgM peaking just after virus shedding and clinical signs, closely followed by rising levels of IgG. However both serum IgG and VN levels in group LS015 rose more slowly than in LS027 which to some extent may account for the increased virus shedding in the LS015 group. The good correlation in both groups between the IgG response and the VN response suggests that IgG probably constitutes the bulk of neutralising antibodies, although IgM probably does contribute to the early neutralising response.
For LS027 both serum and salivary lgA responses were similar rising slowl\ d u r i n g the course of infection: although serum lgA is probably of no protective significance, it m a y be useful as a m a r k e r for mucosal IgA responses.
Although the pattern o f l g G and lgM responses for LS015 were generall~ similar to LS027, serum and salivary lgA for LS015 were much more ~ariablc. The reasons for this are unclear, although for salivary lgA, sampling difficulties may have contributed to the variable nature of the response. It has been suggested that most of the neutralising activity in the respiratory secretions of FCV infected cats is due to IgG (Johnson, 1981 ). ('ON('LUSION
Chronic stomatitis was not induced in these studies with FCV alone. Future work will investigate the role of other possible co-factors in the induction of chronic stomatitis in cats, for example FIV ( Knowles et al., 1989 ). ~('KNOWLEDGEMENTS
We thank Dr. Chris Baldwin from the London School of Tropical Hygiene and Medicine for the mouse monoclonal antibodies to cat IgG and lgM. J.K. was supported by a Research Training Scholarship from the Wellcome Trust and R.M.G. was supported by the Robert Daubney Fellowship (RCVS) and the Whitley Animal Protection Trust. We are also grateful to lntervet Laboratories Ltd. for additional support.
REFERENCES Cocker, F.M., Gaskell, C.,I., Gaskell, R.M., Newby, T.,I. and Stokes, C.R., 1984. Efficac} of carl} (48 and 96 hours) protection againsl feline viral rhinotracheitis following inlranasal vaccination with live temperature sensitive mutant. Vet. Rec., 114: 353-354. (}askell, R.M., 1985. Viral-induced respiratory disease. In: E.A. Chandler, ('..I. Gaskell and A.I).R. Hilbery ( Editors }, Feline Medicine and Therapeutics. Blackwcll Scientific'. pp. 257270. Gaskell. C.J., Gaskell, R.M., Dennis, P.E. and Wooldridge, M.J.A., 1982. Efl]cacy of an inactivated feline calicivirus ( FCV ) vaccine against challenge with Uniled Kingdom field strains and its interaction with the F(TV carrier state. Res. Vet. Sci., 32: 23-26. Hosic, M.]., Roberison, (7. and ,larrett, O., 1989. Prevalence of feline leukaemia virus and antibodies 1o feline immunodeficiency virus in cats in the United Kingdom. Vet. Rec., 128: 293-297. ,larreU, O., Laird, H.M. and Hay, D., 1973. Determinants of the host range of lL-linc leukacmia viruses. J. Gcn. Virol., 20:169-175. ,lohnson, R.P,, 1981. Immunity to feline calicivirus in kittens. Diss. Abslr. Int., 41B: 2c)22. Kahn, I).E. and Gillespie, J.H., 1971. Feline viruses: Pathogenesis ofpicornavirus infection in the cat. Am. J. Vet. Res.. 32: 521-531.
FELINE CALICIVIRUS IN CHRONIC STOMATITIS IN CATS
2 19
Kahn, D.E. and Hoover, E.A., 1976. Feline caliciviral disease: experimental immunoprophylaxis. Am. J. Vet. Res., 37: 279-283. Kahn, D.E., Hoover, E.A. and Bittle, J.L., 1975. Induction of immunity to feline caliciviral disease. Infect. Immun., 11 : 1003-1009. Kalunda, M., Lee, K.M., Holmes, D.F. and Gillespie, J.H., 1975. Serologic classification of feline caliciviruses by plaque-reduction neutralisation and immunodiffusion. Am. J. Vet. Res., 36: 353-356. Knowles, J.O., 1988. Studies on feline calicivirus with particular reference to chronic stomatitis in the cat. PhD Thesis, University of Liverpool. Knowles, J.O., Gaskell, R.M., Gaskell, C.J,, Harvey, C.E. and Lutz, H., 1989. Prevalence of feline calicivirus, feline leukaemia virus and antibodies to FIV in cats with chronic stomatitis. Vet. Rec., 124: 336-338. Mallonee, D.H., Harvey, C.E., Venner, M. and Hammond, B.F., 1988. Bacteriology of periodontal disease in the cat. Arch. Biol., 33: 677-683. Ormerod, E. and Jarrett, O., 1978. A classification of feline calicivirus isolates based on plaque morphology. J. Gen. Virol., 39: 537-540. Ormerod, E., McCandlish, I.A.P. and Jarrett, O., 1979. Diseases produced by feline caliciviruses when administered to cats by aerosol or intranasal instillation. Vet. Rec., 104: 65-69. Orr, C.M., Gaskell, C.J. and Gaskell, R.M., 1980. Interaction of an intra-nasal combined feline viral rhinotracheitis, feline calicivirus vaccine and the FVR carrier state. Vet. Rec., 106: 164. Pederscn, N.C,, Laliberte, L. and Ekman, S., 1983. A transient 'limping syndrome' of kittens caused by two different strains of feline calicivirus. Feline pract., 13: 26-35. Povcy, R.C. and Hale, C.J., 1974. Experimental infections with feline caliciviruses (picornaviruses) in specific-pathogen-free kittens. J. Comp. Path., 84: 245-256. Povey, R.C. and Ingersoll, J., 1975. Cross-protection among feline caliciviruses. Infect. Immun., 11: 877-885. Thompson, R.R., Wilcox, G.E., Clark, W.T. and Jansen, K.L., 1984. Association of calicivirus infection with chronic gingivitis and pharyngitis in cats. J. Small Anita. Pract., 25:207-210. Wardlcy, R.C., 1974. Studies on feline caliciviruses with particular reference to persistent infections. PhD Thesis, University of Bristol. Wardley, R.C., 1976. Feline calicivirus carrier state. A study of the host/virus relationship. Arch. Virol., 52: 243-249. Wardley, R.C. and Povey, R.C., 1977. The clinical disease and patterns of excretion associated with three different strains of feline caliciviruses. Res. Vet. Sci., 23: 7-14. Wardley, R.C., Gaskell, R.M. and Povey, R.C., 1974. Feline respiratory viruses - their prevalence in clinically healthy cats. J. Small Anita. Pract., 15: 579-586. Yamamoto, J.K., Hansen, H., Ho, E.W., Morishita, T.Y., Okuda, T., Sawa, T.R.,Nakamura, R.M. and Pedersen, N.C., 1989. Epidemiologic and clinical aspects of feline immunodeficiency virus infection in cats from the continental United States and Canada and possible mode of transmission. J. Am. Vet. Med. Assoc., 194: 213-220.
205
Studies on the role of feline calicivirus in chronic stomatitis in cats J.O. Knowles a'b, F. McArdle b, S. Dawson a'b, S.D. Carter% C.J. Gaskell a and R.M. G a s k e l l b Departments of a Veterinary Clinical Science and b Veterinary Pathology, University ~/Livet7)ool, Liverpool, UK (Accepted 10 January 1991)
,ABSTRACT Knowles, J.O., McArdle, F., Dawson, S., Carter, S.D., Gaskell, C.J. and Gaskell, R.M., 1991. Studies on the role of feline calicivirus in chronic stomatitis in cats. Vet. Microbiol., 27:205-219. Two groups of cats were inoculated oro-nasally with one of two isolates of feline calicivirus ( FCV ) from clinical cases of chronic stomatitis. All cats developed signs typical of acute FCV infection: namely, ocular and nasal discharge, conjunctivitis, and marked oral ulceration. None of the cats shed virus beyond 28 days. Seronegative control cats were then infected with a lower dose of one isolate, but again only acute signs were seen and no carriers produced. The original cats were then re-infected with lhe heterologous isolate. As before, only signs of acute disease were seen, but the range of clinical signs and severity was reduced. Virus shedding patterns in one group were similar to those seen originally, but in the other the duration was reduced. No chronic stomatitis developed over the 10 months of the study. Serum virus neutralising and serum and salivary class specific immunoglobulin responses were investigated. Although long-term carriers were not induced, no relationship between cessation of virus shedding in an individual animal and systemic and local antibody responses was seen.
INTRODUCTION
Feline calicivirus is a common cause of upper respiratory disease and acute oral ulceration in cats (Gaskell, 1985 ). It has also been implicated in chronic stomatitis. Recent studies have shown that approximately 85% of cats with chronic stomatitis in the U K are carriers of feline calicivirus, compared to 20% controls (Knowles et al., 1989 ), confirming an earlier smaller Australian survey by Thompson et al. (1984). An association of feline immunodeficiency virus (FIV) infection with chronic stomatitis in cats has also been found (Yamamoto et al., 1989; Hosie et al., 1989; Knowles et al., 1989). However, the role of these agents either individually or in combination in inducing the disease has not been established. In both field and experimental infections, the carrier state appears to be a
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© 1991 - - Elsevier Science Publishers B.V.
c o m m o n sequel to feline calicivirus ( F C V ) infection (Wardley cl al., 1974: Wardley and Povey, 1977: Orr el al., 1980: Gaskell ctal., 1982 ). Wardlcy and Povey (1977) have shown that although the duration of excretion is highl~ variable between individuals, in a group of infected cats the infective-animal half-life appeared to be approximately 75 days. Thus at 30 days after infection, the majority of cats were still shedding virus whereas at 75 days, 51)% had ceased. The possible immunological events involved in this apparenll) sudden cessation of virus shedding tbr an individual cat have not been examined. The purpose of this study was, firstly, to examine the role of feline calicivirus in chronic stomatitis in cats. There are a number of feline calicivirus isolates which differ slightly in their antigenicity and pathogenicity ( Kalunda et al., 1975; Povey and Hale, 1974 ). However, pathogenesis studies have been done with isolates of feline calicivirus from a variety of clinical syndromes, rather than isolates originating from cases of chronic stomatitis. The second aim of the study was to relate local and systemic humoral immune responses to the course of infection and to the cessation of virus shedding in carriers. MATERIALS AND METHODS
('ell culture A feline embryo-derived cell line (FEA; Jarrett et al., 1973 ) grown in B H K media (Wellcome) with antibiotics, sodium bicarbonate and Hepes buffers, and 4% (growth) or 1% (maintenance) foetal calf serum was used. [ ~II'IISCS Two isolates, from cats LS015 and LS027 with chronic stomatitis, and persistently infected with FCV, were used. Isolates were selected on the basis of their differing in vitro characteristics. Isolate LS027 was significantly neutralised by antiserum to FCV F9, and LS015 was not: F9 is a widely used FCV vaccine strain (Kahn and Hoover, 1976). LS015 formed large plaques (4.8 ram. diameter) under agar overlay, whereas LS027 produced small plaques (0.8 mm. ) (Ormerod and Jarrett, 1978; Knowles, 1988 ). Viral stocks were prepared and titrated in FEA cells as described previously (Wardley, 1976).
Virus neutralisation (VN) tests VN tests were performed using 100 TCID~o of homologous virus and serial two-fold dilutions of serum each contained in 50 microlitres in 96 well plates. Virus-serum mixtures were incubated at 37°C for one hour prior to the addition of FEA cells. The results were recorded after 72 hours.
FELINE C~L1CIVIRUS IN CHRONIC STOMATITIS IN CATS
207
Class-specific enzyme linked immunosorbent assay (ELISA) A single dilution ELISA assay was used with homologous virus precipitated with 7% polyethylene glycol (PEG) 6000 and then spun for two hours at 10 000 g, and resuspended in 1/ 10th of the original tissue culture volume in PBSA. ELISA plates (Flow) were coated at 10 #g/ml at 37°C for two hours. After washing the plates, the test cat sera ( 1 : 200 in PBSA+ 0.05% Tween 20) or test saliva ( 1:20 in PBSA+0.05% Tween 20) were incubated in duplicate at 37°C for one hour. Bound antibodies were detected with mouse monoclonal antibodies to IgM and IgG cat immunoglobulins and a monoclonal to human IgA which cross-reacts with cat IgA (Oxoid). The products were then developed with goat anti-mouse IgG (alkaline phosphatase-conjugated) and the substrate, p-nitrophenyl phosphate (Sigma). The colour conversion was read at 405 nm. Sera and saliva were also tested against ELISA plates previously coated with the PEG precipitate from a non-infected culture and any resulting Optical density (OD) subtracted from the antigen specific data. The data was expressed as relative ODs (i.e. values were expressed relative to the highest result within an individual immunoglobulin class for each virus group). CaIs Eight 16 week old SPF cats of mixed sexes were used. They were housed in three groups of two or three in barrier maintained rooms with positive pressure ventilation.
Experimental design Experiment 1. Two groups of three cats were inoculated with 10ss TCIDs0 in 2 ml of either LS015 or LS027, half given intranasally and half orally. Two control cats were similarly inoculated with 2 ml of uninfected cell culture fluid. The cats were monitored daily for the first nine days and thereafter at least twice weekly until 63 days after inoculation. Clinical signs were scored on a 0-5 scale, where 0 indicated a clinical sign was absent and 5 indicated the cat was severely affected. However oral ulcers were scored by their diameter in mm. Nasal and oropharyngeal swabs were placed in viral transport medium and stored at - 7 0 ° C prior to attempted virus isolation (Wardley et al., 1974; Cocker et al., 1984). Oropharyngeal swabs for class specific anti-FCV immunoglobulin estimation were taken into 1 ml PBSA containing 0.05% Tween 20 and stored at - 20 ° C. Serum samples were also obtained for VN tests and for class specific antibody estimations.
Experiment 2. Sixty-five days after the start of Experiment l, the two control cats were inoculated as above with a lower dose of LS015 (104 TCIDs0 in
"~(}N
ii)
b, N I ~ V , [ I ' - , [
I \i
2 ml). These cats were monitored for clinical signs, virus shedding, and serology as in experiment 1, although sampling was slightly less frequent.
k\vperimenl 3. At the same time as Experiment 2, each of the two FCV-infected groups were then re-inoculated with the heterologous isolate, using an identical dose and route of infection as in Experiment 1. These cats were monitored for clinical signs, virus shedding, and serology as in Experiment 1. Clinical observation was carried out on all cats in these studies for a total of 10 months after the start of Experiment 1. RESULTS
E:vperirnenl 1 Clinical signs After an incubation period of one to four days, both isolates produced an acute disease of approximately two to three weeks duration (Table 1 ). This I.\BLtl I Q u a n t i t a l i x c scores o f c o m m o n clinical signs in cats i n f e c t e d oronasally with either F C V l.S015 or t_S027 ( Expert mcnl I ) ( l i n i c a l signs
Days post infection 0
I
l_S015 l_S027
0"* 0
1 0
Rcduccdappetite
LS015 l_S027
0 0
(l (1
Nasal d i s c h a r g e
LS015 LS027
0 0
()cular d i s c h a r g e
LS015 LS027
('onjunctivitis
()ral ulcers
Letharg 3
t ) t h e r signs +
2
3
Fotal* 4
6
7
£
9
l]
14
I¢,
IN 63
14
(1 0
1
(~
3
2
[
(t
(I
II
0
I)
2
5
(l
(t
(~
0
(I
(J
!)
~)
0 0
2 3
6 4
5 2
3 ()
(I ()
(i 0
0 0
t~ 0
0 o
IJ ()
16 9
t) I
1 1
2 1
3 1
1.5 1
1.5 1
0.5 1
0.5 0
0 ()
0 0
t) II
I0 7
0 0
2.5 1
I
2
1.5 1
1
I 0.5
I 0
0
1
3.5 0.5
I
1.5
0
I) ()
(I O
14.5 7.5
kS015 LS027
0 0
1.5 2
2.5 1.5
3.5 3.5
3.5 3
3.5 3
3.5 2
2.5 2
2.5 1.5
1.5 I
0.5 I
0 I)
26 22.5
LSOI5 LS027
0 0
15
LSOI5 LS027
0 0
0 0
7 20 25
38 38
37 51
37 50
37 48
39 49
38 46
31 20
4 0
4 0
5 3
2 5
2 5
7 5
~ 3
6 0
17 8 . I 0
I0 ~ ' 7~+ 0 (I
301 345 43 21
* ( ' u m u l a t i v c total w i t h 13 o b s e r v a t i o n d a y s o v e r an 18 day period. P a r a m e t e r s were scored on a 0 5 basis w h e r e 0=paramelcrabsentand5 severe. **Total clinical scores lot all three cats per g r o u p per day. Oral ulcer sizes w e r e r e p r e s e n t e d by total d i a m e t e r o f all ulcers in ram. +See text. + + R e s o l v e d between days 24 a n d 28.
FELINECALICIVIRUSIN CHRONICSTOMATITISIN CATS
209
was characterised predominantly by oral ulceration which was generally most severe between days four and fourteen post-infection and persisted in three cats until days 24 to 28 (Table 1; Figs. la and lb - Experiment 1 ). Lingual ulceration was present in all six cats and one cat from each group also developed labial ulcers. Other relatively common, but generally milder signs which occurred in most cats were transient pyrexia (up to 41 ° C between days one and six ), lethargy, reduced appetite, nasal and ocular discharges and
20+ o u~
(el
~
-a- Expt.1 -4- Expt.2 Expt,3
p..__~c~-,--=~ t ,
10, I-
=
=E
0
i
I
10
20
Days Post-Infection
Fig. la. Mean ulcer score in cats infected with FCV LS015. Expt. l, three seronegative cats infected oronasally with 108-8 LS015 on day 0. Expt. 2, two seronegative cats infected oronasally with 104 LS015 on day 0 (see text). Expt. 3, three cats previously infected with LS027, and reinfected with 108.5 LS015 on day 0 (see text). *Ulcers present in two cats until days 24-28. + O r a l ulcer sizes were represented by total diameter of all ulcers in ram.
20-
(b)
+
-0- Expt.1 --e.- Expt.3
o o
,8 .=
0
,
10
•
i
20
Days Post Infection
Fig. lb. Mean ulcer score in cats infected with FCV LS027. Expt. 1, three seronegative cats infected oronasally with 108.8 LS027 on day 0. Expt. 3, three cats previously infected with LS015, and reinfected with 108.8 LS027 on day 0 (see text). *Ulcers present in one cat until days 2428. + O r a l ulcer sizes were represented by total diameter of all ulcers in ram.
.
ill
K%'~','
conjunctivitis (Table I 1 days posl-infection ation ( LSOI 5, one cat (LS015. one cat) and
",i
I
~,
I ). Less frequent clinical signs which devclopcd up lo included sneezing, b l e p h a r o s p a s m and ear pinna ulccr), sneezing, lameness and joint swelling and diarrhoea ulceration of the skin of lhe right tarsus ( 1.Si)27, one
,5
', c, i
~
4
o
C=
0
!
!
!
10
3O
20
days post-infection
Fig. 2a. Mean titres F C V in o r o p h a r y n g c a l swabs f l o m cats infected wilh one o r lyre strains o f F('\,' (Expt. 1). [~ • kSO15, t h r c c c a t s i n f c c l c d oronasall.\, day 0 ~ i l h 10'~'~ LS()IS. 0 " LS027, three cats infected oronasally da~ 0 with 1()~ s 1_S027.
5 5 m o
b
4
ta,3
o
3
k-
t
0
f
I
i
10
20
30
days post infection Fig. 2b. M e a n titres FCV in nasal swabs from cats infected with one o f two strains o f FCV (Expl. 1). [] : LS015, three cats infected oronasally day 0 with l0 ss LS015. 0 : 1,S027, three cats infected oronasally da} 0 with 10 a~ LS027.
FELINE CALICIVIRUS IN CHRONIC STOMATITIS IN CATS
21 [
cat). Overall there was no significant difference in the clinical scores between the two groups (total cumulative score, LS015 = 424.5; LS027 = 419 ). Beyond day 28 no clinical signs were seen except for mild transient inflammation of the gingival margins in association with eruption of some of the permanent teeth. This was also present in the control cats. No other clinical signs were seen at any time in the control cats.
Virus shedding For the two isolates, and for both oropharyngeal and nasal swabs, the level and duration of shedding was broadly similar (Figs. 2a and 2b and Table 2). Maximal shedding occurred between days one and nine with generally slightly higher levels being shed by cats infected with LS015. Detectable shedding in all six cats ceased between days 16 and 28 (Table 2). No virus was isolated from any of the cats on ten occasions between days 28 and 65. FCV was also isolated from the two ulcerated skin lesions which developed in two of the cats. No virus was isolated at any time from either of the control cats. TABLE 2
Virus shedding in cats infected oronasally with F C V L S 0 1 5 o r L S 0 2 7 Days postinfection
Virus group LS015
LS027
Cal. 1
Cat. 2
Cat. 3
Cat. 4
Cat. 5
Cat. 6
O* N
ON
ON
ON
ON
ON
O
--
--
1
+
+
2 3 4 6 7 8 9
11
+ + + + + + + +
NT + NT + NT + NT +
++ +NT ++ +NT ++ +NT ++ +NT ++
++ +NT ++ +NT ++ +NT ++ +NT ++
++ +NT ++ +NT ++ +NT ++ +NT ++
++ +NT ++ +NT ++ +NT ++ +NT ++
++ +NT ++ +NT ++ +NT ++ +NT ++
14 16
+ +
NT +
+ NT +-
+ NT --+
+ NT +--
+ NT +-
+ NT ++
18
+
NT
+NT
-NT
+NT
-NT
21 24 28-65
+ -
NT
+ . -NT
.
+ + _ _
-NT .
. . -NT
. --
* + / - F C V isolated/not-isolated from oropharyngeal (O) and nasal ( N ) swabs. N T = Not tested.
//#l/#ltlllo/oLTi('d[ l'd57]o/l.vCs Despite minor differences seen between cats in virus shedding patterns, there were no significant differences seen between their individual immunological responses which could be related to this. Mean data is therefore presented for each group. IW antibody re,v~onses. In both groups the serum VN antibody was first detected between days four and eight (Fig. 3a). Peak levels were attained in group LS027 by day eleven in contrast to group LS015 where there was a slower response and maximal levels were not reached until days 20-24.
108 -'=I-
4
--e- LS015 "~- LS027
2
O
'
0
I
•
2
1
'
I
4
'
8
I
'
11
I
'
14
I
•
18
I
'
21
I
'
24
I
'
28
Days Post-Infection
Fig. 3a. Homologous serum virus neutralising antibody response in cats infected with F ( V ( Expl. I ). LS015. seronegative cats int'ccted with LS015 on day 0. LS027, scronegative cats intO'trod with LS027 on day 0.
10
b)
8
= I-
4 /
._.J/
2
O
Rei f. -.e,- LSO27Reinf
/
I
0
'
I
2
•
I
4
'
I
8
'
I
11
'
I
14
'
I
I
18
!
21
'
1
24
'
!
'
28
Days Post Infection
Fig. 3b. Homologous serum virus neutralising response to reinfecting isolate in cats in Expt. 3. LS015, cats previously infected with LS027, and reinfected with LS015 on day 0 (see text }. LS027, cats previously infected with LS015, and reinfected with LS027 on day 0 (see text ).
FELINE CALICIVIRUSIN CHRONIC STOMATITIS IN CATS
2 13
Class specific anti-FCV immunoglobulin responses. In both groups the serum IgM response increased sharply by day eight, peaked at day twelve and returned to lower levels by day twenty-five (Figs. 4a and 4b). Salivary IgM showed similar response but was delayed by two or three days relative to serum (Figs. 4a and 4b). The IgG responses (Figs. 5a and 5b) were earlier in the LS027 group than in the LS015 group; serum IgG rose sharply by day 12 in the LS027 group compared to a slower rise in the LS015 group up to day 60 and beyond. The salivary IgG response to LS015 also took longer to develop fully, peaking around day 55. There is a good correlation in both groups (r> 0.75 ) between the IgG response and the VN response, the slower rise in LS015 VN antibody being mirrored by the slower rise in LS015 serum IgG. IgA responses to LS015 were very variable (Fig. 6a). In LS027 (Fig. 6b) serum and saliva responses were similar to each other, although maximal lev12
(a)
1.0 0
.,J
"o- Ser.um IgM
0.8-
ary IgM
0.6-
0.4IP
m
0.20.0
I
!
I
20
40
60
I
80
Days Post Infection
Fig. 4a. Mean salivary a n d serum IgM levels in LS015 infected cats (Expt. 1 ).
1.2
(b)
~
1.00
0.8" Serum IgM Salivary IgM
0.6IXl
0.4 0.2 0.0
I
0
I
0
20
i
I
30
40
I
•
50
i
60
•
|
70
•
i
80
Days Post Infection
Fig. 4b. M e a n salivary a n d serum IgM levels in LS027 infected cats (Expt. 1 ).
12 10
¸
08l/) .m
•-e- Serum IgG Salivary IgG
o6-
W
0.4O.20.0
I
i
I
I
20
40
60
80
day post infection
Fig. 5a. Mean salixary and serum lgG le',cls in LS015 infected cats (Expt. I ). 1.2 1.0
8
08 -~.-*-
0.6-
Serum IgG Salivary IgG
,-1
Lid
I1}
0.4 02
t.r 0002
i
~
10
20
I
I
30
T
i
40
•
i
50
[
I
60
70
•
i
80
Days Post Infection
Fig. 5b. Mean salix,ary and serum lgG le\els in LS027 infected cats (Expl. I ).
cls were reached more quickly in saliva (approx. 20 days ) compared to serum (approx.
4 0 d a y s ).
E.q~erimen! 2. Further attempts m induce FCl'carricrs Since no cat shed detectable virus beyond 28 days post int~ction, two further attempts to induce carriers were made ( Experiments 2 and 3 ). In Experiment 2, cats were infected by the same routes but with a significantly lower dose of LS015. The onset, severity, duration and range of clinical signs seen were generally similar to the previous experiment except that ulcers were less extensive and generally resolved earlier (Fig. la). In addition, no nasal discharge or any of the more infrequent clinical signs were seen. The pattern of oropharyngeal virus shedding was similar to that in cats infected with a high dose of virus with neither cat shedding virus beyond day 28, although one of
FELINE CALICIVIRUS IN CHRONIC STOMATITIS IN ('ATS
2 15 (Q)
1.2 1.0 O
u~
0.8
.J
0.6
14J
.~ '6
~-
Serum IgA
•- ~
Salivary IgA
0.4 0.2 0.0
1
i
i
i
20
40
60
80
Days Post-Infection.
Fig. 6a. Mean salivary and serum IgA levels in LS015 infected cats (Expt. 1 ). 1.2
==
(b)
1.0
u~
0,8
,,~
0.6
-.e.- Salivary IgA Serum IgA
0.4 0.2 0.0
i
0
10
•
i
i
I
20
30
40
•
I
i
50
60
•
i
70
•
i
80
Days Post Infection
Fig. 6b. Mean salivary and serum IgA levels in LS027 infected cats (Expt. 1 ).
the cats shed relatively higher levels of virus up until day 28. The serum V N response was also similar to that of cats in the previous experiment.
Experiment 3. Reinfection studies Both groups of cats reinfected with the heterologous isolate showed a more limited range of clinical signs than the n o n - i m m u n e cats, consisting of ocular discharge, conjunctivitis and oral ulceration. The severity and duration of ocular discharge and conjunctivitis was similar between Experiments 1 and 2 ( p > 0.05 for both groups, M a n n - W h i t n e y test), but mean ulcer size was significantly less ( p < 0 . 0 1 for both groups) and the duration slightly reduced (Figs. 1a and 1b). For LS015, both the overall level and duration oforopharyngeal virus shedding was similar to the non-immune cats and no cat shed virus beyond day
-~](}
it~
b. N I t \ \ I I ~ , l
I \1
28. For LS027, initial shedding patterns were similar, although shedding ceased earlier, by day 16. The serum neutralising response to the reinfection isolate was similar to the response in non-immune cats (Figs. 3a and 3b), with no evidence of prior cross-reactivity and no anamnestic response. There was a slight increase in neutralisation titres to the original infecting isolate (approximately 2 ( - l o g 2 ) ) in at least two of the three cats in each group (data not shown). No signs of chronic stomatitis were seen in any of the cats throughout the 10 months of the study. DISCUSSION
Neither of the two isolates from clinical cases of chronic stomatitis induced chronic stomatitis in experimental cats observed for the 10 months of the study. Both isolates produced clinical signs characteristic of acute FCV infection following experimental oronasal inoculation, namely mild malaise, nasal and ocular discharge and conjunctivitis, and severe oral ulceration, similar to those seen in previous studies (Povey and Hale, 1974; Ormerod et al., 1979; Gaskell et al., 1982). It is possible that the syndrome may have developed if the cats had been monitored for longer. Alternatively, the isolates chosen may have been unrepresentative of chronic stomatitis isolates, or, more likely, other cofactors may be involved, such as FIV (Knowles et al., 1989), or bacterial flora possibly not present in SPF cats (Mallonee et al., 1988). Other host factors may also have been involved. Another consideration is that virus shedding ceased by day 28 and no long term carriers were produced. As approximately 85% of cats with chronic stomatitis appear to be long term carriers, this state may be a prerequisite for the induction of chronic stomatitis. The reason why no carriers were produced in this study is not clear. In other work with experimental FCV infections, a significant proportion of cats excreted virus beyond 30 days (Wardley and Povey, 1977; Orr et al., 1980; Gaskell et al., 1982), although in an earlier study Povey and Hale (1974) found virus shedding normally ceased by three weeks post-infection. It is possible that some cats in the present study may have been very low level shedders only detectable by continuous swabbing (Wardley, 1976 ). However the sampling regime used would normally be sufficiently sensitive to detect carriers; although there might be some variation in sensitivity of different cell cultures. It is also possible that different strains have a different propensity to induce a carrier state. However, the two isolates used in the present study were from persistently infected cats, and in subsequent studies using one of these isolates and cats of the same age and source with a similar infection protocol, carriers have been produced (Dawson et al., 1991, unpublished data). In order to determine whether a lower infecting dose might influence the
FELINE CALICIV1RUS IN CHRONIC STOMATITIS IN CATS
217
outcome of infection with respect to chronic stomatitis or the development of carriers, the two control cats were inoculated with a significantly lower dose (4.8 ( - log~o) ) of LS015. However the level and duration of virus shedding was similar, and virus shedding again ceased by day 28. Despite similar amounts of virus being shed, clinical signs were milder in this group and the range of signs seen was reduced. In a previous study Povey and Hale (1974) reported reduced severity of clinical signs, but shorter duration of virus shedding with a lower infecting dose. In addition, heterologous reinfection of cats produced neither carriers nor chronic stomatitis, but resulted in signs typical of acute FCV infection. A more limited spectrum of signs was seen than in the non-immune cats, and signs were generally less severe. In some previous cross-infection studies with FCV, greater clinical cross-protection has been seen (Kahn et al., 1975; Povey and Ingersoll, 1975). However in the present study isolates were specifically selected for their divergent in vitro characteristics, and this may be why crossprotection was less evident. More recently, other workers have also noted an apparent lack of cross-protection with some isolates of FCV (Pedersen et al., 1983 ) and this may be of importance in terms of vaccination. In the heterologous reinfection study, virus shedding patterns were similar to those seen in non-immune cats, except for a slight reduction in duration of shedding in LS027. This is broadly in agreement with previous challenge studies, where some workers have found the duration of shedding remains unchanged (Gaskell et al., 1982), and some have found the duration to be reduced (Povey and Ingersoll, 1975 ). As no long term virus carriers were produced it was not possible to relate the cessation of the carrier state in an individual cat to immunological events. Even though minor differences in virus shedding patterns were seen up to 28 days post-infection no differences were seen in the immunological responses of individual animals to account for this. Overall, virus neutralising antibodies rose more rapidly in the LS027 group compared to the LS015. Previous investigators have also found that the VN response can vary considerably between different isolates (Wardley, 1974) and in some instances between cats infected with the same isolate (Kahn and Gillespie, 1971; Povey and Ingersoll, 1975 ). Class specific anti-FCV i m m u n e responses in both virus groups demonstrated a characteristic response to antigenic stimulation, with IgM peaking just after virus shedding and clinical signs, closely followed by rising levels of IgG. However both serum IgG and VN levels in group LS015 rose more slowly than in LS027 which to some extent may account for the increased virus shedding in the LS015 group. The good correlation in both groups between the IgG response and the VN response suggests that IgG probably constitutes the bulk of neutralising antibodies, although IgM probably does contribute to the early neutralising response.
For LS027 both serum and salivary lgA responses were similar rising slowl\ d u r i n g the course of infection: although serum lgA is probably of no protective significance, it m a y be useful as a m a r k e r for mucosal IgA responses.
Although the pattern o f l g G and lgM responses for LS015 were generall~ similar to LS027, serum and salivary lgA for LS015 were much more ~ariablc. The reasons for this are unclear, although for salivary lgA, sampling difficulties may have contributed to the variable nature of the response. It has been suggested that most of the neutralising activity in the respiratory secretions of FCV infected cats is due to IgG (Johnson, 1981 ). ('ON('LUSION
Chronic stomatitis was not induced in these studies with FCV alone. Future work will investigate the role of other possible co-factors in the induction of chronic stomatitis in cats, for example FIV ( Knowles et al., 1989 ). ~('KNOWLEDGEMENTS
We thank Dr. Chris Baldwin from the London School of Tropical Hygiene and Medicine for the mouse monoclonal antibodies to cat IgG and lgM. J.K. was supported by a Research Training Scholarship from the Wellcome Trust and R.M.G. was supported by the Robert Daubney Fellowship (RCVS) and the Whitley Animal Protection Trust. We are also grateful to lntervet Laboratories Ltd. for additional support.
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