Histopathological alterations of lymphatic tissues in cats without feline infectious peritonitis after long-term exposure to FIP virus

Introduction of feline infectious peritonitis virus (FIPV)-infected animals into a group of coronavirus (CoV)-free cats lead to the outbreak of FIP. Many cats survived the long-term FIPV exposure without even developing clinical symptoms. The purpose of this study was to examine healthy cats which survived long-term natural FIPV exposure in order to evaluate the effects of exposure and to gain possible explanations as to why these animals did not succumb to FIP.

The study was performed on four groups of cats, consisting of FIPV-exposed SPF cats without FIP (n  = 7; Group Ia) and with FIP (n  = 3; Group IIa), 2 cats from a breeding colony where FIP cases had recently occurred (Group Ib), 9 routinely necropsied cats with FIP (Group IIb), and 38- (n  = 9) and 73-week-old (n  = 5) SPF cats (Group III) as FIPV-negative controls. Eleven naturally FIPV-infected cats from various animal shelters served as source of infection (Group IV) for SPF cats (Table 1 ). These animals had been tested positive for circulating FCoV immune complexes (see below) and were housed with SPF cats (Groups Ia and IIa) for 30 weeks.

Table 1.

Animals

Group	n	Origin	Age	Comments
Ia	7	SPF cats	51 weeks	FIPV exposure since 21 weeks of age, housed together with Group IV cats
Ib	2	routinely necropsied	11 weeks	from breeding colony eith FIP, ELISA-pos.a
IIa	3	SPF cats	FIPV exposure since 21 week of age, housed together with Group IV cats
IIb	9	routinely necropsied	6 months
−7 years
IIIa	9	SPF cats	38 weeks	control cats
b	5	SPF cats	73 weeks	control cats
IV	11	animal shelters	6 months	ELISA-pos.
−4 years

^aELISA-pos., positive result for circulating FCoV immune complexes.

Sections from snap-frozen or formalin-fixed and paraffin-embedded samples from spleen, mesenteric lymph nodes, and thymus were either stained with hematoxylin-eosin or used for immunohistological examinations.

Immunohistology was used for the demonstration of cell populations (T-cells, B-cells, macrophages/neutrophils, follicular dendritic cells), CoV antigen as well as anti-CoV antibodies in situ (Kipar et al., 1998), and the cellular turnover (proliferating (PCNA-positive) and apoptotic (TUNEL method) cells) in situ (Table 2 ).

Table 2.

Immunohistology

Demonstration of T cells	Method, antibody
CD3	rabbit anti-human CD3 (Dako Diagnostika, GmbH, Hamburg, Germany)
CD4	vpg 33 (B. Willett)
CD8	vpg 9 (B. Willett)
B-cells (CD45R)	Ly5, B220 (Cedarlane Lab. Ltd., Hornby, Canada)
Macrophages/neutrophils (myeloid/histiocyte AGa)	MAC 387 (Dako)
FDCb	RFB-6 (K. Ivory)
FCoV AG	FCV3-70 (Custom Monoclonals Int. W. Sacramento, USA)
Anti-FCov ABc in situ	(Kipar et al., 1998)
PCNAd	PC10 (Dako)
Apoptotic cells in situ	TUNEL method (ApopTag®, Oncor, Heidelberg, Germany)

^aAG: antigen.

^bFDC: follicular dentridic cell.

^cAB: antibodies.

^dPCNA: proliferating cell nuclear antigen.

In SPF cats, CoV antibody titres (Osterhaus et al., 1977) and circulating FCoV immune complexes were demonstrated at time points (TP) 0 (age: 9 weeks), 1 (age: 21 weeks), 2 (10 weeks of FIPV exposure), 3 (20 weeks of FIPV exposure), and 4 (30 weeks of FIPV exposure). For demonstration of circulating FCoV immune complexes, a competitive ELISA was performed (Pfeiffer, 1991). Test serum was treated with polyethylenglycole for precipitation of immune complexes. The resolubilized precipitate was applied to a microtitre plate coated with FIPV (FIPV-DF2). FCoV-containing immune complexes inhibit binding of a mouse monoclonal antibody against FCoV to the FIPV-coated microtiter plate. Thereby, binding of peroxidase-labelled goat anti-mouse IgG is reduced, resulting in lower extinction levels. The threshold value for a positive result had been statistically evaluated (Schroo, 1994).

Virus was isolated from buffy coat monocytes of SPF cats at TP 3 and 4, and cultivated on whole feline embryo (WFE) cells (Gunn-Moore et al., 1998).

At necropsy, ingesta was taken for ultrastructural demonstration of CoV particles by negative staining technique (Vieler and Herbst, 1995), and faeces were investigated for CoV genome by nested RT-PCR (Herrewegh et al., 1995).

All FIPV-exposed SPF cats (Group Ia) developed positive CoV antibody titres, demonstrable at TP 2 (n  = 5) and 3 (n  = 7). Circulating FCoV immune complexes were seen at TP 2 (n  = 3) and 3 (n  = 6). At the end of the experiment (TP 4), all FIPV-exposed SPF cats were negative for FCoV immune complexes; 2 cats still showed positive antibody titres (Fig. 1 ).

Fig. 1.

Coronavirus antibody titres and circulating FCoV immune complexes in FIPV-exposed SPE cats without FIP (Group Ia, n = 7).

CoV particles were demonstrated in the ingesta of 3 FIPV-exposed SPF cats (Group Ia), both 11-week-old FIPV-exposed cats without FIP (Group Ib), and 8 cats with FIP (Group II) (Table 3, Table 4 ). Six FIPV-exposed SPF cats (Group Ia), both 11-week-old FIPV-exposed cats without FIP (Group Ib), and 10 cats with FIP (Group II) were positive in the CoV PCR (Table 3, Table 4).

Table 3.

Virus demonstration in FIPV-exposed cats without FIP (Group I, a: cat No. 1–7, b: cat No. 8 and 9)

Cat No.	EMa	RT-PCRb	Cultivationc
1	−d	+d	+
2	(+/−)d	+	+
3	–	+	+
4	–	+	+
5	–	–	+
6	+	+	+
7	+++	+	+
8	++	+	n.d.e
9	+	+	n.d.e

^aEM, electron microscopical demonstration of coronavirus particles in ingesta.

^bRT-PCR, nested FCoV RT-PCR on faeces (Herrewegh et al., 1995).

^cCultivation, cultivation of buffy coat monocytes on whole feline embryo cells (Gunn-Moore et al., 1998).

^d–: negative result (no viral particles); +/−: single viral particles; +: few viral particles; ++: numerous viral particles; +++: abundant viral particles.

^en.d.: not done.

Table 4.

Virus demonstration in cats with FIP (Group II; a: cat No. 1–3, b: cat No. 4–12)

Cat No.	Ema	RT-PCRb
1	n.d.c	+d
2	(+/−)d	+
3	n.d.	n.d.
4	n.d.	+
5	n.d.	+
6	+	+
7	–d	+
8	–	+
9	+/−	–
10	+++	+
11	+++	+
12	+++	+

^aEM: electron microscopical demonstration of coronavirus particles in ingesta.

^bRT-PCR: nested FCoV RT-PCR on faeces (Herrewegh et al., 1995).

^cn.d: not done.

^d: negative result (no viral particles); +/−: single viral particles; +: few viral particles: ++: numerous viral particles; +++: abundant viral particles.

Coronavirus was isolated from buffy coat monocytes of all FIPV-exposed SPF cats (Group Ia). The cytopathic effect on WFE cells was consistent with that usually seen with Serotype 2 FCoV.

Compared to control cats (Group III), all FIPV-exposed cats without FIP (Group I) showed extremely large follicles with extended hypercellular germinal centers and hypercellular T-cell zones. Follicles exhibited a high number of PCNA-positive proliferating cells. Simultaneously, the number of apoptotic cells, mainly visible as apoptotic bodies within tingible body macrophages, was increased. In FIPV-exposed cats without FIP (Group I), macrophages accumulated around lymphoid follicles and T-cell zones, but were rare in the red pulp which was dominated by B and T-cells in approximately equal amounts. In control cats (Group III), the number of macrophages was generally low.

In comparison, cats with FIP (Group II) exhibited moderately depleted follicles and reduced T-cell zones. The red pulp was dominated by macrophages and showed a higher rate of proliferation than the follicles. Both in FIPV-exposed cats without FIP (Group I) and control cats (Group III), mesenteric lymph nodes were very active and exhibited numerous secondary follicles and broad T-cell zones. In cats with FIP (Group II), moderate to distinct lymphoid depletion was observed. In comparison to both 38- and 72-week-old control cats (Group III), FIPV-exposed cats without FIP (Group I) showed a broad hypercellular cortex with numerous tingible body macrophages and a large medullary zone with inconspicious Hassal’s corpuscles. The proliferating activity was increased as numerous PCNA-positive thymocytes indicated in both the outer and inner cortex as well as in the medulla. In control cats (Group III), PCNA-positive cells were mostly restricted to the outer cortex. Simultaneously, the number of apoptotic cells was increased in FIPV-exposed cats without FIP (Group I). B-cells, mainly located along the corticomedullary junction, were more numerous in FIPV-exposed cats without FIP (Group I). In general, cats exhibited focal B-cell accumulations at various sites which occasionally formed follicles with follicular dendritic cells. Regardless of age, cats with FIP (Group II) generally exhibited massive to complete thymic atrophy. Thymic tissue was at least reduced to a hypocellular medulla.

In cats with FIP (Group II), CoV antigen was detected within intact macrophages in granulomatous-necrotizing lesions. In FIPV-exposed cats without FIP (Group I), positive staining for CoV antigen was restricted to single detached intestinal epithelial cells in both 11-week-old kittens (Group Ib). Lymphatic tissues were completely negative in all FIPV exposed cats without FIP (Group I).

Both in cats with FIP and FIPV-exposed cats without FIP (Groups I and II), plasma cells with CoV-specific antibodies could be demonstrated in variable amounts in the intestinal mucosa, as well as in capsule and medullary cords of mesenteric lymph nodes. In some cats, they were occasionally observed in the splenic red pulp and around blood vessels in the thymic interstitium. In cats with FIP (Group II), they were also seen among infiltrating cells close to areas with CoV antigen-positive macrophages.

Results show that long-term naturally FIPV-exposed cats develop CoV infection and start a CoV-specific immune response regardless of the development of FIP: animals exhibit CoV antibody titres and circulating FCoV immune complexes during exposure. The presence of FCoV immune complexes can be regarded as indicative of an FIPV infection as experimental studies have shown that in FECV-infected cats, circulating FCoV immune complexes only occur occasionally during a short time period after about 3 weeks postinfection. Cats which develop FIP, however, remain positive for a longer time span (Pfeiffer, 1991). Although virus shedding, indicated by demonstration of CoV particles in the ingesta and CoV genome in faeces (Addie et al., 1996, Foley et al., 1997), was not consistently detected, CoV isolation supports that all exposed animals were viraemic (Gunn-Moore et al., 1998).

FIPV-exposed cats without FIP exhibit an intensely stimulated immune system, represented by distinct follicular and T-cell hyperplasia in spleen and mesenteric lymph nodes, and an hyperplastic thymus. Active mesenteric lymph nodes, however, are also observed in control cats, indicating that already the normal intestinal bacterial flora induces an intense immune activity. Nevertheless, the demonstration of plasma cells positive for CoV-specific antibodies in situ in FIPV-exposed cats support that the observed systemic immune response is CoV-specific (Kipar et al., 1998).

In comparison, lymphatic tissues of cats with FIP generally show distinct atrophic changes. T-cell depletion has already been described in cats with FIP (Haagmans et al., 1996, Kipar et al., 1998). However, our findings indicate that reduction of B-cell areas is also present.

The development of granulomatous-necrotizing lesions in FIP is thought to be mediated by immune complexes (Jacobse-Geels et al., 1982). On the other hand, our findings indicate that prevention of FIP obviously requires an even more intense B-cell immune response, accompanied by an increased and effective T-cell response. This does not rule out that lymphocyte depletion also may depend on virus variants which can evolve independently in each infected individual (Herrewegh et al., 1997, Vennema et al., 1998).