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Delayed-type hypersensitivity skin responses associated with feline infectious peritonitis in two cats
Research in Veterinary Science 1988, 44, 396-398
Delayed-type hypersensitivity skin responses associated with feline infectious peritonitis in two cats R. C. WEISS, N. R. COX, Scott Ritchey Research Program and the Department of Pathobiology, College of Veterinary Medicine, Auburn University, Alabama 36849, USA
Two cats previously challenge-exposed and seropositive to feline infectious peritonitis virus (FlPV) were evaluated for delayed-type hypersensitivity (DTH) skin responses to intradermal FlPV. Before testing, cat 1 (nr-reslstant) had survived a severe experimental FIPV challenge-exposure and had remained asymptomatic, whereas cat 2 (FIP-susceptible) developed acute fulminant FIP after a considerably smaller virus challenge-exposure. Cat 1 developed a focal thickened plaque at the FlPv~injected skin site at 48 hours after injection. Histological examinations of serial punch biopsies from virus-inoculated skin revealed perivascular and diffuse dermal infiltrations of macrophages, lymphocytes and polymorphonuclear leucocytes which were maximal at 48 to 72 hours after injection. In contrast, cat 2 did not react grossly and showed only very mild dermal infiltrates at 72 hours after injection. The present findings of strong DTH responses to FIPV hi a resistant cat and minimal responses in a cat with acute fulminant np suggest that certain in vivo cellular immune reactions may be associated with disease resistance. FELINE infectious peritonitis (fIP) is a fatal, immunopathological coronavirai disease of domestic and exotic cats (Pedersen 1985a). The lesions in flP frequently involve blood vessels and consist of disseminated fibrinonecrotising or pyogranulomatous inflammation in abdominal and thoracic viscera, parietal and visceral serosae, eyes and central nervous system (Weiss and Scott 1981a,b, Pedersen 1985a). Histologically, the vascular lesions can resemble an arthus-type reaction and suggest immune complex formation and complement activation (Pedersen and Boyle 1980, Weiss and Scott 198Ia,b). Humoral immunity to flP virus (fIPV) is not protective; antiviral antibodies can, in fact, enhance disease and mediate immunopathological lesions (Pedersen and Boyle 1980, 1985a, Weiss and Scott 1981 b). The specific host immunological mechanisms which can protect cats against flPv-induced immunopathological disease are not known. This report documents the occurrence of a delayed-type hypersensitivity (DTH) skin response, an in vivo correlate of cellular immunity (Greene et al 1984) in two cats previously exposed to virulent fIPV. One of the cats was resistant to a severe experimental challenge with flPV and subsequently demonstrated strong microscopic DTH skin responses, whereas the second cat was terminally ill with disseminated flP and demonstrated only mild DTH skin responses. Two adult, random-source mixed breed cats were studied. The cats were selected serendipitously from a group of 18 FIPV challenge-exposed cohorts on the basis of their individual resistance or susceptibility to flP. Cat I (fIPresistant) previously had been inoculated intraperitoneally
with approximately 5000 lethal doses (LDIOO) of flPV (DF2 strain, American Type Culture Collection, ATCC No VR2004, Rockville, Maryland) and was asymptomatic at the time of DTH testing five weeks later. By the virus neutralisation assay for flPV serum antibodies described previously (Pedersen and Black 1983), a fourfold increase in virus neutralising antibody titres occurred between post challenge-exposure days 0 and 24 (640 and 2560, respectively). The finding of intrinsic flP disease resistance in a cat challenge-exposed to such a massive virus dose was itself an extremely fortuitous event (R. C. Weiss, unpublished observations) and prompted study of potential immune mechanisms. Cat 2 (rtr-susceptible) previously had been inoculated intraperitoneally with only 5 LDIOO (or about onethousandth the concentration of virus given to cat 1) and developed the typical clinical signs of acute experimental flP (Weiss and Scott 1981b) two weeks after virus inoculation. Cat 2 was seronegative for virus neutralising antibodies to flPV on post challenge-exposure day 0, and subsequently demonstrated an antibody titre of 640 on day 12. Cats 1 and 2 were tested for DTH skin responses on post challenge-exposure day 34 and 18, respectively. At those times, cat I was asymptomatic, and cat 2 was dying from acute flP. Each cat was mildly sedated with ketamine hydrochloride (Vetalar; Parke, Davis), and the hair over the left and right flanks was clipped. The skin was scrubbed with a disinfectant soap (Betadine surgical scrub; Purdue Fredrick), rinsed in water, and wiped with 70 per cent ethyl alcohol. Skin tests were performed by intradermally injecting either 0·1 ml of fIPv-DF2 (containing approximately 1()6 tissue culture infectious units, TCIDSO) or 0'1 ml of tissue culture medium used to propagate the fJPV (media control) into each of 10sites on the left or right flank, respectively. At hour 12, 24, 48, 72 and 96 (for cat 1) and hour 24, 48 and 72 (for cat 2) after injection, each cat was sedated as described previously and biopsies were taken from the virus-inoculated or control skin of each cat using a 6 mm biopsy punch (Baker's Biopsy Punch; Key Pharmaceuticals). The biopsy samples were fixed in 10 per cent neutral buffered formalin, embedded in paraffin, sectioned at 6 JAm, and stained with haernatoxylin and eosin. Cat I subsequently developed a circumscribed 3 mm 2 slightly thickened' plaque in the rn-v-iniected site at 48 hours after injection. Gross changes were not observed in fIPVinjected sites at other examination times. Media-injected control sites did not show gross changes at 12, 24, 48, 72, or 96 hours after injection. Cat 2 did not demonstrate gross changes either in the virus-inoculated or control sites. The histological findings in sequential skin biopsies from cats I and 2 are recorded in Table I. Photomicrographs of dermal
396
Delayed-type sensitivity and F1P
397
TABLE 1: Histological evaluation of DTH skin responses to FIPVin two cats· Cat
History FIP-resistantt
2
FIP-susceptible:l:
Intradermal antigen
12
Time after injection (hours) 24 48 72
FIPV media
Neg Neg
+
Neg
+++
+++ Neg
Neg
FIPV media
NO NO
Neg Neg
Neg Neg
± Neg
NO NO
Neg
96
+
Perivascular. perifollicular and diffuse dermal infiltrates of macrophages. lymphocytes and polymorphonuclear cells. graded in severity as follows: ± rare foci; + mild; + + moderate; + + + marked t Survived challenge-exposure of 5000 L0100 FIPV :I: ) Developed FIP and died after challenge-exposure of 5 L0100 OTH Delayed-type hypersensitivity FIPV Feline infectious peritonitis virus Neg Negative NO Not determined
responses to FIPV in cats I and 2 at 48 hours or 72 hours after injection, respectively, are shown (Figs I and 2). Cat I was still asymptomatic several months after FIPV challengeexposure; cat 2 died on day 21 after challenge-exposure (ie, at 72 hours after injection). Necropsy examination of cat 2 demonstrated the typical gross and histopathological lesions of non-effusive FIP (Weiss and Scott 1981a, Pedersen 1985a). Elicitation of DTH responses occurred after intradermal injection of virus in the FIP-resistant cat. Previous studies showed that classic DTH skin reactions are difficult to demonstrate grossly in cats and can be transient (Schultz and Adams 1978, Legendre et al 1979). In the present study, gross manifestations of DTH, with the exception of one positive reaction at 48 hours after injection (cat I), were not apparent. Histological examinations of sequential biopsies from FIPv-inoculated skin at 24 to 96 hours after injection in cat I, however, demonstrated an admixture of predominantly mononuclear cells (lymphocytes, macrophages) and also polymorphonuclear cells (mononuclear:polymorphonuclear ratio, 3:1) surrounding dermal adnexa, veins and subcutaneous blood vessels (Fig I). This mixed
FIG 1: Skin biopsy of cat 1 (FIP-resistantl showing strong OTH response 48 hours after intradermal inoculation of FIPV. Note the prominent perivascular and oiffuse dermal infiltration of mixed inflammatory cells and also the marked dilatation of a dermal vein (v). Haematoxylin and eosin x 424 .
inflammatory infiltrate, which was maximal in intensity at 48 to 72 hours after injection, was characteristic of the DTH response previously described in cats and dogs (Schultz and Adams 1978, Legendre et al 1979). Thus, both the dermal histopathology and also the characteristic reaction times observed in cat I were consistent with. a DTH response, in contrast to an arthus-type (antibody-mediated) or JonesMote (cutaneous basophil hypersensitivity) lesion (Sell 1975). Except for some scattered, mild mononuclear cell foci around dermal veins at 72 hours after injection (Fig 2), histological changes suggestive of DTH were not observed in cat 2 (rre-susceptible). Natural resistance to FIP is believed to be a function, predominantly, of cell-mediated immunity (Pedersen 1985a). In a previous study (Pedersen 1985b), healthy FIPV challenge-exposed cats demonstrated specific lymphocyte blast transformation to FIPV antigen in peripheral lymphocyte cultures or demonstrated gross DTH responses to F1PV injected into their nictitating membranes. Seemingly, the present findings of DTH skin responses in a healthy cat previously challenge-exposed and resistant to numerous lethal doses of F1PV also suggests involvement of cellmediated immunity in FIP disease resistance. The relatively weak DTH responses in cat 2 (Fig 2) may, as suggested
FIG 2: Skin biopsy of cat 2 (FIP-susceptible)at 72 hours after inoculation. There is a mild inflammatory cell infiltrate around a large dermal vein. Haematoxylin and eosin x 424
398
R. C. Weiss, N. R. Cox
previously for non-effusive (granulomatous) FIP (Pedersen 1985a). represent a partial and ineffectual cell-mediated immunity directed against viral foci. Accordingly. necropsy examination of cat 2 did reveal predominantly non-effusive FIP. It is known that certain viral infections (eg, measles. mumps. rabies. Newcastle disease and lactic dehydrogenase virus) can suppress DTH reactions or other cell-mediated immune responses by inhibiting T cell function either directly (by destruction of cells) or indirectly (by alteration of normal lymphocyte traffic or impairment of effector cell maturation) (Drutz and Mills 1978). Cats with FJP can develop severe lymphopenia and also widespread lymphoid necrosis. particularly in T cell dependent areas in spleen and peripheral lymph nodes (Weiss and Scott 198Ia.b). Conceivably. the weak DTH responses observed in the FIPdiseased cat might reflect either inadequate numbers of circulating effector lymphocytes. poor Iymphokine production. or perhaps some functional change in the effector cells. The difficulty in eliciting macroscopic DTH reactions to virus in a resistant animal (cat I) which. none the less. showed prominent DTH lesions in skin biopsies. suggests that specific DTH to FIPV can develop in cats in the absence of classic. tuberculin-type gross skin reactions. References DRUTZ. D. J. & MtLLS, J. (1978) Basic and Clinical Immunology.
Eds H. H. Fudenberg, D. P. Stites, J. L. Caldwell and J. V. Wells. Los Altos. Lange Medical Publications. pp 217-236 GREENE, M. I., SCHATTEN. S. & BROMBERG, S. (1984) Fundamental Immunology. Ed W. E. Paul. New York. Raven Press. pp 685-696 LEGENDRE, A. M., EASELY, R. J. & BECKER, P. U. (1979) American Journal of Veterinary Research 40, 1613-1619 PEDERSEN, N. C. & BOYLE, J. F. (1980) American Journal of Veterinary Research 41. 868-876 PEDERSEN, N. C. & BLACK, J. W. (1983) American Journal of Veterinary Research 44, 229-234 PEDERSEN, N. C. (l985a) Comparative Pathobiology of Viral Diseases. Vol 2. Eds R. G. Olsen. S. Krakowka and J. R. Blakeslee. Boca Raton. Florida. CRC Press. pp 115-136 PEDERSEN. N. C. (1985b) Compendium on Continuing Education for the Practising Veterinarian 7, JOOI-IOII SCHULTZ, R. D. & ADAMS, L. S. (1978) Veterinary Clinics of North America 8. 721-753 SELL, S. (1975) Immunology, Immunopathology and Immunity, 2nd edn. Hagerstown. Maryland, Harper & Row. pp 216-217 WEISS, R. C. & SCOTT, F. W. (l98Ia) American Journal of Veterinary Research 42. 2036-2048 WEISS, R. C. & SCOTT. F. W. (l98Ib) Comparative Immunology, Microbiology and Infectious Diseases 4, 175-189
Received September 30, 1987 Accepted January 12, 1988
Delayed-type hypersensitivity skin responses associated with feline infectious peritonitis in two cats R. C. WEISS, N. R. COX, Scott Ritchey Research Program and the Department of Pathobiology, College of Veterinary Medicine, Auburn University, Alabama 36849, USA
Two cats previously challenge-exposed and seropositive to feline infectious peritonitis virus (FlPV) were evaluated for delayed-type hypersensitivity (DTH) skin responses to intradermal FlPV. Before testing, cat 1 (nr-reslstant) had survived a severe experimental FIPV challenge-exposure and had remained asymptomatic, whereas cat 2 (FIP-susceptible) developed acute fulminant FIP after a considerably smaller virus challenge-exposure. Cat 1 developed a focal thickened plaque at the FlPv~injected skin site at 48 hours after injection. Histological examinations of serial punch biopsies from virus-inoculated skin revealed perivascular and diffuse dermal infiltrations of macrophages, lymphocytes and polymorphonuclear leucocytes which were maximal at 48 to 72 hours after injection. In contrast, cat 2 did not react grossly and showed only very mild dermal infiltrates at 72 hours after injection. The present findings of strong DTH responses to FIPV hi a resistant cat and minimal responses in a cat with acute fulminant np suggest that certain in vivo cellular immune reactions may be associated with disease resistance. FELINE infectious peritonitis (fIP) is a fatal, immunopathological coronavirai disease of domestic and exotic cats (Pedersen 1985a). The lesions in flP frequently involve blood vessels and consist of disseminated fibrinonecrotising or pyogranulomatous inflammation in abdominal and thoracic viscera, parietal and visceral serosae, eyes and central nervous system (Weiss and Scott 1981a,b, Pedersen 1985a). Histologically, the vascular lesions can resemble an arthus-type reaction and suggest immune complex formation and complement activation (Pedersen and Boyle 1980, Weiss and Scott 198Ia,b). Humoral immunity to flP virus (fIPV) is not protective; antiviral antibodies can, in fact, enhance disease and mediate immunopathological lesions (Pedersen and Boyle 1980, 1985a, Weiss and Scott 1981 b). The specific host immunological mechanisms which can protect cats against flPv-induced immunopathological disease are not known. This report documents the occurrence of a delayed-type hypersensitivity (DTH) skin response, an in vivo correlate of cellular immunity (Greene et al 1984) in two cats previously exposed to virulent fIPV. One of the cats was resistant to a severe experimental challenge with flPV and subsequently demonstrated strong microscopic DTH skin responses, whereas the second cat was terminally ill with disseminated flP and demonstrated only mild DTH skin responses. Two adult, random-source mixed breed cats were studied. The cats were selected serendipitously from a group of 18 FIPV challenge-exposed cohorts on the basis of their individual resistance or susceptibility to flP. Cat I (fIPresistant) previously had been inoculated intraperitoneally
with approximately 5000 lethal doses (LDIOO) of flPV (DF2 strain, American Type Culture Collection, ATCC No VR2004, Rockville, Maryland) and was asymptomatic at the time of DTH testing five weeks later. By the virus neutralisation assay for flPV serum antibodies described previously (Pedersen and Black 1983), a fourfold increase in virus neutralising antibody titres occurred between post challenge-exposure days 0 and 24 (640 and 2560, respectively). The finding of intrinsic flP disease resistance in a cat challenge-exposed to such a massive virus dose was itself an extremely fortuitous event (R. C. Weiss, unpublished observations) and prompted study of potential immune mechanisms. Cat 2 (rtr-susceptible) previously had been inoculated intraperitoneally with only 5 LDIOO (or about onethousandth the concentration of virus given to cat 1) and developed the typical clinical signs of acute experimental flP (Weiss and Scott 1981b) two weeks after virus inoculation. Cat 2 was seronegative for virus neutralising antibodies to flPV on post challenge-exposure day 0, and subsequently demonstrated an antibody titre of 640 on day 12. Cats 1 and 2 were tested for DTH skin responses on post challenge-exposure day 34 and 18, respectively. At those times, cat I was asymptomatic, and cat 2 was dying from acute flP. Each cat was mildly sedated with ketamine hydrochloride (Vetalar; Parke, Davis), and the hair over the left and right flanks was clipped. The skin was scrubbed with a disinfectant soap (Betadine surgical scrub; Purdue Fredrick), rinsed in water, and wiped with 70 per cent ethyl alcohol. Skin tests were performed by intradermally injecting either 0·1 ml of fIPv-DF2 (containing approximately 1()6 tissue culture infectious units, TCIDSO) or 0'1 ml of tissue culture medium used to propagate the fJPV (media control) into each of 10sites on the left or right flank, respectively. At hour 12, 24, 48, 72 and 96 (for cat 1) and hour 24, 48 and 72 (for cat 2) after injection, each cat was sedated as described previously and biopsies were taken from the virus-inoculated or control skin of each cat using a 6 mm biopsy punch (Baker's Biopsy Punch; Key Pharmaceuticals). The biopsy samples were fixed in 10 per cent neutral buffered formalin, embedded in paraffin, sectioned at 6 JAm, and stained with haernatoxylin and eosin. Cat I subsequently developed a circumscribed 3 mm 2 slightly thickened' plaque in the rn-v-iniected site at 48 hours after injection. Gross changes were not observed in fIPVinjected sites at other examination times. Media-injected control sites did not show gross changes at 12, 24, 48, 72, or 96 hours after injection. Cat 2 did not demonstrate gross changes either in the virus-inoculated or control sites. The histological findings in sequential skin biopsies from cats I and 2 are recorded in Table I. Photomicrographs of dermal
396
Delayed-type sensitivity and F1P
397
TABLE 1: Histological evaluation of DTH skin responses to FIPVin two cats· Cat
History FIP-resistantt
2
FIP-susceptible:l:
Intradermal antigen
12
Time after injection (hours) 24 48 72
FIPV media
Neg Neg
+
Neg
+++
+++ Neg
Neg
FIPV media
NO NO
Neg Neg
Neg Neg
± Neg
NO NO
Neg
96
+
Perivascular. perifollicular and diffuse dermal infiltrates of macrophages. lymphocytes and polymorphonuclear cells. graded in severity as follows: ± rare foci; + mild; + + moderate; + + + marked t Survived challenge-exposure of 5000 L0100 FIPV :I: ) Developed FIP and died after challenge-exposure of 5 L0100 OTH Delayed-type hypersensitivity FIPV Feline infectious peritonitis virus Neg Negative NO Not determined
responses to FIPV in cats I and 2 at 48 hours or 72 hours after injection, respectively, are shown (Figs I and 2). Cat I was still asymptomatic several months after FIPV challengeexposure; cat 2 died on day 21 after challenge-exposure (ie, at 72 hours after injection). Necropsy examination of cat 2 demonstrated the typical gross and histopathological lesions of non-effusive FIP (Weiss and Scott 1981a, Pedersen 1985a). Elicitation of DTH responses occurred after intradermal injection of virus in the FIP-resistant cat. Previous studies showed that classic DTH skin reactions are difficult to demonstrate grossly in cats and can be transient (Schultz and Adams 1978, Legendre et al 1979). In the present study, gross manifestations of DTH, with the exception of one positive reaction at 48 hours after injection (cat I), were not apparent. Histological examinations of sequential biopsies from FIPv-inoculated skin at 24 to 96 hours after injection in cat I, however, demonstrated an admixture of predominantly mononuclear cells (lymphocytes, macrophages) and also polymorphonuclear cells (mononuclear:polymorphonuclear ratio, 3:1) surrounding dermal adnexa, veins and subcutaneous blood vessels (Fig I). This mixed
FIG 1: Skin biopsy of cat 1 (FIP-resistantl showing strong OTH response 48 hours after intradermal inoculation of FIPV. Note the prominent perivascular and oiffuse dermal infiltration of mixed inflammatory cells and also the marked dilatation of a dermal vein (v). Haematoxylin and eosin x 424 .
inflammatory infiltrate, which was maximal in intensity at 48 to 72 hours after injection, was characteristic of the DTH response previously described in cats and dogs (Schultz and Adams 1978, Legendre et al 1979). Thus, both the dermal histopathology and also the characteristic reaction times observed in cat I were consistent with. a DTH response, in contrast to an arthus-type (antibody-mediated) or JonesMote (cutaneous basophil hypersensitivity) lesion (Sell 1975). Except for some scattered, mild mononuclear cell foci around dermal veins at 72 hours after injection (Fig 2), histological changes suggestive of DTH were not observed in cat 2 (rre-susceptible). Natural resistance to FIP is believed to be a function, predominantly, of cell-mediated immunity (Pedersen 1985a). In a previous study (Pedersen 1985b), healthy FIPV challenge-exposed cats demonstrated specific lymphocyte blast transformation to FIPV antigen in peripheral lymphocyte cultures or demonstrated gross DTH responses to F1PV injected into their nictitating membranes. Seemingly, the present findings of DTH skin responses in a healthy cat previously challenge-exposed and resistant to numerous lethal doses of F1PV also suggests involvement of cellmediated immunity in FIP disease resistance. The relatively weak DTH responses in cat 2 (Fig 2) may, as suggested
FIG 2: Skin biopsy of cat 2 (FIP-susceptible)at 72 hours after inoculation. There is a mild inflammatory cell infiltrate around a large dermal vein. Haematoxylin and eosin x 424
398
R. C. Weiss, N. R. Cox
previously for non-effusive (granulomatous) FIP (Pedersen 1985a). represent a partial and ineffectual cell-mediated immunity directed against viral foci. Accordingly. necropsy examination of cat 2 did reveal predominantly non-effusive FIP. It is known that certain viral infections (eg, measles. mumps. rabies. Newcastle disease and lactic dehydrogenase virus) can suppress DTH reactions or other cell-mediated immune responses by inhibiting T cell function either directly (by destruction of cells) or indirectly (by alteration of normal lymphocyte traffic or impairment of effector cell maturation) (Drutz and Mills 1978). Cats with FJP can develop severe lymphopenia and also widespread lymphoid necrosis. particularly in T cell dependent areas in spleen and peripheral lymph nodes (Weiss and Scott 198Ia.b). Conceivably. the weak DTH responses observed in the FIPdiseased cat might reflect either inadequate numbers of circulating effector lymphocytes. poor Iymphokine production. or perhaps some functional change in the effector cells. The difficulty in eliciting macroscopic DTH reactions to virus in a resistant animal (cat I) which. none the less. showed prominent DTH lesions in skin biopsies. suggests that specific DTH to FIPV can develop in cats in the absence of classic. tuberculin-type gross skin reactions. References DRUTZ. D. J. & MtLLS, J. (1978) Basic and Clinical Immunology.
Eds H. H. Fudenberg, D. P. Stites, J. L. Caldwell and J. V. Wells. Los Altos. Lange Medical Publications. pp 217-236 GREENE, M. I., SCHATTEN. S. & BROMBERG, S. (1984) Fundamental Immunology. Ed W. E. Paul. New York. Raven Press. pp 685-696 LEGENDRE, A. M., EASELY, R. J. & BECKER, P. U. (1979) American Journal of Veterinary Research 40, 1613-1619 PEDERSEN, N. C. & BOYLE, J. F. (1980) American Journal of Veterinary Research 41. 868-876 PEDERSEN, N. C. & BLACK, J. W. (1983) American Journal of Veterinary Research 44, 229-234 PEDERSEN, N. C. (l985a) Comparative Pathobiology of Viral Diseases. Vol 2. Eds R. G. Olsen. S. Krakowka and J. R. Blakeslee. Boca Raton. Florida. CRC Press. pp 115-136 PEDERSEN. N. C. (1985b) Compendium on Continuing Education for the Practising Veterinarian 7, JOOI-IOII SCHULTZ, R. D. & ADAMS, L. S. (1978) Veterinary Clinics of North America 8. 721-753 SELL, S. (1975) Immunology, Immunopathology and Immunity, 2nd edn. Hagerstown. Maryland, Harper & Row. pp 216-217 WEISS, R. C. & SCOTT, F. W. (l98Ia) American Journal of Veterinary Research 42. 2036-2048 WEISS, R. C. & SCOTT. F. W. (l98Ib) Comparative Immunology, Microbiology and Infectious Diseases 4, 175-189
Received September 30, 1987 Accepted January 12, 1988