Effect of recombinant human interferon-alpha in vitro and in vivo on mitogen-induced lymphocyte blastogenesis in cats

The effect of recombinant human interferon-alpha (rHuIFN-alpha) in vitro and in vivo on mitogen-induced lymphocyte blastogenesis was evaluated in specific-pathogen-free cats. Pre-incubation of isolated feline peripheral blood lymphocytes (PBL) in vitro with either 10(4) or 10(3) International Units (U) of rHuIFN-alpha for 24 h significantly suppressed (P less than 0.001 and 0.01, respectively) blastogenic responses to the phytomitogens concanavalin A (Con A) and pokeweed mitogen (PWM). Lower doses of IFN (range, 10-10(-3) U/ml) neither suppressed nor enhanced mitogenesis. In the absence of phytomitogens, incubation of PBL with 10(4) - 10(2) U (P less than 0.001) or 10 U (P less than 0.05) of rHuIFN-alpha/ml resulted in a significant decrease in incorporation of [methyl-3H] thymidine into newly synthesized cellular DNA. Cultures of PBL exposed continuously for 4 days to rHuIFN-alpha doses of 10(4) U/ml or less did not demonstrate specific reductions in cell viability, indicating that the observed antiproliferative actions of IFN apparently were independent of any direct cytotoxic effects. To investigate the dose-response effects of rHuIFN-alpha in vivo on lymphocyte blastogenesis, individual groups of cats were evaluated on 3 consecutive days before and then 24 h after each cat was inoculated intramuscularly with either a high dose (10(6) U/kg), moderate dose (10(4) U/kg), or a relatively low dose (10(2) U/kg) of rHuIFN-alpha. Cats inoculated with 10(6) U of rHuIFN-alpha/kg had significantly reduced (P = 0.037) blastogenic responses to Con a at 24 h postinoculation compared to preinoculation values; mean PWM responses were also decreased, but this effect was not statistically significant. In contrast, inoculation of cats with either 10(4) or 10(2) U of rHuIFN-alpha/kg significantly enhanced (P = 0.05 or 0.008, respectively) Con A-induced blastogenesis and had no discernible effect on PWM responses. These findings suggest that very high doses of rHuIFN-alpha given parenterally may be associated with suppression of certain T-cell responses in cats; conversely, much lower doses may be immunoenhancing.     

Inhibitory effects of ribavirin alone or combined with human alpha interferon on feline infectious peritonitis virus replication in vitro

The antiviral activities of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide; virazole), either alone or in combination with recombinant human leukocyte (alpha) interferon (rHuIFN-alpha), were evaluated against feline infectious peritonitis virus (FIPV) in feline kidney-cell cultures. The 50% inhibitory dose (ID50) of ribavirin for uninfected, rapidly dividing cells was approximately 17 micrograms ml-1 whereas the ID50 for FIPV was 2.5 micrograms ml-1. The therapeutic index (TI) of ribavirin (i.e. the ratio of the minimum cell-toxic dose to minimum virus-inhibitory dose) was 6.8. Although a dose-dependent inhibition of viral infectivity occurred at non-toxic doses, maximum antiviral effects (greater than or equal to 4 log10 reduction in FIPV) occurred at cytotoxic doses. When low or moderate doses of ribavirin were combined with either 10 or 100 U of rHuIFN-alpha ml-1, the resulting antiviral effects were significantly greater than the sum of the observed effects from either ribavirin or rHuIFN-alpha alone. Significant synergistic interactions with rHuIFN-alpha occurred at ribavirin doses of 1, 5, 12.5 and 25 micrograms ml-1. Synergistic combinations of rHuIFN-alpha and ribavirin produced up to an 80-fold or a 200-fold relative increase in FIPV antiviral activities compared with that produced by equivalent doses, respectively, of ribavirin or rHuIFN-alpha alone. In cell growth studies, the addition of either 10 or 100 U of rHuIFN-alpha ml-1 to test doses of ribavirin did not increase the anticellular effect observed with ribavirin alone; seemingly, the potentiation of ribavirin antiviral activity by rHuIFN-alpha was independent of any additive cytotoxic effects. Potentially, synergistic combinations of the two antiviral agents in vivo may decrease the therapeutic dose of ribavirin required for inhibition of FIPV and thus reduce drug toxicity.     

Evaluation of immunity to feline infectious peritonitis in cats with cutaneous viral-induced delayed hypersensitivity

Delayed-type hypersensitivity (DTH)-like reactions to feline infectious peritonitis (FIP) virus (FIPV) were induced in the skin of nine cats that were asymptomatic after a previous challenge-exposure with FIPV. Four of the nine previously challenge-exposed cats were negative for virus-neutralizing antibodies against FIPV at the time of intradermal (ID) testing for DTH. Two other cats tested for DTH when acutely ill with clinical FIP did not have cutaneous DTH responses to FIPV. Gross skin reactions to FIPV injected ID were observed in six of nine asymptomatic cats (67%) at postintradermal inoculation hours (PIH) 24, 48, and/or 72. The reactions consisted of focal, 1-5-mm to 2.5-cm diameter indurated or semi-firm, nonerythematous, slightly raised nodules. Microscopically, DTH-like reactions were observed in biopsies taken from the FIPV-inoculated skin of asymptomatic cats at PIH 24 to 72. The lesions consisted of perivascular and diffuse dermal infiltrations by macrophages, lymphocytes, and polymorphonuclear leukocytes (PMN). The dermal infiltrates, which were maximal at PIH 48 or 72, were predominantly mixed inflammatory cells (five of nine cats) or PMN (four of nine cats) at PIH 24, but later were predominantly mononuclear cells (six of nine cats) or mixed inflammatory cells (two of nine cats) at PIH 72. Five of nine cats (56%) with positive DTH skin responses had increased survival times after lethal ID challenge-exposure with FIPV compared to mean survival times in FIPV-naive, non-immune control cats that were DTH-negative when ID challenge-exposed. Four of nine DTH-positive cats (44%) resisted an ID challenge-exposure dose of FIPV that was fatal in both control cats, and two of the four remaining DTH-positive cats survived a third challenge-exposure with highly lethal doses of FIPV given intraperitoneally. Four of the six DTH-positive cats (67%) that died after re-challenge and were necropsied had lesions of noneffusive FIP, suggesting that cellular immunity may also be involved in the pathogenesis of noneffusive disease, whereas both control cats and both DTH-negative cats with clinical disease succumbed to effusive FIP. Seemingly, DTH responses to FIPV can be associated with an increased level of resistance to disease; however, this state of immunity is variable and apparently can be lost with time in some cats.     

Induction and enhancement of feline infectious peritonitis by canine coronavirus.

Preexisting antibody to feline infectious peritonitis virus (FIPV) causes acceleration and enhancement of disease on subsequent infection of cats with FIPV. Other workers have shown that canine coronavirus (CCV) can infect cats subclinically, but have found no evidence of enhancement of, or protection against, subsequent FIPV infection. With various isolates of CCV, we determined that 1 strain of CCV can induce transient mild diarrhea in cats and, furthermore, that previous infection with CCV causes acceleration and enhancement of subsequent infection with FIPV. In addition, sequential inoculation of cats with another strain of CCV caused lesions indistinguishable from those of FIP, without exposure at any time to FIPV.     

Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV)

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats.     

Synergistic antiviral activities of acyclovir and recombinant human leukocyte (alpha) interferon on feline herpesvirus replication

The antiviral activities of 9-(2-hydroxyethoxymethyl)guanine (acyclovir; ACV) either alone or combined with recombinant human leukocyte (alpha) A/D interferon (rHuIFN-alpha) against feline herpesvirus type 1 (FHV-1) were evaluated in feline embryo cell cultures, using an infectivity-inhibition assay. In ACV-treated cultures, the 50% inhibitory dose (ID50) was approximately 10 to 20 micrograms of ACV/ml. Maximal inhibition of FHV-1 infectivity (range, 3.4 to 4.2 log10 TCID50) was observed when high test doses of ACV (125 or 250 micrograms/ml) were given 1 to 6 hours after infection. Although mild inhibition (range, 0.3 to 1.6 log10 TCID50) of virus was observed at lower drug doses (10 to 62.5 micrograms/ml), FHV-1 was relatively resistant to ACV and required higher minimal inhibitory doses than those reported for other herpesviruses. However, when ACV was combined with 10 or 100 U of rHuIFN-alpha/ml, synergistic antiviral effects were associated with ACV dosage of 10 to 62.5 micrograms/ml. Antiviral activities resulting from use of the combined drugs permitted nearly eightfold reduction in the dose of ACV required to achieve maximal inhibition of FHV-1. Significant (P less than 0.01) synergistic interactions with ACV resulted when the rHuIFN-alpha was given before or after infection; at the lower doses of ACV, however, rHuIFN-alpha pretreatment was more effective. Although dosages of either greater than or equal to 62.5 micrograms of ACV/ml or 100 U of rHuIFN-alpha/ml were cytosuppressive in control cell cultures, additive anticellular effects were not observed at synergistic combinations of ACV and 10 U of rHuIFN-alpha/ml.     

Humoral immune responses of cats to feline infectious peritonitis virus infection.

Immunoperoxidase antibody (IPA) method as a titrating method of feline infectious peritonitis (FIP) virus (FIPV) was developed for titrating antibody to FIPV (IPA-titer). By this method the immune responses of the cats that had been infected with FIPV, were traced. The infected cats could be grouped into three types by their immune response to FIPV and clinical appearances. Type I cats lived for a long time, formed a major group among infected cats, had 160 to 1 x 10(4) IPA-titers, and showed healthy appearances without any changes both on autopsy and histopathologically. From among type I cats, type II cats appeared sporadically with rapid elevation of IPA titers to 3.2 x 10(5) and showing clinical signs of FIP, and died. Type III cats lived healthily for a long time with gradual elevation of IPA-titers to a plateau of about 1 x 10(5), then showed neuronal disorder of hind leg paralysis with the descending IPA-titers to 2 x 10(4), and died. Thus, typical FIP appeared as a hyper-immune disease. Other related problems are discussed.     

Vaccine efficacy of a cell lysate with recombinant baculovirus-expressed feline infectious peritonitis (FIP) virus nucleocapsid protein against progression of FIP

The Type II feline infectious peritonitis virus (FIPV) infection of feline macrophages is enhanced by a monoclonal antibody (MAb) to the S protein of FIPV. This antibody-dependent enhancement (ADE) activity increased with the MAb that showed a neutralizing activity with feline kidney cells, suggesting that there was a distinct correlation between ADE activity and the neutralizing activity. The close association between enhancing and neutralizing epitopes is an obstacle to developing a vaccine containing only neutralizing epitopes without enhancing epitopes. In this study, we immunized cats with cell lysate with recombinant baculovirus-expressed N protein of the Type I FIPV strain KU-2 with an adjuvant and investigated its preventive effect on the progression of FIP. Cats immunized with this vaccine produced antibodies against FIPV virion-derived N protein but did not produce virus-neutralizing antibodies. A delayed type hypersensitivity skin response to N protein was observed in these vaccinated cats, showing that cell mediated immunity against the FIPV antigen was induced. When these vaccinated cats were challenged with a high dose of heterologous FIPV, the survival rate was 75% (6/8), while the survival rate in the control group immunized with SF-9 cell-derived antigen was 12.5% (1/8). This study showed that immunization with the cell lysate with baculovirus-expressed N protein was effective in preventing the progression of FIP without inducing ADE of FIPV infection in cats.     

Attempted immunisation of cats against feline infectious peritonitis using canine coronavirus

Specific pathogen free kittens were vaccinated with an unattenuated field isolate of canine coronavirus (CCV) either by aerosol or subcutaneously, and received boosting vaccinations four weeks later. Aerosolisation elicited a homologous virus-neutralising (VN) antibody response that increased steadily over a four-week period and levelled off one to two weeks after revaccination. The initial aerosolised dose produced an asymptomatic infection with excretion of CCV from the oropharynx up to eight days after vaccination; virus shedding was not detected, however, after the second inoculation. Cats vaccinated subcutaneously developed low VN antibody titres after the first CCV dose and experienced a strong anamnestic response after the second dose. Neutralising antibody titres then levelled off one to two weeks after revaccination at mean values somewhat lower than in cats vaccinated by aerosol. CCV was not isolated from the oropharynx after either subcutaneous dose. Four weeks after CCV boosting inoculations, vaccinated cats and sham-vaccinated control cats were divided into three subgroups and challenged by aerosol with the virulent UCD1 strain of feline infectious peritonitis virus (FIPV UCD1) at three different dosage levels. Five of six cats (including sham-vaccinated controls) given the lowest challenge dose showed no signs of disease, while all other cats developed lesions typical of feline infectious peritonitis (FIP). The five surviving cats developed FIP after subsequent challenge with a fivefold higher dose of FIPV. Thus heterotypic vaccination of cats with CCV did not provide effective protection against FIPV challenge.     

An outbreak of feline infectious peritonitis in a Taiwanese shelter: epidemiologic and molecular evidence for horizontal transmission of a novel type II feline coronavirus

Feline infectious peritonitis (FIP) is a fatal disease caused by feline coronavirus (FCoV) infection. FCoV can be divided into serotypes I and II. The virus that causes FIP (FIPV) is believed to occur sporadically and spread infrequently from cat to cat. Recently, an FIP outbreak from an animal shelter was confirmed in Taiwan. FCoV from all the cats in this shelter were analyzed to determine the epidemiology of this outbreak. Thirteen of 46 (28.2%) cats with typical signs of FIP were identified. Among them, seven cats were confirmed by necropsy and/or histopathological examinations. Despite the fact that more than one FCoV was identified in this multi-cat environment, the eight FIP cats were invariably found to be infected with a type II FCoV. Sequence analysis revealed that the type II FIPV detected from fecal samples, body effusions and granulomatous tissue homogenates from the cats that succumbed to FIP all harbored an identical recombination site in their S gene. Two of the cats that succumbed to FIP were found to harbor an identical nonsense mutation in the 3c gene. Fecal shedding of this type II virus in the effusive form of FIP can be detected up to six days before death. Taken together, our data demonstrate that horizontal transmission of FIPV is possible and that FIP cats can pose a potential risk to other cats living in the same environment.     

Inhibition of feline infectious peritonitis virus replication by recombinant human leukocyte (alpha) interferon and feline fibroblastic (beta) interferon

Replication of feline infectious peritonitis virus (FIPV) in feline cell cultures was inhibited after incubation of cells with either human recombinant leukocyte (alpha) interferon (IFN) or feline fibroblastic (beta) IFN for 18 to 24 hours before viral challenge exposure. Compared with virus control cultures, FIPV yields were reduced by ranges of 0.1 to 2.7 log10 or 2 to 5.2 log10 TCID50 in cultures treated with human alpha- or feline beta-IFN, respectively; yield reductions were IFN dose dependent. Sensitivity to the antiviral activities of IFN varied with cell type; feline embryo cells had greater FIPV yield reductions than did similarly treated feline kidney or feline lung cells. Comparison of the virus growth curves in IFN-treated and virus control cultures indicated marked reduction in intracellular and extracellular FIPV in IFN-treated cultures. Compared with virus control cultures, intracellular and extracellular infectivity in IFN-treated cultures was delayed in onset by 12 and 30 hours, respectively, and FIPV titers subsequently were reduced by 3 to 3.5 and 5 log10 TCID50, respectively. Frequently, immunofluorescent and electron microscopy of IFN-treated cells or cell culture fluids did not reveal virus; however, even in cultures without viral cytopathic changes, small amounts of virus occasionally persisted in cells.     

Disseminated intravascular coagulation in experimentally induced feline infectious peritonitis

Disseminated intravascular coagulation was induced in kittens by intraperitoneal inoculation of feline infectious peritonitis virus (FIPV). Kittens seronegative to FIPV survived significantly (P less than 0.05) longer than those seropositive to FIPV. Pyrexia, anemia, icterus, hyperbilirubinemia, and elevated concentrations of liver-specific enzymes were detected in the inoculated cats. Lesions induced included disseminated fibrinonecrotic and pyogranulomatous inflammation, hepatic necrosis, and widespread phlebitis and thrombosis. Localization of FIP viral antigen and immunoglobulin G was demonstrated in foci of heptic necrosis by immunofluorescence miroscopy. Lymphopenia, thrombocytopenia, hyperfibrinogenemia, and increased quantities of fibrin-fibrinogen degradation products were present in cats after the onset of clinical illness. Depression of factor VII, VIII, IX, X, XI, and XII plasma activities and prolongation of prothrombin and partial thromboplastin times also developed in infected cats. The accelerated onset of clinical disease and mortality in seropositive kittens vs seronegative kittens and the association of virus and antibody in multiple foci of hepatic necrosis suggest an immune-mediated component is involved in the pathogenesis of this disease.     

Pathogenicity studies of feline coronavirus isolates 79-1146 and 79-1683

Two feline coronavirus isolates were characterized by their disease-causing potential in cats. The 79-1683 feline coronavirus isolate caused an inapparent-to-mild enteritis when given oronasally to specific-pathogen-free kittens and was not a cause of feline infectious peritonitis (FIP). Target tissues for the virus were the mature apical epithelium of the small intestine, mesenteric lymph nodes, tonsils, thymus, and (to a lesser extent) the lungs. Inoculated kittens shed high numbers of virus in their feces for 14 to 17 days, but remained infectious to susceptible kittens for longer periods of time, as evidenced by contact-exposure studies. Because the 79-1683 isolate induced only enteritis, it was designated feline enteric coronavirus (FECV) 79-1683. The 79-1146 feline coronavirus isolate induced effusive abdominal FIP in specific-pathogen-free kittens after oronasal and intraperitoneal inoculation. Clinical signs of disease appeared within 12 to 14 days in almost all inoculated kittens. Because this isolate caused FIP, it was designated FIP virus (FIPV) 79-1146. Cross-protective immunity was not induced by the various coronavirus infections. Kittens preimmunized with the UCD strain of FECV (FECV-UCD) or with FECV-79-1683 were not immune to infection with FIPV-79-1146. Likewise, kittens previously inoculated with FECV-79-1683 were not immune to infection with FIPV-UCD1. In fact, preexisting heterologous FECV-79-1683 immunity often accelerated and enhanced the severity of disease caused by inoculation with FIPV-UCD1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of antigenic heterogeneity in feline coronavirus nucleocapsid in feline pyogranulomatous meningoencephalitis

A new monoclonal antibody (mAb), CCV2-2, was compared with the widely used FIPV3-70 mAb, both directed against canine coronavirus (CCoV), as a diagnostic and research tool. Western blot showed that both anti-CCoV mAbs only reacted with a protein of 50 kD, a weight consistent with the feline coronavirus (FCoV) viral nucleocapsid. A competitive inhibition enzyme-linked immunosorbent assay showed that the 2 recognized epitopes are distinct. Preincubation of CCV2-2 mAb with FCoV antigen suppressed the immunostaining. Formalin-fixed, paraffin-embedded sections from brains of 15 cats with the dry form of feline infectious peritonitis (FIP) were examined by immunohistochemistry. Immunohistochemistry was performed with both anti-CCoV mAbs, either on consecutive or on the same sections. A myeloid-histiocytic marker, MAC 387, was also used to identify FIP virus-infected cells. In all regions where MAC 387-positive cells were present, positive staining with the CCV2-2 mAb was systematically detected, except at some levels in 1 cat. In contrast, none or only a few cells were positive for the FIPV3-70 mAb. Double immunostaining showed macrophages that were immunopositive for either CCV2-2 alone or alternatively for CCV2-2 and FIPV3-70 mAbs. This reveals the coexistence of 2 cohorts of phagocytes whose FIP viral contents differed by the presence or absence of the FIPV3-70-recognized epitope. These findings provide evidence for antigenic heterogeneity in coronavirus nucleocapsid protein in FIP lesions, a result that is in line with molecular observations. In addition, we provide for the first time morphologic depiction of viral variants distribution in these lesions.     

Virus shedding and immune responses in cats inoculated with cell culture-adapted feline infectious peritonitis virus

Eight specific pathogen-free cats were inoculated orally or parenterally with a cell culture-adapted strain of feline infectious peritonitis virus (FIPV). Faeces and oropharyngeal swabs were monitored daily for infectious virus by inoculation of feline embryo lung cells. Virus was recovered from both sites for approximately 2 weeks after inoculation, before clinical signs of disease developed. Peripheral blood lymphocytes collected from these cats were tested in an in-vitro blastogenic assay using concanavalin A (con A) and FIPV antigen. All cats showed a profound suppression of the response to con A which only recovered to pre-inoculation levels in 2 cats, one of which survived. These 2 cats also responded to FIPV antigen on the 21st day after infection, the greater response being in the survivor. The other cats, surviving 16-18 days, developed no response to FIPV antigen. Antibody titres, measured by immunofluorescence and by virus neutralization, rose rapidly to very high levels in all cats, regardless of the route of inoculation.     

Using direct immunofluorescence to detect coronaviruses in peritoneal in peritoneal and pleural effusions

Twenty-one cases of feline infectious peritonitis (FIP) were diagnosed using a direct immunofluorescence test on cytocentrifuged pleural and peritoneal effusions from cats sampled in vivo (11 cases) and at necropsy (10 cases). A commercial fluorescent polyclonal antiserum of feline origin reacting with FIPV and cross reacting with transmissible gastroenteritis virus and canine coronavirus was used. Eleven cats with ascites of a different origin were used as negative controls. The direct immunofluorescence test was 97 per cent reliable (31 cases of 32) and can be used in routine diagnosis.     

Suppression of feline coronavirus replication in vitro by cyclosporin A

ABSTRACT: The feline infectious peritonitis virus (FIPV) is a member of the feline coronavirus family that causes FIP, which is incurable and fatal in cats. Cyclosporin A (CsA), an immunosuppressive agent that targets the nuclear factor pathway of activated T-cells (NF-AT) to bind cellular cyclophilins (CyP), dose-dependently inhibited FIPV replication in vitro. FK506 (an immunosuppressor of the pathway that binds cellular FK506-binding protein (FKBP) but not CyP) did not affect FIPV replication. Neither cell growth nor viability changed in the presence of either CsA or FK506, and these factors did not affect the NF-AT pathway in fcwf-4 cells. Therefore, CsA does not seem to exert inhibitory effects via the NF-AT pathway. In conclusion, CsA inhibited FIPV replication in vitro and further studies are needed to verify the practical value of CsA as an anti-FIPV treatment in vivo.     

Disease outcome and cytokine responses in cats immunized with an avirulent feline infectious peritonitis virus (FIPV)-UCD1 and challenge-exposed with virulent FIPV-UCD8

Eight cats were immunized with an avirulent strain of feline infectious peritonitis virus (FIPV)-UCD1, then challenge-exposed to a highly virulent cat passaged strain (FIPV-UCD8). Th1 and Th2 cytokine profiles in the peripheral blood mononuclear cells (PBMCs) were measured throughout in the experiment. No clinical signs of FIP were evident in the experimental cats after immunization. After challenge, the immunized cats demonstrated one of four clinical outcomes: (1) classical effusive FIP; (2) accelerated FIP; (3) non-effusive FIP, or (4) resistance to challenge. Only minor cytokine changes were observed following immunization, however, several cytokine changes occurred following challenge-exposure. The most noteworthy changes were in tumor necrosis factor-alpha (TNF-alpha) and interferon gamma (IFN-gamma) levels. Our preliminary findings suggest that immunity against FIP is associated with TNF-alpha and IFN-gamma response imbalance, with high TNF-alpha/low IFN-gamma mRNA responses favouring disease and low TNF-alpha/high IFN-gamma mRNA responses being indicative of immunity.