Serum alpha1-acid glycoprotein (AGP) concentration in non-symptomatic cats with feline coronavirus (FCoV) infection

Previous studies have demonstrated that the concentration of alpha1-acid glycoprotein (AGP) transiently increases in asymptomatic cats infected with feline coronavirus (FCoV). In order to establish whether these fluctuations depend on the FCoV status, the serum concentration of AGP and anti-FCoV antibody titres and/or faecal shedding of FCoVs in clinically healthy cats from catteries with different levels of prevalence of FCoV infection were monitored over time. Serum AGP concentrations fluctuated over time in clinically healthy cats from the cattery with the highest prevalence of feline infectious peritonitis (FIP) and significantly increased just before an outbreak of FIP. Further studies are required to clarify whether the observed increase of AGP concentration is a consequence of the increased viral burden or a protective response against mutated viral strains. Nevertheless, the results of the present study suggest that AGP might be useful in monitoring FCoV-host interactions in FCoV-endemic catteries.     

Total sialic acid: An acute phase reactant in cats with a possible role in feline coronavirus infection

The aims of this study were to validate a colorimetric method to measure total sialic acid (TSA) in feline serum and to investigate the serum concentration of TSA in clinically healthy cats seronegative (n = 9) and seropositive (n = 48) for feline coronavirus (FCoV), and in cats affected by feline infectious peritonitis (FIP, n = 28), tumors (n = 20), or inflammation (n = 16). The correlation between TSA and α₁-acid glycoprotein (AGP) was also investigated. The method employed in this study is precise and accurate at TSA levels (in mg/L) commonly encountered in feline serum. No significant differences between seropositive (385.6 ± 192.2 mg/L) and seronegative (433.5 ± 179.0 mg/L) cats were detectable, suggesting that the simple infection by FCoVs does not influence TSA levels. Compared with seropositive controls, the concentration of TSA was higher in cats with FIP (556.7 ± 268.3 mg/L, P = 0.003), tumors (522.5 ± 294.4 mg/L, P = 0.028), and inflammation (546.8 ± 208.3 mg/L, P = 0.018). The discriminating power of TSA for FIP is moderate (area under the ROC curve = 0.65) and the likelihood ratio is higher than 3.0 only at high TSA levels. Consequently, TSA could support a diagnosis of FIP only at extremely high serum concentration (> 800 mg/L) or when the pre-test probability of FIP is high. No correlations were found between the TSA and AGP concentrations in cats with FIP, suggesting that sialylated proteins other than AGP are present. Both the antibody titre and the degree of AGP sialylation were negatively correlated with TSA levels, suggesting that increased TSA may contribute to reduce the burden of FCoVs.     

An immunoturbidimetric assay for rapid quantitative measurement of feline alpha-1-acid glycoprotein in serum and peritoneal fluid

BACKGROUND: Alpha-1-acid glycoprotein (AGP) is an acute phase protein that increases in concentration in infectious and inflammatory conditions. The serum and peritoneal fluid concentrations of AGP may be useful in the diagnosis of feline infectious peritonitis (FIP), a lethal disease of cats. Currently AGP can be measured by radioimmunodiffusion (RID) assays, which are time consuming and difficult. OBJECTIVES: The objectives of this study were to develop a rapid immunoturbidimetric assay for measurement of AGP in feline serum and peritoneal fluid and to compare the results with those obtained by RID. METHODS: AGP was purified by perchloric acid precipitation and ion-exchange chromatography from a pool of peritoneal fluid obtained from cats with FIP, as determined by a panel of laboratory tests, including serum AGP concentration, albumin: globulin ratio, and total protein concentration, anti-coronavirus antibody titers, and effusion analysis. The purified AGP in a complete Freund's adjuvant and Tween 20 mixture was injected into a sheep and blood was collected at monthly intervals. Anti-AGP antiserum, as confirmed by ELISA and Western blot techniques, and a pool of peritoneal fluid from cats with FIP were used to prepare standards. Clinical samples of feline peritoneal fluid (n=55) and serum (n=59) were assayed for AGP and results from the immunoturbidimetric and RID methods were compared. RESULTS: Significant correlation (P < .001) was obtained between methods for both peritoneal fluid (R2=.9259) and serum (R2=.9448) samples. Coefficients of variation for the immunoturbidimetric method were <5%. CONCLUSIONS: This rapid immunoturbidimetric assay for measurement of feline AGP in serum and peritoneal fluid may be of value in the diagnosis of FIP and possibly other inflammatory diseases in cats.     

Changes in some acute phase protein and immunoglobulin concentrations in cats affected by feline infectious peritonitis or exposed to feline coronavirus infection

The possible role of some acute phase proteins (APPs) and immunoglobulins in both the pathogenesis and diagnosis of feline infectious peritonitis (FIP) has been investigated. Serum protein electrophoresis and the concentration of haptoglobin (Hp), serum amyloid A (SAA), alpha(1)-acid glycoprotein (AGP), IgG and IgM were evaluated in cats exposed to feline coronavirus (FCoV) and in cats with FIP. The highest concentration of APPs was detected in affected cats, confirming the role of these proteins in supporting a clinical diagnosis of FIP. Repeated samplings from both FIP affected and FCoV-exposed cats showed that when FIP appeared in the group, all the cats had increased APP levels. This increase persisted only in cats that developed FIP (in spite of a decrease in alpha(2)-globulins) but it was only transient in FCoV-exposed cats, in which a long lasting increase in alpha(2)-globulins was observed. These results suggest that changes in the electrophoretic motility of APPs or APPs other than Hp, SAA and AGP might be involved in the pathogenesis of FIP or in protecting cats from the disease.     

Evaluation of antibodies against feline coronavirus 7b protein for diagnosis of feline infectious peritonitis in cats

OBJECTIVE: To determine whether expression of feline coronavirus (FCoV) 7b protein, as indicated by the presence of specific serum antibodies, consistently correlated with occurrence of feline infectious peritonitis (FIP) in cats. SAMPLE POPULATION: 95 serum samples submitted for various diagnostic assays and 20 samples from specific-pathogen-free cats tested as negative control samples. PROCEDURES: The 7b gene from a virulent strain of FCoV was cloned into a protein expression vector. The resultant recombinant protein was produced and used in antibody detection assays via western blot analysis of serum samples. Results were compared with those of an immunofluorescence assay (IFA) for FCoV-specific antibody and correlated with health status. RESULTS: Healthy IFA-seronegative cats were seronegative for antibodies against the 7b protein. Some healthy cats with detectable FCoV-specific antibodies as determined via IFA were seronegative for antibodies against the 7b protein. Serum from cats with FIP had antibodies against the 7b protein, including cats with negative results via conventional IFA. However, some healthy cats, as well as cats with conditions other than FIP that were seropositive to FCoV via IFA, were also seropositive for the 7b protein. CONCLUSIONS AND CLINICAL RELEVANCE: Expression of the 7b protein, as indicated by detection of antibodies against the protein, was found in most FCoV-infected cats. Seropositivity for this protein was not specific for the FCoV virulent biotype or a diagnosis of FIP.     

Tissue distribution of a feline AGP related protein (fAGPrP) in cats with feline infectious peritonitis (FIP)

Feline alpha(1)-acid glycoprotein (fAGP) increases during feline infectious peritonitis (FIP). We have recently identified a 29 kDa protein that we named feline AGP-related protein (fAGPrP) due to its cross-reactivity with an anti-human AGP monoclonal antibody. In this work we describe the tissue distribution of fAGPrP during FIP, and its relationship with feline coronavirus (FCoV) and myeloid cells. Tissues from five control cats and from 15 cats with FIP were examined by immunohistochemistry using monoclonal antibodies against human AGP, FCoV and myeloid antigens. Diffuse fAGPrP positivity within the lesions, likely due to vascular plasma leakage, endothelial and epithelial lining were detectable. Compared to controls, fAGPrP-expressing cells often increased in number and were diffusely distributed in lymph nodes, as usually occurs for IgM-producing plasma cells during early immune responses. These findings did not depend on the presence of FCoVs or of myeloid cells, suggesting that fAGPrP is not directly involved in the pathogenesis of FIP.     

Association between faecal shedding of feline coronavirus and serum alpha1-acid glycoprotein sialylation

The sialylation pattern of serum alpha1-acid glycoprotein (AGP) in non-symptomatic cats infected by feline coronavirus (FCoV) and its possible relationship with the amount of FCoVs shed in faeces were investigated. Blood from three specific pathogen-free cats (group A) and from 10 non-symptomatic FCoV-positive cats from catteries with low (group B, three cats) or high (group C, seven cats) levels of faecal shedding were collected monthly. AGP was purified from serum and Western blotting followed by lectin-staining of alpha(2,3)-linked and alpha(2,6)-linked sialic acid. Faecal shedding was quantified in group C by quantitative polymerase chain reaction. Variations of AGP sialylation were recorded only in cats from group C, on which viral shedding peaked before the occurrence of feline infectious peritonitis (FIP) in the cattery, and decreased 1 month later, when serum AGP had an increase of alpha(2,3)-linked sialic acid. These results suggest that hypersialylation of AGP may be involved in host-virus interactions.     

Long-term impact on a closed household of pet cats of natural infection with feline coronavirus, feline leukaemia virus and feline immunodeficiency virus

A closed household of 26 cats in which feline coronavirus (FCoV), feline leukaemia virus (FeLV) and feline immunodeficiency virus (FIV) were endemic was observed for 10 years. Each cat was seropositive for FCoV on at least one occasion and the infection was maintained by reinfection. After 10 years, three of six surviving cats were still seropositive. Only one cat, which was also infected with FIV, developed feline infectious peritonitis (FIP). Rising anti-FCoV antibody titres did not indicate that the cat would develop FIP. The FeLV infection was self-limiting because all seven of the initially viraemic cats died within five years and the remainder were immune. However, FeLV had the greatest impact on mortality. Nine cats were initially FIV-positive and six more cats became infected during the course of the study, without evidence of having been bitten. The FIV infection did not adversely affect the cats' life expectancy.     

Interferon-gamma in the serum and effusions of cats with feline coronavirus infection

The aim of this study was to quantify and compare interferon-γ (IFN-γ) concentrations in the serum of clinically normal cats infected with feline coronavirus (FCoV) with its concentration in the sera and effusions of cats with feline infectious peritonitis (FIP), a disease associated with infection with a mutated form of FCoV.Clinically normal FCoV-infected cats living in catteries with a high prevalence of FIP had the highest serum IFN-γ concentrations. The serum concentration of IFN-γ was not significantly different in cats with FIP compared with clinically normal FCoV-infected animals living in catteries with a low prevalence of the disease. Moreover, the concentration of IFN-γ was significantly higher in the effusions than in the serum of cats with FIP, probably due to IFN-γ production within lesions. These findings support the hypothesis that there is a strong, ‘systemic’ cell mediated immune response in clinically normal, FCoV-infected cats and that a similar process, albeit at a tissue level, is involved in the pathogenesis of FIP.     

Shifts in circulating lymphocyte subsets in cats with feline infectious peritonitis (FIP): pathogenic role and diagnostic relevance

Cats with feline infectious peritonitis (FIP) are usually lymphopenic and have lymphoid depletion evident in spleen and lymph nodes. In particular, the number of CD4+ lymphocytes in tissues decreases during the evolution of FIP lesions. This decrease is most likely due to increased lymphocyte apoptotic rate. In contrast, cats infected with the Feline Coronavirus (FCoV) develop a follicular hyperplasia in the peripheral lymph nodes. The current study was devised to evaluate the possible pathogenic role of shifts in circulating lymphocyte subsets in FIP. Peripheral blood from cats with FIP was evaluated and compared with peripheral blood from clinically healthy cats living in both FCoV-free and FCoV-endemic catteries. Blood from cats with diseases other than FIP was also examined in order to define the diagnostic relevance of the changes. Lymphocyte subsets were analysed by flow cytometry, using a whole blood indirect immunofluorescence technique and mAbs specific for feline CD5, CD4, CD8, CD21. The results of the current study suggest that cats recently infected with FCoV that do not develop the disease have a transient increase in T cells; cats from groups with high prevalence of FIP have a moderate but persistent decrease in T cell subsets; cats with FIP have a very severe decrease in all the subsets of lymphocytes. Moreover, during FIP many lymphocytes do not express any membrane antigen, most likely due to early apoptosis. Cats with diseases other than FIP also had decreased number of lymphocytes: as a consequence, the diagnostic relevance of these findings is very low. Nevertheless, the lack of flow cytometric changes had a high negative predictive value (NPV), thus allowing to exclude FIP from the list of possible diagnoses in cats with normal cytograms.     

Prevalence of feline coronavirus types I and II in cats with histopathologically verified feline infectious peritonitis

Feline coronaviruses (FCoV) vary widely in virulence causing a spectrum of clinical manifestations reaching from subclinical course to fatal feline infectious peritonitis (FIP). Independent of virulence variations they are separated into two different types, type I, the original FCoV, and type II, which is closely related to canine coronavirus (CCV). The prevalence of FCoV types in Austrian cat populations without FIP has been surveyed recently indicating that type I infections predominate. The distribution of FCoV types in cats, which had succumbed to FIP, however, was fairly unknown. PCR assays have been developed amplifying parts of the spike protein gene. Type-specific primer pairs were designed, generating PCR products of different sizes. A total of 94 organ pools of cats with histopathologically verified FIP was tested. A clear differentiation was achieved in 74 cats, 86% of them were type I positive, 7% type II positive, and 7% were positive for both types. These findings demonstrate that in FIP cases FCoV type I predominates, too, nonetheless, in 14% of the cases FCoV type II was detected, suggesting its causative involvement in cases of FIP.     

Natural FCoV infection: cats with FIP exhibit significantly higher viral loads than healthy infected cats

Natural feline coronavirus (FCoV) infection has been shown to not only induce intestinal infection with viral shedding, but also systemic infection which either remains without clinical signs or leads to feline infectious peritonitis (FIP). As systemic infection is not the key event in the development of FIP, the question arises as to whether a potential difference in viral load might be of importance. Therefore, the purpose of this study was to quantitatively assess feline coronavirus (FCoV) RNA loads in haemolymphatic tissues of healthy, long-term FCoV-infected cats and cats with FIP. In cats that died from FIP, viral loads were significantly higher, indicating a higher rate of viral replication or a reduced capacity for viral clearance in cats developing and/or suffering from FIP.     

Production of IFN-γ in feline whole blood after incubation with potential T-cell epitopes of the nucleocapsid protein of feline coronavirus

Interferon gamma (IFN-γ) plays an important role in cell mediated responses against mutated feline coronavirus strains (FCoV) involved in the pathogenesis of feline infectious peritonitis (FIP). The aim of this study was to establish a combined in silico and in vitro approach to assess feline leukocyte production of IFN-γ in response to selected peptides of the nucleocapside protein (N) of FCoVs. To this aim, we designed, through a bioinformatic approach, 8 potentially immunogenic peptides from the protein N corresponding to sequences of residues 14, 182, 198 detected only in FCoVs from FIP cats (virulent strains), only in FCoVs from healthy cats (avirulent strains) and both in FIP and in healthy cats (mixed strains). The peptides or a sham solution were incubated with whole blood from 16 cats (7 healthy and 9 with chronic diseases other than FIP) and IFN-γ concentration was measured on plasma using an ELISA system. RT-PCR expression of IFN-γ mRNA was also evaluated after incubation of the peptides or a sham solution with whole blood from 4 clinically healthy cats. The mean plasma concentration of IFN-γ in samples incubated with peptides decreased and the expression of IFN-γmRNA did not change compared with the sham solution, except for some cats with chronic diseases (which probably have a "pre-activated" immune response). These cats responded to "avirulent" or "mixed" peptides by increasing the concentration of IFN-γ and the expression of IFN-γ mRNA. The combined approach employed in this study allowed us to identify potentially immunogenic peptides of FCoV N protein that can modulate the production of IFN-γ especially in cats with a "pre-activated" cell mediated response.     

The feline acute phase reaction

The acute phase reaction (APR) is a response to potentially pathogenic stimuli. It begins with the release of interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF)-alpha from inflammatory cells. These cytokines induce fever, leucocytosis and release of serum acute phase proteins (APPs). In this review, the characteristics of the feline APR are described. In cats with inflammatory conditions, fever is a common finding, with leucocytosis due to the release of cells from the marginal pool, followed by activation of myelopoiesis. Because excitement frequently causes leucocytosis in cats, a diagnosis of inflammation should therefore be supported by additional findings such as the presence of toxic neutrophils. The major APPs are serum amyloid A and alpha(1)-acid glycoprotein (AGP), which both increase a few hours after the inflammatory stimulus and remain elevated for as long as the inflammation persists. AGP plays an important role in the diagnosis of feline infectious peritonitis (FIP) and may also be useful also in studies of FIP pathogenesis.     

Feline leucocyte antigen class II polymorphism and susceptibility to feline infectious peritonitis

There are four outcomes to feline coronavirus (FCoV) infection: the development of feline infectious peritonitis (FIP, which is immune-mediated), subclinical infection, development of healthy lifelong carriers and a small minority of cats who resist infection (Addie and Jarrett, Veterinary Record 148 (2001) 649). Examination of the FCoV genome has shown that the same strain of virus can produce different clinical manifestations, suggesting that host genetic factors may also play a role in the outcome of infection. FIP is most prevalent amongst pedigree cats, although how much of this is due to them living in large groups (leading to higher virus challenge and stress which predisposes to FIP) and how much is due to genetic susceptibility is not known. If host genetics could be shown to play a role in disease, it may allow the detection of cats with a susceptibility to FIP and the development of increased population resistance through selective breeding. The feline leucocyte antigen (FLA) complex contains many genes that are central to the control of the immune response. In this preliminary study, we used clonal sequence analysis or reference strand conformational analysis (RSCA) to analyse the class II FLA-DRB of 25 cats for which the outcome of FCoV exposure was known. Individual cats were shown to have between two and six FLA-DRB alleles. There was no statistically significant association between the number of alleles and the outcome of FCoV infection. No particular allele appeared to be associated with either the development of FIP, resistance to FCoV, or the carrier status. However, the analysis was complicated by apparent breed variation in FLA-DRB and the small number of individuals in this study.