Feline Infectious Peritonitis
Updated March, 2021
The Feline Infectious Peritonitis guidelines were first published by D. Addie et al. in the Journal of Feline Medicine and Surgery (2009) 11, 594-604; the present update was drafted by Séverine Tasker et al.
- Feline coronavirus (FCoV) is a ubiquitous virus of domestic and large cats.
- Most FCoV-infected cats either stay healthy or show only mild enteritis.
- Only a small proportion of FCoV-infected cats goes on to develop feline infectious peritonitis (FIP).
- FCoV transmission is faecal-oral via litter trays and fomites.
- FCoV infection of monocytes is the key event in FIP pathogenesis.
- Most likely, internal mutations of FCoV (to mutants with a switch of cell tropism arising in an individual cat) are the reason for the development of highly pathogenic FIP-inducing FCoV (internal mutation theory).
- Coronaviral genomes possess a high level of genetic variation due to the error rate of RNA polymerase leading to different types of mutations.
- FIP disproportionately affects pedigree cats under two years old.
- Sampling the effusion, when present, is the most useful diagnostic step.
- The definitive diagnosis of FIP relies on consistent histopathological changes in affected tissues and this, together with FCoV antigen immunostaining, is considered the gold standard for diagnosis.
- Faecal RT-PCR is not useful for diagnosis of FIP but for identification of FCoV shedders within a cat colony.
- A positive FCoV antibody test is not confirmatory of FIP (it is not a „FIP-test”) but absence of FCoV antibodies makes FIP less likely.
- Without treatment with new potentially curative anti-coronaviral drugs (not yet widely available), FIP has a very poor prognosis.
- The ABCD considers the FIP vaccine to be non-core and it is not recommended in FCoV antibody-positive cats. However, FCoV antibody-negative kittens could potentially benefit from vaccination.
Some key aspects of virus properties are shown in Figs 1 and 2. Feline Coronavirus (FCoV) is a large, spherical, enveloped virus particle and is classified in the order Nidovirales; family Coronaviridae; genus Alphacoronavirus; species Alphacoronavirus 1, which also includes canine coronavirus (CCoV), transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCoV) (De Groot et al., 2012). The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is very distinct and different to FCoV, belonging to a different genus; SARS-CoV-2 is a member of the genus Betacoronavirus (Haake et al., 2020) and separate guidelines on SARS-CoV-2 in cats have been published (Hosie et al., 2021) together with updated information available in the ABCD Guideline on “SARS-CoV-2 and cats” available here.
Virus genome and structure
Being an enveloped virus, FCoV is readily inactivated by most disinfectants, steam and washing at 60°C. It has been suggested it preserves its infectivity for days to a few weeks, depending on environmental conditions (Scott, 1988).
The 5’ two-thirds of the positive sense coronavirus (CoV) genome consist of two overlapping open reading frames (ORFs 1a and 1b) that encode non-structural polyprotein (pp) 1 (pp1a and pp1b) (Fig. 1). The polyproteins are cleaved into individual non-structural proteins (nsps), including RNA-dependent RNA polymerase, that plays a role in viral replication. ORF 1a also encodes for viral proteases including the viral 3C-like protease being a potential target for antiviral therapy (see treatment section). The other third of the genome consists of ORFs encoding structural proteins, spike [S], matrix [M], nucleocapsid [N] and envelope [E] (Fig. 2) and some non-structural accessory proteins 3a, 3b, 3c, 7a and 7b (Fig. 1) (Terada et al., 2014). The non-structural proteins are involved in the replication of the virus, and modification of the host immune response, but are not incorporated into the mature virus particle.
Type II FCoV strains arise from recombination with CCoV (Fig. 3), usually including the spike of CCoV and varying amounts of adjacent ORF 3 genes (Herrewegh et al., 1998; Le Poder et al., 2013; Terada et al., 2014). The RNA-dependent RNA polymerase makes a full-length negative-strand RNA copy of the genome as well as a nested set of smaller subgenomic RNAs (RNAs) with a common 3’ end. These negative strand RNAs serve as templates for new positive sense genomes and positive sense subgenomic mRNAs. The subgenomic mRNAs have a nested-set structure with sequences starting at the 3’ terminus and extending to various distances toward the 5’ end. If a real-time reverse-transcriptase (RT)-PCR assay is designed to amplify 3’ subgenomic mRNAs, this can influence the quantitative results for apparent FCoV load (see reverse-transcriptase (RT)-PCR section in diagnosis). In general, only the 5’ most ORF of each subgenomic mRNA is used for encoding of the proteins even though the subgenomic mRNAs have more than one coding sequence (except of the smallest one).
FCoV genome mutations
Coronaviral genomes possess a high level of genetic variation due to the error rate of RNA polymerase leading to different types of mutations including insertions, deletions, and introduction of stop codons as well as recombinations. The hypothesis is that genetic variation and subsequent selection also facilitates switching of cell tropism within an FCoV-infected cat that develops feline infectious peritonitis (FIP).
FCoVs are assigned to two pathotypes or biotypes generally referred to as feline enteric coronavirus (FECV), which mainly replicates in the enteric epithelium, and feline infectious peritonitis virus (FIPV), which results in a mostly lethal infection with efficient replication in monocytes or macrophages (Barker and Tasker, 2020b). Since it is known that all FCoV can be found and replicate systemically (Meli et al., 2004; Kipar et al., 2006; Fish et al., 2018) (even in cats without FIP), we prefer to call both biotypes “FCoV” but distinguish them as a “less virulent FCoV” and an “FIP-associated FCoV”. Therefore, these terms will be used when possible in this guideline to underline the real differences in biological behaviour between the FCoV.
Although the genes involved in the FCoV virulence shift are still unknown, mutations in different genes have been postulated to be associated with the switch of the less virulent FCoV into the virulent FIP-associated FCoV, including the spike gene and accessory genes 3c and 7b (Pedersen et al., 2012) (see Fig. 2).The spike proteins are the main determinant of entry into host cells (Belouzard et al., 2012), as they possess both receptor binding and fusion functions (Millet and Whittaker, 2015). Two alternative amino acid differences in the putative fusion peptide of the S protein (called M1058L and S1060A – nomenclature based on position and nature of amino acid change i.e. methionine to leucine at position 1058 and serine to alanine at position 1060) have been detected that together distinguished FIP-associated from less virulent FCoV in 95.8% of cases in one study (Chang et al., 2012) and 93% of cases in another (Decaro et al., 2021).
Another mutation was detected in the cleavage site between the receptor binding (S1) and the fusion domain (S2) of the spike protein. While all less virulent FCoV had a conserved furin cleavage site, in most FIP-associated FCoV at least one substitution was found (Licitra et al., 2013). Mutations in the heptad repeat 1 region of the S gene are also said to be associated with FIP (Bank-Wolf et al., 2014; Lewis et al., 2015).
The ORF 3 gene encodes for a protein for which the function is still unknown. Interestingly, mutations leading to a truncated protein were detected in approximately two-thirds of the 3c genes of FCoV found in cats with FIP (Pedersen et al., 2009; Chang et al., 2010; Hsieh et al., 2013), while the ORF 3 gene was intact in all FCoV in faecal samples. This suggests that an intact 3c is an absolute requirement for infection of the gut epithelial cells (Chang et al., 2010; Pedersen at al., 2012), but is not necessary for replication in monocytes. FIP-associated FCoV with an intact 3c will replicate in the gut but this virus does not seem to be transmitted to other cats (Pedersen et al., 2012).
There is a general consensus that the less virulent FCoV converts to the FIP-associated FCoV in the individual cat by modifications that include a cell tropism change from enterocytes to monocytes/macrophages (Pedersen et al., 2009; Chang et al., 2012; Barker et al., 2013). This so-called internal mutation theory is supported by several studies showing a close genetic relationship between the FIP-associated FCoV and FCoV from faecal samples of cats living in the same environment (Decaro et al., 2021), representing a much closer relationship than to FCoV collected from cats of other environments. The internal mutation theory was questioned at one point based on the results of a single study that indicated that “FECV” and “FIPV” were two distinct types of FCoV circulating independently in the population (Brown et al., 2009). However, in that study, samples were derived from a population of shelter cats, a population in which introduction of different genetically unrelated FCoV can be expected because of their different geographic origin (Pedersen, 2014). The internal mutation theory has now been widely accepted amongst researchers.
In addition to distinguishing the two pathotypes, the less virulent FCoV and the FIP-associated FCoV, there is another classification based on differences in antigenic and genomic properties in type I and type II FCoV. Both, type I and type II FCoV can occur as less virulent FCoV and as FIP-associated FCoV (Pedersen, 2014). Type I FCoV is most prevalent worldwide (Addie et al., 2003; Lin et al., 2009; Soma et al., 2013; Terada et al., 2014; Wang et al., 2014; Decaro et al., 2021). Type II FCoV results from double recombination between type I FCoV and CCoV (Herrewegh et al., 1998; Terada et al., 2014) within the feline cell, which permits entry of both FCoV and CCoV (Tusell et al., 2007; Terada et al., 2014). Recombination events have occurred many times across the world, with individual type II FCoV containing CCoV spike genes and variable amounts of 3abc, and envelope genes, but not the nucleocapsid gene, which remains of FCoV origin (Herrewegh et al., 1998; Terada et al., 2014) (see Fig. 3). Most research has focused on type II FCoV strains since they, unlike type I FCoV, can be readily propagated in vitro (Pedersen et al., 1984), facilitating experimental studies, despite most field infections being type I FCoV. Experimental studies have tried to develop culture methods for type 1 FCoVs using both permanent feline intestinal epithelial cell cultures of ileocyte and colonocyte origin (Desmarets et al., 2013) and colonic organoid preparations (Tekes et al., 2020), but neither are currently routinely available for use.
Transmission of FCoV
FCoV is a contagious virus, and transmission is often indirect, such as contact with objects (e.g. via litter trays, scoops, brushes, vacuum cleaners, shoes, hands and clothes), handling at cat shows, shelters or in a veterinary practice. Faeces are the main source of FCoV, with litter boxes representing the principal source of infection in groups of cats. Cats are most likely to be infected orally following contact with FCoV in faeces. Thus, the major route of transmission is faecal-oral.
A case report (Andre et al., 2020), documenting FIP-associated rhinitis, suggested that the respiratory tract might be a place of entry for transmission for FCoV, but further studies are required. Since virus is found only rarely in the saliva of healthy cats, close contact or sharing feeding bowls are not major routes of infection (Addie and Jarrett, 2001). Transplacental transmission has been described from a queen that developed FIP during pregnancy (Pastoret and Henroteaux, 1978), but this phenomenon is extremely rare (Addie and Jarrett, 1990). A study (Stranieri et al., 2020a) evaluated testicular tissue and semen in tom cats for FCoV by RT-PCR to evaluate the risk of venereal transmission of FCoV. FCoV RNA was amplified from around 15% (6 of 39) of testicles in the study and none of the 17 semen samples tested, suggesting venereal transmission was unlikely. Transmission of FCoV via blood transfusion has not been reported.
In FCoV infected breeding catteries, kittens commonly become infected at a young age within a few weeks of age (Lutz et al., 2002) (see also “Pathogenesis and Immunity”).
After natural infection, cats begin to shed virus in the faeces within one week (Meli et al., 2004) and continue to shed for weeks, months, and a few even for life (persistent) (Addie and Jarrett, 2001; Addie et al., 2003; Pedersen et al., 2008). Shedding is usually intermittent and recurrent (although a few cats appear to recover and no longer shed after an initial period of shedding) but some cats have persistent shedding (Addie et al., 2003; Pedersen et al., 2008), which may be influenced by the dose of virus received at inoculation (Vogel et al., 2010). Faecal excretion reaches high levels (Addie and Jarrett, 2001; Addie et al., 2003; Pedersen et al., 2008; Vogel et al., 2010). The higher the FCoV antibody titre, the greater is the chance of the cat shedding FCoV (Addie and Jarrett, 2001; Lutz et al., 2002; Pedersen et al., 2008; Addie et al., 2015; Felten et al., 2020), as well as the greater the frequency of faecal FCoV shedding and FCoV load present (Felten et al., 2020). Due to the suspected short duration of any immunity following infection, failure to separate non-shedding cats from virus shedders could favour spread and persistence of FCoV in a household, which could account for the high antibody prevalence in the multi-cat environment.
Horizontal transmission of FIP, in contrast to FCoV, is not believed to occur very frequently, if at all (see Pathogenesis and Immunity, and Disease Management sections).
Although FCoV and CCoV are closely related, contact with dogs does not appear to be a major predisposing factor for CoV infection of cats (Le Poder et al., 2013). However, Benetka et al. (2006) found feline/canine CoV recombinant viruses in cats of a rescue shelter which housed both cats and dogs. In the M protein gene these strains were more closely related to FCoV-like CCoV than to FCoV, suggesting that infection with CCoV and subsequent recombinations with FCoV had occurred within this environment.
Prevalence of FCoV
With the exception of a few islands of isolated feline populations (e.g. the Falkland Islands) (Horzinek and Osterhaus, 1979; Levy et al., 2008; Addie et al., 2012), FCoV infection has been reported worldwide. FCoV, and therefore FIP, is particularly common where conditions are crowded (Sharif et al., 2009; Felten et al., 2020), while the prevalence is lower in individually housed, stray or feral cats (Addie and Jarrett, 1992; Herrewegh et al., 1995; Addie, 2000; Bell et al., 2006; Cave et al., 2004, Taharaguchi et al., 2012). Wild felids, especially those in zoos, can also be FCoV-infected (Kennedy et al., 2002). FCoV-infected cheetahs are even predisposed to develop FIP (Evermann et al., 1988).
FCoV is highly contagious, and in households where it is present, prevalence of antibodies indicating exposure is often close to 100% (Felten et al., 2020). Cats who spent over 60 days in UK shelters were five times more likely to have antibodies (Cave et al., 2004).
In a Japanese study including 17,392 cats, the antibody prevalence was 66.7% in purebred cats, and 31.2% in domestic cats (Taharaguchi et al., 2012). Prevalence increased greatly in purebreds by three months of age, while it did not fluctuate greatly in non-pedigree breeds with aging, indicating that cattery environments can contribute to FCoV epidemics. Purebred cats from northern regions of Japan were commonly antibody-positive (76.6% in Hokkaido, 80.0% in Tohoku), indicating that cattery cats in cold climates might be more closely confined. Among purebred cats in Japan, the breeds American shorthair, Himalayan, Oriental, Persian, and Siamese showed low antibody prevalence, while the breeds American curl, Maine coon, Norwegian forest cat, Ragdoll and Scottish fold had high antibody prevalence (Taharaguchi et al., 2012).
The prevalence of FCoV in various countries is given in the table below.
|Country||What detected||Number of cats||Prevalence||Reference|
|Australia (Sydney)||Antibodies||49 feral cats|
306 owned cats
|Bell et al., 2006|
|Austria||Antibodies||157 cats without FIP:|
17 single cats
31 multi-cat household
99 shelter cats
10 feral cats
|Posch et al., 2001|
|Falkland Islands||Antibodies||10 feral cats|
95 pet cats
|Addie et al., 2012|
FCoV RNA in faeces
|82 cats from 19 breeding catteries||78%|
|Felten et al., 2020|
|Japan||Antibodies||17,392||66.7 % in purebred cats|
31.2 % in domestic cats
|Taharaguchi et al., 2012|
107 pet cats
105 shelter cats
129 healthy cats
83 sick cats
|An et al., 2011|
|Korea||FCoV RNA in faeces||212||6.6 %||An et al., 2011|
|Malaysia||FCoV RNA in faeces||24 cats in a Persian cattery|
20 cats in a rescue cattery
|Sharif et al., 2009|
|Sweden||Antibodies||209||17 % domestic|
65 % pedigree
|Ström Holst et al., 2006|
|Switzerland||Antibodies||466 DHS and DLH cats|
143 purebred cats
|49 % |
|Fehr et al., 1997|
|Taiwan||Antibodies||760 healthy cats||28.2 %||Wang et al., 2014|
|Turkey (Bursa province)||Antibodies||100||21 %||Pratelli et al., 2009|
|Turkey (Istanbul)||Antibodies||169||31 %||Tekelioglu et al., 2015|
2207 shelter cats
|Cave et al., 2004|
Prevalence and risk factors for FIP
The prevalence of FIP within a cat population as a whole was 0.52% (60 of 11,535) of all the cats examined at the North Carolina State University College of Veterinary Medicine (1986 – 2002), which is high considering the fact that US university teaching hospitals are tertiary referral centres (Pesteanu-Somogyi et al., 2006). In multi-cat environments, such as at breeders and shelters, up to 12% of FCoV-infected cats can succumb to FIP (Addie et al., 1995). The incidence of FIP in a household or cattery increases with the number of cats (Kass and Dent, 1995).
While cats of any age or breed can develop FIP, FIP disproportionately affects pedigree cats under two years old (Rohrbach et al., 2001; Norris et al., 2005; Pesteanu-Somogyi et al., 2006; Tsai et al., 2011; Worthing et al., 2012; Soma et al., 2013; Riemer et al., 2016). In Australia, 71% of cats with FIP were purebred and 55% less than two years old (Norris et al., 2005). In a North Carolina study, 67% of cats with FIP were less than two years, and pedigree cats were also over-represented: FIP was present in nearly 1.3% of the pedigree cats compared to 0.35% in mixed breed cats, and breed predisposition was statistically significant in the Abyssinian Bengal, Birman, Himalayan, Ragdoll and Rex breeds (Pesteanu-Somogyi et al., 2006). In a study in Australia, domestic crossbred, Persian and Himalayan cats were significantly under-represented in the FIP cohort, while several other breeds were over-represented, including British Shorthair, Devon Rex and Abyssinian (Worthing et al., 2012).
Percentage of effusions that were positive by FCoV reverse-transcriptase (RT)-PCR varied with the cat’s breed and age in a study in Japan (Soma et al., 2013). In this study RT-PCR was used on effusions to indicate FIP, although the diagnosis was not confirmed. In cats up to one year, 95% of effusions of pedigree cats were RT-PCR positive, whereas FCoV RNA was only found in 79% of the effusions of domestic cats, and up to the age of five years, effusions of purebred cats were more likely to be FCoV RT-PCR-positive than were those from domestic cats.
Some authors have noted a predisposition for FIP in male over female cats (Rohrbach et al., 2001; Benetka et al., 2004; Norris et al., 2005; Worthing et al., 2012; Riemer et al., 2016), while others found no sex predisposition (Pedersen, 1976). Pedigrees of cats that die of FIP can often be traced back to the stud cat, rather than the queen (Foley and Pedersen, 1996).
As noted above, the major route of FCoV infection is faecal-oral. Following ingestion of the virus, such as by grooming paws contaminated during litter tray use or from eating fomite-contaminated food, the virus first enters and replicates within the epithelial cells of the small intestinal villi. Type II FCoV uses the feline aminopeptidase-N receptor (fAPN) present on the intestinal villi and the monocyte (Tusell et al., 2007; Tekes et al., 2010). The receptor for type I FCoV remains unknown (Dye et al., 2007; Tekes et al., 2010).
FCoV shedding occurs in the faeces from two to three days post-infection (Meli et al., 2004; Kipar et al., 2010). This infection is not usually associated with clinical signs, but sometimes is accompanied by enteritis (Sabshin et al., 2012). Occasionally, very severe, indeed fatal, coronavirus enteritis has been reported (Kipar et al., 1998). As described earlier, virus shedding of type I FCoV in faeces follows two patterns. (1) Most transiently infected cats shed virus for two to three months (Addie and Jarrett, 2001) - immunity is short-lived because these cats can be re-infected by the same or a different strain of FCoV, within a few weeks (Addie et al., 2003), showing recurrent infections. (2) Around 13% of cats infected with type I FCoV become persistently infected carrier cats. However, in contrast, cats experimentally infected with type II FCoV shed virus for around two weeks (Stoddart et al., 1988) and no carrier cat has been reported. Only a small proportion of FCoV-infected cats goes on to develop FIP (Pedersen, 1987; Kipar et al., 2005).
From two weeks post infection, the virus is found in the colon (Kipar et al., 2010). In persistently infected asymptomatic carrier cats, the ileo-caecocolic junction is the main site of viral replication (Herrewegh et al., 1997).
The mesenteric lymph nodes (MLNs), as the most likely first site of FCoV spread from the intestine regardless of subsequent viraemia, have been evaluated for mediators of the innate immune response, and evidence for toll-like receptor involvement has been found in the response to FCoV infection (Malbon et al., 2019).
Efficient FCoV replication in activated monocytes and macrophages is a key event in FIP pathogenesis (Malbon et al., 2020a): whether or not the cat will go on to mount a successful immune response and eliminate the virus; whether the cat will mount a semi-successful immune response, remaining clinically well, but shedding FCoV in the faeces for months to years; or whether the cat will mount a deleterious immune response, resulting in a widespread pyogranulomatous vasculitis and ultimately premature death without effective antiviral treatment. The outcome of infection of the monocytes and macrophages is partially dependent on the cell; however, virulent strains do replicate more efficiently within permissive monocytes and macrophages (Dewerchin et al., 2005). Monocytes from an outbred population of cats varied in their ability to sustain FCoV replication regardless of whether the strain of FCoV was deemed very virulent or relatively avirulent, with the monocytes of some cats not sustaining replication of either FIP-associated FCoV or less virulent non-FIP-associated FCoV (Dewerchin et al., 2005). What happens in monocytes and macrophages following FCoV infection is quite extraordinary: usually an infected cell will display viral antigens in association with feline leucocyte antigen (the feline version of the major histocompatibility complex) on its surface to enable antibody-mediated, or cell-mediated, lysis; but in cats with FIP, infected macrophages lacked surface expression of viral antigens (Cornelissen et al., 2007).
FCoV viraemia, when it occurs, is short-lived, peaking about seven to 14 days post-infection and declining thereafter (Kipar et al., 2010; Mustaffa-Kamal et al., 2019), thus by the time clinical signs of FIP appear, viraemia will likely usually be over, so that performing RT-PCR on blood samples to detect FCoV RNA is often negative in cats with FIP.
The virulence of the virus, the viral load and the cat’s immune response determine whether or not FIP will develop. Thus, both viral genetics and host immunity are likely to play a role in the development of FIP (Addie and Jarrett, 1995; Dewerchin et al., 2005; Rottier et al., 2005; Hsieh and Chueh, 2014; Pedersen et al., 2014; Mustaffa-Kamal et al., 2019; Malbon et al., 2020b). Resistance in terms of the ability to fight off FCoV infection increases between six and 12 months of age (Pedersen et al., 2014).
In those cats in which FCoV is able to replicate freely within the monocytes, infected monocytes attach to the walls of small and medium sized veins, releasing matrix metalloproteinase-9 (MMP-9) which destroys the collagen of the basal lamina of affected vessels. This event permits extravasation of the monocytes, where they differentiate into macrophages, and allows plasma to leak out of the vessels (Kipar et al., 2005). It is believed that death of virus-laden macrophages (apoptosis) plays a key role in FCoV dissemination (Watanabe et al., 2018). In more acute forms of FIP, many blood vessels are affected, and this leakage becomes apparent clinically as an effusion. In more chronic forms of FIP, fewer blood vessels are affected, but the perivascular pyogranulomata can become quite large, even easy to mistake for a tumour on gross examination, at exploratory laparotomy or necropsy. The FCoV-infected macrophages release cytokines such as tumour necrosis factor alpha (TNF-alpha) (Takano et al., 2007a): TNF-alpha upregulates fAPN (Takano et al., 2007a), causes lymphopenia (Takano et al., 2007b) and inhibits neutrophil apoptosis (Takano et al., 2009). The role of TNF-alpha is important in the development of FIP, such that anti-TNF-alpha antibodies have been used as a possible therapy (Doki et al., 2013; Doki et al., 2020b).
As described above, FIP arises in a small percentage of FCoV-infected cats in vivo, following FCoV infection, and horizontal transmission of FIP, via a FIP-associated FCoV strain, is not believed to occur very frequently, if at all. Several experimental and field observations support the assumption that cats do not become infected with FIP-associated FCoV via the natural route. First, FIP-associated FCoV strains from different cats of the same household show mostly unique genetic characteristics suggesting that these viruses developed independently in individual cats (Chang et al., 2012; Licitra et al., 2013, Barker et al., 2013). Only a very small percentage of cats with FIP may shed FIP-associated FCoV, most likely because these mutated viruses cannot replicate in enterocytes (Pedersen et al., 2009; Pedersen et al., 2012; Wang et al., 2013; Porter et al., 2014). Furthermore, faecal samples of cats with FIP do not cause disease after oral inoculation (Pedersen et al., 2012). Also, in multi-cat households, FIP cases are often limited to a single (or few) cat and additional cases might not occur for several years. However, a few cases have been reported in which a higher number of cats (>10%) developed FIP in multi-cat environments (Graham et al., 2012; Wang et al., 2013; Barker et al., 2013). In these so-called mini-outbreaks other factors than direct horizontal transmission likely play a role (see chapter “Control of FCoV and FIP in specific situations”)
FIP is associated with severe suppression of natural killer cells and regulatory T cells, central players in the innate and adaptive cell-mediated immunity (CMI), respectively (Vermeulen et al., 2013). Until the study on FCoV replication in monocytes by Dewerchin et al. (2005), the outcome of FCoV infection had been mainly attributed to virulence factors (mutations, deletions) in the virus (Pedersen, 2014), although host factors obviously play a role in pathogenesis. One of the most investigated cytokines important in FCoV infection has been interferon gamma (IFN-γ), which is an important modulator of CMI. The expression of IFN-γ mRNA by leucocytes in the circulation or in tissues has been investigated in many studies using RT-PCR and immunohistochemistry (Gunn-Moore et al., 1998; Dean et al., 2003; Kiss et al., 2004; Berg et al., 2005; Gelain et al., 2006). Some studies (Gunn-Moore et al., 1998; Kiss et al., 2004; Gelain et al., 2006) found high IFN-γ mRNA expression in the peripheral blood leucocytes of clinically normal cats with FCoV infection, but low expression in cats with FIP. In contrast, IFN-γ mRNA is abundant within FIP lesions (Berg et al., 2005). Giordano and Paltrinieri (2009) concluded in their paper that although cats resistant to FIP have strong CMI, which can be measured by high serum IFN-γ production, CMI is also likely to be involved in the pathogenesis of FIP, albeit at a tissue level, as evidenced by high IFN-γ concentration of the FIP effusions. These findings could be the basis of further studies into the mechanisms through which IFN-γ production could prevent the onset of FIP. The importance of CMI in the resistance to FIP was further investigated in an experimental study (Mustaffa-Kamal et al., 2019) in which the antiviral T cell responses were measured during primary and secondary exposure to FIP-associated FCoV. Definitive adaptive T cell responses predictive of disease outcome were not detected during the early phase of primary infection with FIP-associated FCoV but recovery antiviral T cell responses were seen later in primary infection for a subset of cats showing slow progression to FIP or resistance to FIP compared to those showing fast progression to FIP. The emergence of antiviral T cell responses after secondary exposure (rechallenge) to FIP-associated FCoV in cats that were resistant to FIP after primary infection also suggested a role of CMI in the later control of infection with FIP-associated FCoV and disease progression.
Hsieh and Chueh (2014) investigated whether single nucleotide polymorphisms (SNP) in the feline IFN-γ gene (fIFNG) were associated with the outcome of FCoV infection. Some “FIP-resistant” and “FIP-susceptible” alleles have been suggested, and a subsequent study found an increased frequency of documented fIFNG SNPs in pedigree cats, but small numbers limited statistical analysis (Kedward-Dixon et al., 2020). A larger study (Barker et al., 2020) published on the prevalence of fIFNG SNPs in non-pedigree cats did find a statistical association between presence or absence of FIP and genotype; however the presence of the ‘protective’ genotype in 16% of the cats with FIP and its absence in 66% of the cats without FIP limits its use in individual cats or to guide breeding.
The role of humoral immunity in protecting against FIP is ambiguous. Maternally derived antibodies have been suggested to provide protection until about five to six weeks of age (Addie and Jarrett, 1992) until they decline and become undetectable by six to eight weeks of age. However, infection at two weeks of age has also been detected (Lutz et al., 2002), questioning protection by maternally derived antibodies. On the other hand, cats with active enteric FCoV infections have strong systemic IgG and mucosal secretory IgA responses that wane after FCoV clearance, with no evidence of a mucosal IFN-γ T cell response, suggesting that humoral responses can control infection (Pearson et al., 2019).
Antibody production to FCoV takes 10-28 days post-infection (Meli et al., 2004; Vogel et al., 2010). Clearance of natural infections has been associated with antibodies directed against the FCoV S protein (Gonon et al., 1999). Conversely, in experimental infections, antibodies directed against the S protein can be detrimental (Vennema et al., 1990). In cats with pre-existing antibodies, ‘antibody dependent enhancement’ (ADE) has been observed experimentally, resulting in a more rapid disease course and earlier death. This enhancement was observed irrespective of whether cats had acquired antibodies through passive or active immunization using some experimental vaccine studies (Weiss and Scott, 1981; Vennema et al., 1990; De Groot and Horzinek, 1995). However, in field studies cats developed FIP on first exposure to FCoV (and thus, had not have pre-existing antibodies) and cats experienced repeated infections by FCoV without developing FIP, leading to the conclusion that ADE is a laboratory phenomenon which is not important in the real world (Addie et al., 1995, 2003). Additionally, an experimental study (Mustaffa-Kamal et al., 2019) documented that nine of ten cats that had not developed FIP following primary infection with a FIP-associated FCoV strain resisted development of disease following rechallenge.
Clinical signs associated with FCoV infection
FCoV does not usually cause any clinical signs in cats following infection. Occasionally it is accompanied by enteritis (Sabshin et al., 2012) with clinical signs of diarrhoea and/or vomiting. Occasionally, very severe, indeed fatal, coronavirus enteritis has been reported (Kipar et al., 1998).
Clinical signs associated with FIP
The clinical picture of FIP varies considerably, reflecting the variability in the distribution of the vasculitis and granulomatous lesions. The vasculopathy can result in (‘wet’) effusions whilst the granuloma formation results in (‘dry’) mass lesions. A form which includes the development of effusions is regarded as being most common (Sparkes et al., 1991; Tsai et al., 2011; Riemer et al., 2016): 78% of 224 cases of FIP had effusions (Riemer et al., 2016). The distinction between so-called ‘effusive’ and ‘non-effusive’ forms of FIP is important for diagnostic purposes; however, there is considerable overlap between the two forms, and indeed FIP cases with effusions also have pyogranulomatous lesions visible at post-mortem examination and, similarly, many cats with a non-effusive form will eventually develop effusions. Clinical signs of FIP can change over time, therefore repeated physical examinations are important to detect newly apparent clinical signs; for example, an effusion can develop or ocular changes can become visible on ophthalmoscopic examination.
Non-specific clinical signs can occur in both cats with effusion or without effusion and include lethargy, anorexia and weight loss (or failure to gain weight/stunted growth in kittens), although occasionally some cats remain bright and retain good body condition. A fever, that can be fluctuating and that is moderate and typically <40 °C (but sometimes can be higher) and that is refractory to many drugs and non-responsive to antibiotics, is commonly present. One study describing referral cats with a history of fever found that FIP was the most common diagnosis made, highlighting its importance as a differential diagnosis for fever even at referral level (Spencer et al., 2017). Another study (Riemer et al., 2016), which described the clinical features of FIP, documented fever in 55.8% of FIP cases. Fever was shown to be more common in cats with effusion than in cats without effusion (Riemer et al., 2016).
FIP can be associated with effusion formation in one or more body cavities. Abdominal effusions leading to a clinical presentation of ascites, sometimes with abdominal distension, are the most common effusions seen with FIP (Fig. 4; Riemer et al., 2016).
A pleural effusion can be present concurrently to abdominal effusion. In some cats, the effusion is restricted to the thorax; cats with pleural effusion can present with dyspnoea (Pedersen, 2009; Beatty and Barrs, 2010; Riemer et al., 2016). In a retrospective study including 306 cats diagnosed with pleural effusion of established aetiology, FIP was only diagnosed in 8.5% of cats, while cardiac disease was the most common aetiology (35.3%), followed by neoplasia (30.7%), pyothorax (8.8%) and chylothorax (4.6%). Cats with FIP were significantly younger than those with cardiac disease and neoplasia, and cats with cardiac disease had a significantly lower body temperature, higher serum alanine aminotransferase and alkaline phosphatase activity, and lower protein concentrations and nucleated cell counts in the effusion than cats with FIP (Konig et al., 2019). Pericardial effusions (Fischer et al., 2012b; Baek et al., 2017), with or without effusions in other body cavities, are also occasionally reported. Rarely, effusion in the scrotum is present in intact male cats due to a serositis involving the tunica vaginalis of the testes, leading to scrotal enlargement. When effusions form in FIP, the disease progression is often quite acute in nature, progressing within a few days or weeks and severely limiting survival (Ritz et al., 2007).
When effusions are not present in FIP cases, FIP is often more difficult to diagnose, as fever, anorexia, lethargy and weight loss (or failure to gain weight in kittens) can be the only signs, particularly in the early stages of disease. It also tends to be more chronic than FIP associated with effusions, progressing over a few weeks to months. Additional signs of non-effusive FIP depend on the organs affected by the granulomatous lesions and can include the central nervous system (CNS), eyes and/or abdominal organs (such as the liver, abdominal lymph nodes, kidney, pancreas, spleen and/or gastrointestinal tract) (Norris et al., 2005), but such signs can also be seen in cats with effusions, so they are not restricted to non-effusive FIP. As well as granulomatous lesions, the kidneys can sometimes show immune-mediated glomerulonephritis when affected by FIP; this is caused by immune complexes through the excessive production of non-neutralizing antibodies in FIP (Hartmann et al., 2020).
Renomegaly, but also occasionally a reduction in kidney size, can occur. A diffuse pyogranulomatous pneumonia is occasionally seen (Trulove et al., 1992) causing respiratory signs. Abdominal lymphadenomegaly can be present. Jaundice can occur (Fig. 5), more commonly in cats with effusions, but the degree of hyperbilirubinaemia is often not high enough to result in clinical jaundice (Pedersen, 2009; Riemer et al., 2016).
Hyperbilirubinaemia can occur because of liver involvement, but also can be a result of immune-mediated haemolytic anaemia, which can occur terminally in cats with FIP. Mild hyperbilirubinaemia can be the result of interference with bilirubin transporters caused by high TNF-alpha levels leading to reduced bilirubin transport into and out of liver cells. Rising bilirubin and falling red blood cell counts presaged imminent death in one study (Tsai et al., 2011).
FIP can also manifest in the intestinal tract and/or regional lymph nodes (sometimes called “focal FIP”) presenting typically as a palpable abdominal mass due to primary involvement of the mesenteric lymph nodes and/or intestinal tract. It can be particularly challenging to diagnose as the lesions can be hard to initially differentiate from neoplasia (Kipar et al., 1999), toxoplasmosis (Cohen et al., 2016) or mycobacterial infection (O'Halloran and Gunn-Moore, 2017). FIP involving the intestinal tract can manifest as a protein-losing enteropathy, leading to low total protein and globulin values, in contrast to the usual presentation of FIP. Often these cats present with mesenteric lymph node enlargement due to necrogranulomatous lymphadenitis (Kipar et al., 1999; Hugo and Heading, 2015), or solitary mural intestinal lesions of the colon or ileo-caecocolic junction with associated regional lymphadenopathy (Harvey et al., 1996). Cats with intestinal FIP usually have a history of vomiting and diarrhoea or constipation.
Dermatological signs are occasionally reported in FIP and can manifest as multiple non-pruritic or pruritic nodules or papules (Cannon et al., 2005; Declercq et al., 2008; Bauer et al., 2013; Redford and Al-Dissi, 2019), due to pyogranulomatous-necrotising dermal phlebitis/vasculitis. Skin fragility syndrome has also been reported (Trotman et al., 2007). Priapism has been reported as a result of granulomatous changes in tissues surrounding the penis (Rota et al., 2008).
Neurological FIP can result in clinical signs associated with focal, multifocal or diffuse changes in the brain, spinal cord and meninges. Up to 30% of cats with FIP show neurological signs (Kline et al., 1994; Foley et al., 1998; Foley and Leutenegger, 2001; Negrin et al., 2007, 2010; Kent, 2009; Ives et al., 2013; Doenges et al., 2016). Sometimes cats with FIP present with only neurological disease (Rissi, 2018). Three clinical syndromes were identified in a retrospective study of neurological FIP (Crawford et al., 2017); of 24 cats, three had a T3-L3 myelopathy, seven had central vestibular syndrome and 14 had multifocal CNS disease. Commonly reported signs include ataxia (with varying degrees of tetra- or paraparesis; Figs. 6 and 7), hyperaesthesia, nystagmus, seizures (Timmann et al., 2008), behavioural and mental state changes and cranial nerve deficits. Central vestibular clinical signs can include head tilt, vestibular ataxia, nystagmus, obtunded appearance and postural reaction deficits. Interestingly, a retrospective study (Grapes et al., 2020), that reviewed cats presenting with vestibular disease, did not identify any discrete clinical characteristics that would help differentiate cats with vestibular disease due to FIP from other causes. This was a surprise given that FIP primarily affects younger cats and is often associated with concurrent non-neurological signs. The absence of clinical characteristics specifically associated with FIP may have been because the study included a number of younger cats with other diagnoses (middle ear polyps, thiamine deficiency, intracranial empyema and otitis media/interna) and cats with intracranial empyema can have non-neurological systemic signs. Fever was shown to be less common in cats with neurological FIP compared to those without neurological signs (Riemer et al., 2016). A retrospective study (Mella et al., 2020) of cats referred for investigation of spinal disease found FIP to be the cause in 18 of the 221 cats in the study; concurrent systemic abnormalities and abnormal findings on clinical examination were significantly associated with a diagnosis of FIP, but these features were also associated with a diagnosis of spinal lymphoma (16 cats) and empyema (3 cats).
FIP was the most commonly diagnosed cause of uveitis reported in a study of 120 cats with uveitis, of which 15.8% had FIP (Jinks et al., 2016). A study describing the ocular lesions in 15 cats with FIP found effusions in 13 cats and no effusion in only two cats (Ziolkowska et al., 2017). Ocular manifestations of FIP comprise anterior and/or posterior uveitis (Foley et al., 1998; Norris et al., 2005; Jinks et al., 2016; Doenges et al., 2016). Clinical signs include changes in iris colour, dyscoria or anisocoria secondary to iritis, sudden loss of vision and hyphaema (Figs. 8 and 9). Keratic precipitates can appear as ‘mutton fat’ deposits on the ventral corneal endothelium (Fig. 10). The iris can show swelling and a nodular surface, and aqueous flare can be detected. On ophthalmoscopic examination, chorioretinitis, fluffy perivascular cuffing (representing retinal vasculitis), dull perivascular puffy areas of pyogranulomatous chorioretinitis, linear retinal detachment, vitreous flare and fluid blistering under the retina can be seen.
FIP-associated rhinitis (Andre et al., 2020) has been described in a young cat that presented with some upper respiratory signs as well as other more typical signs of FIP; extensive respiratory panel testing on upper respiratory tract swabs in this cat revealed only a low positive for Mycoplasma felis whilst histopathological examination of lung (and liver and intestine) and nasal samples (including FCoV antigen immunohistochemistry on the nasal samples) confirmed a diagnosis of FIP. Myocarditis associated with FIP has also been described in a cat without effusion (Ernandes et al., 2019); this particular case presented with fever, weight loss and diarrhoea before developing dyspnoea and then neurological and ocular signs of FIP. Histopathology of various organs, including cardiac tissue, was consistent with FIP and FCoV antigen immunohistochemistry of the heart was also positive.
Diagnois of FCoV infection
FCoV infection is not associated with clinical signs in the majority of cats; only a minority develop FIP. Confirmation of FCoV infection in healthy cats is not often indicated unless control of FCoV infection is being attempted by the household owner, either to generally try and reduce the risk of FIP developing in the household or because a case of FIP has been diagnosed in the household. More details on diagnosis of FCoV infection by faecal RT-PCR and FCoV antibody testing can be found later in the sections on ‘General approach to FCoV & FIP control’ and ‘Management of apparently healthy FCoV-infected cats’.
FCoV infection is sometimes accompanied by enteritis (Sabshin et al., 2012). If FCoV infection is suspected in young cats with vomiting and/or diarrhoea, diagnosis of FCoV infection can be achieved via RT-PCR on faecal samples or rectal swabs. However, many other causes of enteritis exist in cats, which should be considered before making a definitive diagnosis of FCoV-associated enteritis.
Diagnosis of FIP
This section will focus on the diagnosis of FIP in sick cats showing clinical signs that could be suggestive of FIP. A cat cannot develop FIP unless it has been infected with FCoV and demonstration of FCoV in affected tissues and effusions can be helpful during diagnostic investigations of FIP.
Signalment and background for FIP
When approaching a case in which FIP is considered a differential diagnosis, one must remember that FIP is more common in young cats [especially under two years old (Riemer et al., 2016)] and that male cats are at slightly higher risk of disease (Rohrbach et al., 2001; Benetka et al., 2004; Norris et al., 2005; Worthing et al., 2012; Riemer et al., 2016). Additionally, most cats that develop FIP have been housed in multi-cat households previously. Although certain breeds have been shown to be predisposed to FIP in certain countries (Pesteanu-Somogyi et al., 2006; Worthing et al., 2012), it is believed that this is due to genetic risk factors being present in those breeds in those countries rather than existing worldwide generalised breed predispositions (Riemer et al., 2016). A recent history of stress (e.g., adoption, being in a shelter, neutering, upper respiratory tract disease, vaccination) is commonly apparent (Rohrer et al., 1993; Riemer et al., 2016) and can contribute to the development of FIP in a FCoV-infected cat.
Approach to the diagnosis of FIP
In cats with FIP that have an effusion, sampling the effusion is the single most useful diagnostic step in the diagnosis of effusive FIP; this is because tests on effusions often have a higher diagnostic value, in comparison to tests on blood (Hartmann et al., 2003) and samples are often relatively easy to obtain. If the effusion is not large in volume, imaging can be used (Pedersen, 2014) to confirm, identify and localize smaller volumes. Ultrasonography is generally regarded as being more sensitive than radiography for the detection of small volumes of fluid in the thorax and abdomen, but this depends on where pockets of fluid reside. Repeated ultrasonography to identify any small volume effusion is recommended and, similarly, ultrasonography can be used to guide sampling of small pockets of fluid (Tasker, 2018). Once an effusion is sampled, the first thing to do is to take note of its appearance: if it is frank blood, or if it can be discerned as urine, FIP is very unlikely. Additionally, purulent exudates are usually not caused by FIP. The presence of chyle will usually indicate other diseases, such as heart failure, lymphoma or a ruptured thoracic duct, but cats with FIP with pure chylous effusion have been reported (Savary et al., 2001). FIP effusions are usually clear, viscous/sticky and straw-yellow in colour (Fig. 11).
Diagnosing FIP if no effusion is present, however, can be very challenging due to the sheer number of possible clinical signs and the non-specificity of most of them (e.g., anorexia, lethargy, weight loss, pyrexia). Aetiological diagnosis of FIP cases that do not have effusions by biopsy collection ante-mortem can be very difficult due to, for example, problems accessing affected tissues, contra-indications (such as the need for general anaesthesia) for the invasiveness of taking biopsies from a sick cat and/or costs involved in tissue collection. Currently, there is no non-invasive, confirmatory test available for cats with FIP that don’t have effusions, although in some cases valuable information can be gained through analysis of fine needle aspirate (FNA) samples collected from affected organs, if accessible, as described later.
The following information on the diagnosis of FIP will consider the merits and drawbacks (and sometimes sensitivity and specificity) of tests available for the diagnosis of FIP, and FCoV infection if relevant. Although each individual test will be described, it should be remembered that when a cat with suspected FIP is being investigated, a veterinarian will be interpreting several test results at the same time, as well as taking into account the signalment and background of the cat. Such interpretation is important in helping determine how likely FIP is as a diagnosis, in the absence of a definitive diagnosis. The advantage of integrating multiple test results during interpretation has been shown in a published study on the diagnosis of FIP (Stranieri et al., 2018) and in an abstract discussing a machine learning approach to the FIP diagnosis (Dunbar et al., 2018b).
Laboratory changes in FIP
Routine haematology in FIP
Routine haematological changes are not specific for FIP but common abnormalities seen include lymphopenia (seen commonly but more in cats with effusions than in cats without), neutrophilia, a left shift and a mild to moderate normocytic, normochromic anaemia (Sparkes et al., 1991, 1994; Rohrer, 1992; Norris et al., 2005; Tsai et al., 2011; Riemer et al., 2016). An association between FIP and microcytosis (with or without anaemia) was reported (Riemer et al., 2016). Immune-mediated haemolytic anaemia occasionally occurs (Norris et al., 2005; Riemer et al., 2016). A decreasing red blood cell count is a poor prognostic sign (Tsai et al., 2011).
Serum biochemistry in FIP
Serum biochemistry changes are also non-specific in cats with FIP, but certain abnormalities can be helpful in making one consider FIP as a differential diagnosis.
Hyperglobulinaemia is often reported in FIP, and can be accompanied by hypoalbuminaemia or low to normal serum albumin (Rohrer, 1992; Riemer et al., 2016). The presence of hypoalbuminaemia alongside hyperglobulinaemia means that hyperproteinaemia does not always occur (Riemer et al., 2016). This combination of changes can cause the albumin to globulin (A:G) ratio to be low, and this ratio can be used to help evaluate how likely FIP is; the A:G ratio has a higher diagnostic value than either total serum protein or globulin concentration (Hartmann et al., 2003). Various A:G ratio cut offs have been suggested; e.g. an A:G ratio of <0.4 makes FIP very likely, whilst an A:G ratio of >0.8 makes FIP very unlikely (Sparkes et al., 1991; Norris et al., 2005; Tsai et al., 2011). One study (Jeffery et al., 2012) using a population of cats with a prevalence of FIP of 4%, reported that a serum A:G ratio of >0.6 was useful in ruling out FIP, but that lower ratios were not helpful in ruling in FIP. Additionally, frequency and extent of hypoalbuminaemia, hyperglobulinaemia and low A:G ratio reported in cats with FIP have decreased in more recent years (Riemer et al., 2016; Stranieri et al., 2017a), which could be due to veterinarians diagnosing FIP earlier, meaning that cases have not progressed to show these changes. Polyclonal and monoclonal elevated γ-globulins have been reported in cats with FIP (Taylor et al., 2010), although polyclonal elevations are far more common.
High bilirubin levels in the absence of both haemolysis and moderate elevations of liver enzyme activity should raise the suspicion of FIP. Hyperbilirubinaemia occurs in 22-63% of cats with FIP (Sparkes et al., 1991; Norris et al., 2005; Tsai et al., 2011; Riemer et al., 2016), and is especially seen in FIP cases that have effusions (Riemer et al., 2016). High bilirubin values are not always correlated with elevated liver enzymes (Riemer et al., 2016), as hyperbilirubinaemia in cats with FIP is not necessarily a reflection of parenchymal liver disease but can be due to excessive erythrocyte fragility leading to haemolysis with reduced clearing of haemoglobin breakdown products (Pedersen, 2014), or altered bilirubin metabolism due to high TNF-alpha levels. ALT, AST and ALP were normal in 86, 66 and 95%, respectively, of cats with FIP (Riemer et al., 2016). It has been found that the level of bilirubin can rise as FIP progresses, and that rising bilirubin levels are a poor prognostic sign (Tsai et al., 2011).
As described earlier, the kidneys can be affected in FIP via granulomatous lesions or glomerulonephritis (Hartmann et al., 2020); these changes can result in azotaemia although this is more commonly seen in cases without effusions (Riemer et al., 2016).
Acute phase proteins (APPs) are produced in the liver in many inflammatory and non-inflammatory diseases in response to cytokines released from macrophages and monocytes. The major APP in cats is Alpha1-acid glycoprotein (AGP), which has an immunomodulatory function, and assays are available for its measurement in some laboratories. The reference range for AGP serum concentrations is <0.48 mg/ml, and a moderately elevated serum AGP concentration of >1.5 mg/ml is frequently reported in cats with FIP (Stranieri et al., 2018). The magnitude of the increase in serum AGP might be helpful in the diagnosis of FIP (Duthie et al., 1997; Paltrinieri et al., 2007a; Giori et al., 2011; Hazuchova et al., 2016). One report (Paltrinieri et al., 2007a) found that markedly elevated serum AGP concentrations of >3 mg/ml could support a diagnosis of FIP in cats with a low pretest probability of disease (i.e. with a history and clinical findings not typical of FIP), whereas less marked elevations were supportive in cats with a higher pretest probability of disease. However another, albeit very small, study of cats with FIP actually found that moderately elevated AGP concentrations of >1.5 mg/ml were still able to discriminate between cats with and without FIP (Giori et al., 2011); interestingly, this study comprised unusual cases of FIP in which some aspects of presentation were atypical although a diagnosis of FIP was confirmed in all cases. However, it must be emphasised that AGP is not specific for FIP and can be increased in other diseases. It has been suggested that an AGP of ≤1.5 mg/ml could be useful to rule out FIP (Stranieri et al., 2018). However, AGP concentrations have been found to increase moderately and transiently in all the cats in a household before the appearance of cases of FIP in an environment with endemic FCoV infection (Paltrinieri et al., 2007b). It has also been found that AGP is hyposialylated in cats with FIP but not usually in clinically healthy FCoV antibody-positive cats or cats with other diseases (Ceciliani et al., 2004; Rossi and Paltrinieri, 2009), but testing for sialylation of AGP is not available routinely.
Cytology and biochemistry on effusions
As described under imaging, ultrasonography or radiography can be used to identify or confirm the presence of effusions and to assist in sample collection (Pedersen, 2014), which can be important as having a sample of effusion to analyse is very helpful in the diagnosis of FIP.
FIP effusions are highly proteinaceous, with a total protein concentration that is usually >35 g/l, consistent with that of an exudate. An early study (Shelly et al., 1988) describing the characteristics of effusions of 12 cats with FIP reported total protein concentrations of 32-99 g/l (median 59 g/l). In contrast, the cell counts of effusions due to FIP are often relatively low, usually <5 x109/l cells (which would be more consistent with a modified transudate); sometimes cell counts are higher, for example up to 20 x109/l cells. Cytology is typically pyogranulomatous in nature with macrophages, non-degenerate neutrophils and few lymphocytes. Thick eosinophilic proteinaceous backgrounds are often described on cytology too. If cytology reveals a septic neutrophilia (typically with degenerate neutrophils containing bacteria), neoplastic cells or a marked lymphocyte population, FIP is highly unlikely (Paltrinieri et al., 1999).
Typical effusions of cats with FIP have low A:G ratios; an A:G ratio of <0.4, has a high positive predictive value, whereas a value of >0.8 has a high negative predictive value (Shelly et al., 1988; Riemer et al., 2016). One study found that elevated effusion AGP concentrations (of >1.55 mg/ml) were more useful (sensitivity and specificity of 93%) in differentiating effusions of cats with FIP from those of cats without FIP when compared with AGP levels in the serum or other acute phase proteins (Hazuchova et al., 2016); however, the diagnosis of FIP in the cats in this study was not always confirmed.
Rivalta’s point-of-care test on effusions
Rivalta’s test is a crude point-of-care assay that was originally developed to differentiate a transudate from an exudate in humans. However, it is important to note that a positive result is not specific for FIP, and positive results have been reported in cats without FIP but also in those with septic peritonitis and lymphoma (Fischer et al., 2012a). The positive predictive value was 58.4% in a study of cats who presented with effusion, in which the prevalence of FIP was 34.6% (Fischer et al., 2012a). If positive, effusion cytology can be helpful to discriminate between these causes (Paltrinieri et al., 1999). However, the test had a high negative predictive value of 93.4% for exclusion of FIP (Fischer et al., 2012a). Thus, a negative Rivalta’s test is useful as it can be used to rule out FIP quickly and cheaply at point-of-care. A positive result needs confirmation of FIP with other tests.
To perform the Rivalta’s test, 8 ml of distilled water at room temperature and one drop of 98% acetic acid (white vinegar can be used instead) (Fischer et al., 2013) are mixed in a test tube, and then one drop of effusion is carefully placed or layered onto the surface of the solution. A positive Rivalta’s test is indicated by the drop staying attached to the surface of the liquid, retaining its shape with a connection to the surface, or floating slowly to the bottom of the tube as a drop or jellyfish-like (Fig. 12). A negative test is indicated by the drop disappearing and the solution remaining clear. However, interpretation of results can be problematic due to subjectivity and difficulties in deciding whether a result is positive or negative (Fischer et al., 2013). A video showing how to perform the test can be accessed at http://www.youtube.com/watch?v=XmOk2veunqA
Fig. 12. This image shows a positive Rivalta’s test; a drop of abdominal effusion has been placed onto the surface of a mixture of 8 ml of distilled water and one drop of 98% acetic acid (or household white vinegar) and it has retained its shape with a connection to the surface. This is not a very specific test for FIP but can be performed in-house; a positive test increases the likelihood of FIP, while a negative test makes FIP very unlikely. Courtesy of Diane Addie, www.catvirus.com
Cerebrospinal fluid (CSF) analysis
CSF is commonly collected from cats with neurological signs, although care should be taken with cisternal CSF sampling as the risk of brain herniation is high (Negrin et al., 2007; Penderis, 2009; Rissi, 2018; Hoey et al., 2020); and thus, ideally advanced imaging, such as CT or MRT, should be performed beforehand to assess the potential risk of herniation.
CSF samples from cats with FIP can show elevated protein concentrations (of >0.3 g/l [>30 mg/dl] in cisternal samples, and >0.46 g/l [>46 mg/dl] in lumbar samples with reference ranges of ≤0.3 g/l and ≤0.46 g/l for cisternal and lumbar CSF samples, respectively); occasionally marked elevations of protein occur (>2 g/l [200 mg/dl]). Additionally, CSF samples of cats with FIP often have an increased cell count (>0.008 x 109/l [>8 cells/µl] in either lumbar and/or cisternal samples; reference range ≤0.008 x 109/l [≤8 cells/µl]); occasionally this pleocytosis is extremely marked in cats with FIP (cell counts of >1x 109/l [>1000 cells/µl]). Cytological examination of the CSF can show the pleocytosis to be predominantly neutrophilic, mononuclear, mixed or pyogranulomatous (Singh et al., 2005; Crawford et al., 2017; Felten et al., 2021). Some cats with neurological FIP have unremarkable CSF analysis results (Foley et al., 1998; Boettcher et al., 2007).
Routine imaging: Ultrasonographic and radiographic findings
Ultrasonography or radiography can be used to identify or confirm the presence of effusions and to assist in sample collection (Pedersen, 2014). A review of abdominal ultrasonographic findings in 16 cats with FIP (Lewis and O'Brien, 2010) showed the presence of peritoneal fluid in seven cases and retroperitoneal fluid was found in one cat. Abdominal lymphadenopathy was documented in nine cats. The liver was of normal echogenicity in 11 cats and variably hypoechoic or hyperechoic in the remainder. Five cats had hypoechoic subcapsular rims in one or both kidneys. The spleen was of normal echogenicity in most cats and hypoechoic in two. A retrospective ultrasonographic study (Ferreira et al., 2020) focused on the significance of the medullary rim sign (MRS) in the kidneys of cats; of 661 cats that had undergone abdominal ultrasonography, 23 cats were diagnosed with FIP; 15 had MRS and eight did not, and this corresponded to a significant association between the presence of MRS and FIP. A diagnosis of FIP was made by the clinician but further details not given. A thick marked intensity type of MRS was most commonly seen with FIP. It is clear that there are no specific ultrasonographic or radiographic findings in FIP. Imaging can also be of use to direct sampling of abnormal tissues, e.g. FNA for cytology can be collected to reveal non-septic pyogranulomatous inflammation, or ultrasound-guided needle core (e.g. Tru-Cut) biopsies (TCB) can be collected and submitted for histopathology. Pneumonia due to FIP that is occasionally reported can be associated with radiographic changes.
Advanced imaging: Magnetic resonance imaging (MRI) and computed tomography (CT)
When a cat is showing neurological signs, imaging of the brain by MRI, if available, can be useful to demonstrate neurological abnormalities due to FIP. Obstructive hydrocephalus, syringomyelia, foramen magnum herniation and marked contrast enhancement of the meninges, third ventricle, mesencephalic aqueduct and brainstem have been reported in cats with FIP (Foley et al., 1998; Negrin et al., 2007; Penderis, 2009; Crawford et al., 2017). Some cats only show abnormalities after administration of contrast (Foley et al., 1998; Negrin et al., 2007), and some cats have normal MRI, even after contrast administration, despite the presence of meningoencephalitis (Negrin et al., 2007). A description of CT findings in cats with neurological FIP has not been published, and although hydrocephalus and/or syringohydromyelia can sometimes be detected by CT, MRI is likely to be more sensitive in the detection of subtle intraparenchymal lesions (Negrin et al., 2009).
Direct detection of the infectious agent
Detection of FCoV antigen
Histopathological examination of tissues with FCoV antigen immunostaining
The definitive diagnosis of FIP relies on consistent histopathological changes in affected tissues and this, together with FCoV antigen immunostaining, is considered the gold standard for diagnosis.
Immunostaining exploits the binding of antibodies to host cell-associated FCoV antigens, which are subsequently visualised by enzymatic reactions producing a colour change in a process called immunohistochemistry (IHC). However, care must be taken to ensure that adequate controls are in place since non-specific staining can occur, leading to false positive results (see below).
The “classical” FIP histopathological lesion is a blood vessel surrounded by an inflammatory lesion dominated by monocytes/macrophages intermingled with a few neutrophils and lymphocytes (Kipar et al., 2005), which are mainly CD4+ (Paltrinieri et al., 1998). Occasionally, monocytes can be seen attached to endothelial cells or emigrating from the vessel (Kipar et al., 2005). Periventricular encephalitis and leptomeningitis are commonly seen in neurological FIP (Mesquita et al., 2016; Rissi, 2018). A useful study (Stranieri et al., 2020b) documented the following patters as being consistent with FIP lesions:
• Pyogranulomas on one or more serosal surfaces;
• Granulomas with or without necrotic areas;
• Lymphocytic and plasmacytic infiltrates in specific sites (e.g., band-like infiltrate in serosal surfaces, perivascular infiltrate in meninges and CNS);
• Granulomatous to necrotizing vasculitis and fibrinous serositis.
Histopathology alone is sometimes used to definitively diagnose FIP (Felten et al., 2017a). In one study analysing 93 tissues from 14 cats with FIP (Stranieri et al., 2020b), histopathological lesions consistent with FIP were found most commonly in the lung (76.9% of samples) then kidneys (64.3%), mesenteric lymph nodes (61.5%), liver (57.1%) and spleen (57.1%).
However, in addition to histopathological changes, a definitive diagnosis of FIP should rely on the demonstration of positive immunostaining for FCoV antigen within appropriate cells (particularly macrophages) within the histopathological lesions, as shown by IHC (Kipar et al., 1998; Kipar and Meli, 2014; Stranieri et al., 2020b). Positive FCoV antigen IHC is highly specific and reliable (Tammer et al., 1995; Rissi 2018; Stranieri et al., 2020b) as long as it is performed with appropriate controls and reagents that prevent non-specific binding of the FCoV antibody to the tissues, as otherwise false positive results occur, although visualization of the pattern of FCoV antigen staining by a pathologist should discern non-specific staining. However, a negative result does not exclude FIP as FCoV antigens can be variably distributed within lesions (Giordano et al., 2005; Emmler et al., 2020; Stranieri et al., 2020b) and might not be detected in all histopathological sections prepared from FIP-associated tissues changes (Kipar and Meli, 2014). If unexpected negative IHC results are obtained, it is worth requesting additional sections of biopsies to be cut and examined by the pathologist (Tasker, 2018; Stranieri et al., 2020b).
Samples of affected tissues (e.g., liver, kidney, spleen, mesenteric lymph nodes) can be collected at necropsy or in vivo by laparotomy, laparoscopy or ultrasound-guided TCB. The samples most likely to be useful are those that are affected by the disease process, and inference of this would hopefully be gained by the results of the diagnostic testing (e.g., imaging results, pyogranulomatous inflammation on FNA cytology) as well as clinical signs.
If cats are euthanised due to suspected FIP, it is important to try to collect samples for IHC staining at post-mortem examination to confirm the disease if possible. Gross findings sometimes are suggestive of FIP (Tasker and Dowgray, 2018) (Figs. 23 and 24), but lesions might not be obvious. Indeed it is known that histopathological changes consistent with FIP can be seen in tissues that have not shown macroscopic changes at post-mortem examination (Stranieri et al., 2020b). Large pyogranulomatous lesions can be mistaken for tumours (Fig. 24). Histopathological examination, with FCoV antigen staining (see below) should ideally be performed to confirm the diagnosis of FIP.
Cytology with FCoV antigen immunostaining on effusions, FNAs, CSF and aqueous humour
FCoV immunostaining can be performed on cytology samples using immunocytochemistry (ICC) or immunofluorescence (IF), where host cell-associated FCoV antigens are detected with FCoV-specific antibodies conjugated with enzymes or fluorescent markers. The presence of FCoV antigens can then be demonstrated by either enzymatic reactions producing a colour change (see Figs. 25 and 26) or by visualization of fluorescence using a UV microscope, respectively. ICC can be a useful test to be performed on effusions, FNAs, CSF and aqueous humour samples that show cytological features consistent with FIP (e.g. the presence of neutrophils and macrophages).
FCoV immunostaining of effusion samples has shown variable sensitivity, ranging from 57 to 100% (Parodi et al., 1993; Hirschberger et al., 1995; Paltrinieri et al., 1999; Hartmann et al., 2003; Litster et al., 2013; Felten et al., 2017c). Since this technique relies on staining FCoV within macrophages in the effusion, false negative results can occur (Hellemans et al., 2020) and can result if the effusion is cell-poor and/or the FCoV antigen is masked by FCoV antibodies in the effusion. Immunostaining was thought to be very specific, although two of seven non-FIP effusions (one of the two cats had heart failure, the other one cholangiocarcinoma) were positive by IF in one study (Litster et al., 2013), and eight (three cats with heart failure and five cats with neoplasia) of 29 non-FIP effusions were positive by ICC in another (Felten et al., 2017c), questioning the specificity of ICC. However, the reported poorer specificity might be due to the methodology, and neither of these studies included negative control slides. Indeed, methodology for ICC that includes dual staining for macrophages as well as FCoV antigen potentially increases specificity by avoiding false positive results from the staining of dead cells or debris, and may be available diagnostically in the future (Howell et al., 2020). Some researchers have used cell pellets prepared from centrifuged effusion samples (Hellemans et al., 2020) for IF, whilst others have suggested using cell pellets prepared from centrifuged effusion samples to prepare formalin-fixed, paraffin embedded samples that can then be treated like a tissue specimen for FCoV antigen IHC (Kipar and Meli, 2014), to improve the reliability of detection of FCoV antigen (Kipar and Meli, 2014), although the processing time required for the latter would be longer than for ICC or IF.
FCoV immunostaining of FNA samples has not yet been described in any large comprehensive studies. Two studies did describe the successful amplification of FCoV RNA from ultrasound-guided FNAs of abnormal tissue in 11 of 11 cats (Freiche et al., 2016), and from mesenteric lymph node FNAs (Dunbar et al., 2018a) in 18 of 20 cats with FIP without effusions. This successful sampling and RNA detection suggest that demonstration of FCoV antigen by immunostaining on cytology samples might also be useful in cats with FIP if abnormal tissues are sampled, but this needs further evaluation.
FCoV immunostaining using ICC has been reported as being successful in detecting FCoV in the CSF of a cat with neurological FIP (Ives et al., 2013). One study evaluated ICC in the CSF of cats with and without FIP, that presented with and without neurological signs, collected at post-mortem examination (Gruendl et al., 2016); this study found that 17 of 20 cats with FIP gave positive ICC results but of 18 cats without FIP, three also had positive results (one cat with mediastinal lymphoma, one with lymphocytic meningoencephalitis and one with hypertensive angiopathy with brain haemorrhage), limiting the test’s specificity. The reasons for the positive ICC results in three cats without FIP are not known but possibilities raised in the study include concurrent presence of FIP alongside the other diseases present (although IHC staining of neurological tissues was also negative), detection of the presence of systemic FCoV antigen in the absence of FIP or non-specific staining and aberrant antibody binding, although these were deemed unlikely. These analyses (Gruendl et al., 2016) excluded those cats that had no cells present in their CSF as ICC could not be performed on these cats. The same group (Felten et al., 2021), performed CSF ICC on two cats with neurological signs that did not have FIP and although one of these was positive, the cytology of the CSF was lymphomonocytic, which would not have been consistent with a diagnosis of FIP. This same study also performed CSF ICC on seven cats with confirmed FIP, three with neurological signs and four without; two of the three cats with neurological FIP were ICC positive whilst three of the four cats with non-neurological FIP were ICC positive. Most of the ICC positive results in the FIP cats in this study showed pyogranulomatous cytology in the CSF, consistent with FIP. Application of ICC to CSF samples collected ante-mortem from a larger number of cats with neurological signs due to FIP and other causes would be desirable to further evaluate the usefulness of this technique.
The use of FCoV antigen immunostaining has also been described in aqueous humour samples collected directly following euthanasia from 26 cats with confirmed FIP and 13 cats with other diseases (Felten et al., 2017b); most (25 with FIP and 11 with other diseases) of these cats were also included in a subsequent study describing both FCoV RT-PCR and FCoV antigen immunostaining in cats with FIP (31 cats) and cats with other diseases (27 cats) (Sangl et al., 2020). These two studies reported sensitivities of 64.0% and 62.5%, respectively, for aqueous humour FCoV antigen immunostaining, but most of the cats with FIP in these studies did not present with uveitis. The specificities were 81.8% and 80%, with positive results occurring in one control cat with lymphoma and one control with a pulmonary adenocarcinoma (in both cats the aqueous humour cytology was not consistent with FIP). Accompanying cytology, therefore, is important to aid interpretation. However, aqueous humour as a target sample is interesting as it could be collected non-invasively from cats with suspected FIP, although the sample collection technique used in the published studies (Felten et al., 2017b; Sangl et al., 2020) might need modification (e.g., a smaller gauge needle) for use ante-mortem. Further evaluation of ICC on aqueous humour samples collected ante-mortem from cats with uveitis due to FIP and other causes are needed to further evaluate the usefulness of ICC in the diagnosis of FIP.
Comparative usefulness of tissue and FNA sampling for immunostaining
A study evaluated the usefulness of hepatic and renal TCBs, and FNAs, collected from cats with FIP confirmed by histopathology and FCoV immunostaining (Giordano et al., 2005). The sensitivity of TCBs and FNAs from hepatic and renal tissues was poor (64% and 82% for hepatic TCB and FNAs respectively, and 39% and 42% for renal TCB and FNAs) although combining analysis of TCB and FNA results for each of the tissues increased sensitivity (to 86% for liver and 48% for kidney). In this study the cytological and histopathological findings of the FNAs and TCBs were classified according to whether they were consistent with FIP for calculation of specificity and sensitivity. However, specific lesions within the liver and kidneys were not targeted for sampling in this study, and targeted sampling under ultrasound-guidance might improve sensitivity. One study, comprising 20 cats with FIP, compared RT-PCR results on FNAs and incisional biopsies (IBs) of popliteal and mesenteric lymph nodes, liver, spleen, omentum and kidneys by RT-PCR. Percentages of positive RT-PCR results were similar or even identical for FNA and IB in intra-abdominal organs. Although immunostaining results for FNA and IB were not reported in the study, it still provides important information in relation to the value of sampling by FNA or IB, since obtaining IB is highly invasive and usually cannot be performed without anaesthesia (Emmler et al., 2020).
Detection of FCoV RNA by reverse-transcriptase polymerase chain reaction (RT-PCR)
PCR is the method by which DNA is exponentially amplified using primers to target a specific sequence, enabling sensitive detection down to a very low starting DNA copy number. Post-PCR amplification processing (e.g., sequencing) can be applied as well if needed. PCR only amplifies DNA so, because FCoV is an RNA virus, a pre-PCR step using a viral enzyme, reverse-transcriptase (RT), is required to generate a strand of cDNA using the original FCoV RNA template, in a process known as reverse transcription. A combination of this process and PCR is known as RT-PCR (Barker and Tasker, 2020b). The RT-PCR assays available to detect FCoV RNA often amplify both cell-associated subgenomic mRNA (RNA produced in feline cells when the FCoV replicates), as well as cell-associated and virus particle-associated genomic RNA (which correlates to the presence of whole FCoV). Where the PCR primers bind to the FCoV genome determines whether subgenomic mRNA is preferentially amplified (Barker et al., 2017; Barker and Tasker, 2020b). Those RT-PCR assays that favour amplification of subgenomic mRNA might overestimate the FCoV viral loads present in the sample (Barker and Tasker, 2020b). Laboratories should be able to report the analytical sensitivity and specificity of their RT-PCRs and also provide details with the positive and negative controls that they use. As an RNA virus, FCoV shows a high rate of errors during replication and any mutations at the site of primer and/or probe binding can result in loss of PCR assay efficiency, and ultimately sensitivity. PCR conditions can be altered to tolerate such mutations, but this can result in a loss of specificity (Barker and Tasker, 2017). Additionally, RT-PCRs designed to target type I FCoV, which represents the majority of field strains found in naturally infected cats, although geographical variation exists (Hohdatsu et al., 1992; Benetka et al., 2004), might not amplify type II FCoV if the PCR primers and probe bind to the region of the FCoV genome which differs between the two (i.e. the spike (S) protein) (Herrewegh et al., 1998; Terada et al., 2014; Decaro et al., 2021).
FCoV RT-PCR has been used to detect FCoV RNA in blood, effusion, tissue (including aspirates), CSF, or aqueous humour samples from suspected cases of FIP, with varying results. Assays used should be quantitative (q in RT-qPCR), and able to report the FCoV load present in the sample, and this information is an important aid to interpretation of results. This is because systemic FCoV infection can occur in healthy cats and cats without FIP (albeit uncommonly) but with lower FCoV viral loads than in cats with FIP (Meli et al., 2004; Kipar et al., 2006, 2010; Desmarets et al., 2016), so a positive RT-PCR result is not specific for FIP, but positive results with a high FCoV load can be used to support a diagnosis.
Running FCoV RNA RT-PCRs can be rapid, although, once the time taken to submit the sample to the laboratory is factored in, reporting of results can still take a few days. This is usually quicker than immunostaining on tissue biopsy samples and often also quicker than immunostaining on effusion samples. More recently rapid molecular techniques (loop mediated isothermal amplification) for detecting FCoV RNA in-house as point-of-care tests have been described (Stranieri et al., 2017b; Gunther et al., 2018), but they suffered from poor sensitivity.
FCoV RT-PCR on blood samples
Samples derived from blood (e.g., whole blood, serum, plasma or peripheral blood mononuclear cells [PBMCs]) can be subjected to RT-PCR for FCoV RNA following RNA extraction but unfortunately these are rarely positive in cats with FIP and so cannot be recommended for the diagnosis of FIP. For example, when FCoV RT-PCR was performed on plasma or serum samples from cats with and without FIP (Doenges et al., 2017; Felten et al., 2017a, 2017d), although none of the cats without FIP cases gave positive results, only 0 to 15.4% of the FIP cases were positive for FCoV RNA. Similarly FCoV RNA was only rarely detected in whole blood of 20 cats with FIP in an experimental study (Pedersen et al., 2015), and although whole blood or PBMCs might be a better target for RT-PCR than serum (Doenges et al., 2017), sensitivity was still poor at 28.6%. One study (Stranieri et al., 2018) amplified FCoV RNA by RT-PCR from pellets derived from whole blood in six of eight (75.0%) cats with FIP but none of eight cats with diseases other than FIP, so results are variable. Despite these studies, specificity of FCoV RT-PCR is an issue as healthy and ill cats without FIP can have detectable viraemia in the blood. One study (Fish et al., 2018) found that nine of 205 (4.4%) healthy US shelter cats were FCoV RNA RT-PCR positive in buffy coats prepared from blood; one of those had replicating virus in the bloodstream, as demonstrated by a positive FCoV mRNA RT-PCR result, and this 8-week old kitten was likely undergoing viraemia. Neither this kitten, nor seven of the nine FCoV RNA RT-PCR positive cats with follow up developed FIP during the subsequent six months.
FCoV RT-PCR on effusions
Effusion samples in cats with FIP often contain FCoV RNA (Pedersen et al., 2015), which can be detected by RT-PCR. Centrifugation of the effusion sample to yield a cell pellet to use for RNA extraction can improve sensitivity (Hellemans et al., 2020). Published studies had amplified FCoV RNA in most (72-100%) effusion samples from cats with FIP (Doenges et al., 2017; Felten et al., 2017d; Longstaff et al., 2017; Stranieri et al., 2018) but usually not in any effusions from cats without FIP (Doenges et al., 2017; Felten et al., 2017d; Longstaff et al., 2017). However, subsequent studies have challenged the specificity of this test. One study (Barker et al., 2017) amplified FCoV RNA, albeit at a low level, in abdominal fluid from one of 29 control cats that did not have FIP. Another study (Felten et al., 2017a) amplified FCoV RNA from three (two of these three had low levels of FCoV RNA) of 24 control cats without FIP that had effusions tested. In the latter study, the control cats that generated positive FCoV RT-PCR results had neoplasia (lymphoma and a malignant round cell tumour) or chronic kidney disease (this cat had higher FCoV RNA levels in the effusion). Additionally, one study (Stranieri et al., 2018) amplified FCoV RNA (levels not reported) from the effusion of one cat with an intestinal carcinoma (out of six control cats with effusions tested). Finally, another study (Hellemans et al., 2020) documented a specificity of 81.2% for RT-PCR on effusions as positive RT-PCR results were obtained in three of 16 samples from cats without FIP; however confirmation of the absence of FIP in these three cats was only based on a negative IF immunostaining result on effusion samples from the cats, and it might well be that these three cats did indeed have FIP. Overall, it can be assumed that the presence of FCoV RNA, particularly in high levels, in an effusion that also has cytological and biochemical features suggestive of FIP, is highly supportive of a diagnosis of FIP.
FCoV RT-PCR on tissue and FNA samples
When tissue biopsy samples are obtained from cats with suspected FIP, the samples should be submitted for histopathology and IHC, as this allows for a definitive diagnosis of FIP. However, if a delay in analysis is expected, tissue can also be submitted for RT-PCR as finding high levels of FCoV RNA in a sample of an affected organ can be helpful and supportive of a diagnosis of FIP. This is because it is known that tissue samples from cats with FIP are significantly more likely to be FCoV RT-PCR positive (Barker et al., 2017; Stranieri et al., 2018), and have significantly higher FCoV RNA loads by RT-PCR (Porter et al., 2014), than tissue samples from cats without FIP. In one study that included 20 cats with FIP confirmed by immunohistochemistry, 70-90% of IBs of popliteal and mesenteric lymph nodes, liver, spleen, omentum and kidneys were positive by RT-PCR (Emmler et al., 2020). However, it is important to remember that cats without FIP can be positive for FCoV RNA by RT-PCR in tissues. One extensive study evaluating FCoV RT-PCR in 260 tissue samples from 57 cats with FIP, and 258 tissue samples from 45 cats without FIP (Barker et al., 2017) found that 90.4% of tissue samples from cats with FIP were FCoV RT-PCR positive compared to only 7.8% of tissue samples from cats without FIP.
In cats with FIP, FCoV RNA loads correlate with histopathological findings suggestive of FIP (Pedersen et al., 2015; Barker et al., 2017). Thus, the presence of high levels of FCoV RNA in tissue samples is highly supportive of a diagnosis of FIP. It is usually suggested that tissue samples should not be formalin-fixed before RT-PCR, as formalin can degrade RNA and decrease PCR sensitivity (Tasker, 2018), although a study has described the successful use of FCoV RT-PCR in formalin-fixed paraffin-embedded tissues in cats with FIP (Sangl et al., 2018).
FNAs, such as obtained by ultrasound guidance, could be a good alternative to tissue samples for FCoV RT-PCR analysis, with the advantage of relatively easy collection. One study (Freiche et al., 2016) described successful amplification of FCoV RNA from ultrasound-guided FNAs of abnormal tissue in 11 cats with FIP without effusions, suggesting that FNAs could be a useful sampling material for RT-PCR in cats with non-effusive FIP. A controlled study of FCoV RNA detection in FNAs collected from the mesenteric lymph nodes from 20 cats with FIP without effusions reported a sensitivity of 90.0% and specificity of 96.1% (Dunbar et al., 2018a). In that study FCoV RNA survived normal mailing as the FNAs tested by RT-PCR were sent by regular mail without ice or an RNA preservative (Dunbar et al., 2018a). As outlined earlier, a study of 20 cats with FIP compared RT-PCR results on FNAs and IBs of popliteal and mesenteric lymph nodes, liver, spleen, omentum and kidneys by RT-PCR. Percentages of positive RT-PCR results were similar or even identical for FNA and IB in intra-abdominal organs (Emmler et al., 2020).
FCoV RT-PCR on CSF samples
Samples of CSF can be submitted for FCoV RT-PCR. Two studies have described the use of FCoV RT-PCR on CSF samples and found it to have 100% specificity for FIP but a sensitivity of only 41.2% (Doenges et al., 2016) or 30% (Felten et al., 2021) in FIP cases. Other studies (Foley et al., 1998; Barker et al., 2017) have also shown poor sensitivity. However, not all cats included in all of these studies had neurological signs, as CSF was collected at post-mortem examination independent of presenting signs (Doenges et al., 2016; Barker et al., 2017; Felten et al., 2021), such that the population tested does not necessarily reflect a population that would have had CSF samples collected for diagnostic purposes. Indeed, in one study (Doenges et al., 2016), the sensitivity of RT-PCR rose to 85.7% when only cats with neurological and ophthalmological signs were considered. The same group found similar findings in a larger number of cats (Felten et al., 2021), where the sensitivity of RT-PCR was only 30% when both neurological and non-neurological FIP cases were included, but rose to 83.3% when only cats with neurological FIP were considered. In one study (Soma et al., 2018) it was found that all CSF samples with a CSF FCoV antibody titre of >640 that were tested for FCoV RNA were positive by RT-PCR. This study was limited by the fact that FIP was not confirmed in all cats, but it does suggest at least an association between high CSF FCoV antibody titres and positive CSF FCoV RT-PCR. In summary, FCoV RT-PCR on CSF appears to be a useful additional test in cats with neurological signs, as a positive result highly supports a diagnosis of FIP, but a negative result does not rule out FIP.
FCoV RT-PCR on aqueous humour samples
Positive FCoV RT-PCR results have been reported in cats with FIP (Barker et al., 2017; Dunbar et al., 2018a; Emmler et al., 2020), although samples have been collected at post-mortem examination rather than for diagnostic purposes ante-mortem. A large study (Sangl et al., 2020) has documented positive FCoV RT-PCR on aqueous humour samples from 11 of 31 cats with confirmed FIP and none of 27 control cats without FIP. Again, these samples were collected at post-mortem examination and, interestingly, only four of the 31 cats with FIP had ocular signs of uveitis with only two of these four cats being FCoV RT-PCR aqueous humour positive. Although FCoV RT-PCR had a specificity of 100% in this study, its sensitive was low at 35.5%.
FCoV RT-PCR on faeces
FCoV RT-PCR can be performed on faecal samples, but this is primarily used to identify cats that are shedding FCoV for the management of infection in a multi-cat household, and it is not used to diagnose FIP, as it is known that many healthy cats without FIP shed FCoV. Two studies found that 75% and 65% of cats with FIP shed virus in their faeces (Addie et al., 1996; Barker et al., 2017). Studies testing faecal samples by RT-qPCR from 50 healthy cats in US shelters (Fish et al., 2018), from 82 healthy cats from German catteries (Felten et al., 2020) and 179 cats from German breeding catteries (all with at least 5 cats per household) (Klein-Richers et al., 2020) found that 56%, 71% and 76.5% of cats, respectively, were positive for FCoV RNA. Although one study (Barker et al., 2017) showed that cats with FIP were more likely to be shedding FCoV in their faeces than cats that were euthanased due to diseases other than FIP, in an individual cat, faecal RT-PCR is not useful for diagnosis of FIP.
Molecular techniques characterizing FCoV spike (S) gene mutations following positive RT-PCR for FCoV RNA
Following detection of FCoV RNA in a sample by RT-PCR, varied molecular techniques can be used to derive sequence data for the FCoV present. However, such techniques are not always successful in FCoV RT-PCR-positive samples as low FCoV levels will preclude sequence analysis and FCoV sequence variability can prevent targeted sequencing techniques from generating sequence data (e.g. some sequence analysis methods only detect mutations in type I FCoV, and not type II FCoV) (Barker et al., 2017; Decaro et al., 2021). As described below, such sequence analysis has focused on the S gene, and techniques employed for analysis include pyrosequencing, sanger sequencing and allelic discrimination (Barker and Tasker, 2020b).
RNA sequence analysis usually focuses on the region of the S gene of type I FCoV in which certain mutations were found in the FCoV in tissues from cats with FIP but not in the FCoV in the faeces of healthy cats without FIP (Chang et al., 2012; Decaro et al., 2021). Subsequent studies (Porter et al., 2014; Barker et al., 2017) analysed FCoV of both tissue and faecal samples from cats with FIP and cats without FIP (confirmed as having other diseases by histopathology). These studies found that these S gene mutations were also found in the FCoV in tissues from cats without FIP; thus they appear to be associated with systemic FCoV infection rather than FIP per se (Porter et al., 2014; Barker et al., 2017). However not all ‘systemic’ derived samples in FCoV-infected cats show these mutations. One study was published describing a cat with neurological FIP (Andre et al., 2019) in which histological changes of FIP were found only in the central nervous system (CNS). The FCoV in the CNS had S gene mutations, whereas those found systemically in other organs did not. Additionally, a study of seven cats that remained healthy following experimental infection with FCoV (Lutz et al., 2020) aimed to document the presence or absence of S gene mutations in samples of tissue (primarily colon, liver, thymus) and faeces obtained from these cats; S gene sequences could only be obtained in five (four colonic, one liver) samples from four of the seven cats, but none of these contained the S gene mutations. Thus, although S gene mutations are likely to be important in the development of FIP, it is proposed that it is via these and/or other mutations that the FCoV acquires its monocyte/macrophage tropism to allow it to spread systemically outside of the gut and contribute to the development of FIP. Other viral factors are likely to then allow effective and sustained replication in monocytes, and activation of infected monocytes, in cats that develop FIP following systemic FCoV infection (Kipar and Meli, 2014). So, the mutations cannot be regarded as being specific for FIP.
S gene mutation analysis on tissue samples
An extensive study (Barker et al., 2017), that included 260 tissue samples from 57 cats with FIP, and 258 tissue samples from 45 cats without FIP, calculated that S gene mutation analysis on tissues, as an additional step to the detection of FCoV RNA alone by RT-qPCR, only slightly increased specificity for the diagnosis of FIP, from 92.6% to 94.6% (equivalent of five tissues), but moderately decreased sensitivity, from 89.8% to 80.9% (equivalent of 20 tissues). The decrease in sensitivity was because of the detection of non-mutated FCoV in cats with FIP (n=4), the presence of type II FCoV in cats with FIP which is not detected by mutation analysis assays that rely on finding the specific S gene mutations seen in type I FCoV by targeted analysis (n=12) and an inability to sequence the FCoV S gene due to only low FCoV copy numbers being present (n=4). The increase in specificity was due to the detection of non-mutated FCoV in cats without FIP (n=2) and an inability to sequence the FCoV S gene due to low FCoV copy numbers (n=3).
Another study (Sangl et al., 2018), that performed S gene mutation analysis on pooled tissue samples (five per cat) from 34 cats with FIP and 30 cats without FIP, reported a much higher specificity of 100% for S gene mutation analysis, as none of the 30 cats without FIP gave positive results on S gene mutation analysis. However, only three cats without FIP were FCoV RT-PCR-positive in this study, and in none of these was S gene mutation analysis successful, so the specificity calculation was not based on detecting non-mutated FCoV as such. Sensitivity in this study was moderate at 70.6%, as only 24 of the 34 FIP cases had mutations detected.
One study performed S gene mutation analysis on FNAs and IBs of popliteal and mesenteric lymph nodes, liver, spleen, omentum, and kidneys in 20 cats with FIP confirmed by immunohistochemistry. FCoV with S gene mutations were present in at least one sample in each cat, but there was variation in which sample was positive. FCoV with mutations in the S gene was most frequently found in effusions (64%), followed by in IBs of the spleen, omentum and kidney (50%), then in mesenteric lymph node IBs and FNAs (45%), and finally in FNAs of spleen and liver and liver IBs (40%). There was a loss in sensitivity in all tissues when compared to RT-PCR for FCoV without mutation analysis (Emmler et al., 2020).
Another mutation analysis study (Stranieri et al., 2018) on tissues (MLN, spleen, small intestine and lung) in 10 cats with confirmed FIP and eight cats with diseases other than FIP reported a sensitivity of 70% (7 of 10 cats with FIP had mutations) and specificity of 88% (1 of eight cats without FIP had a mutation) compared to values of 91% and 50% respectively for RT-PCR alone.
One study (McKay et al., 2020) from Canada documented that only nine of the 20 (45%) S gene sequences that could be obtained from 69 tissue samples showing typical histopathological findings of FIP possessed the previously documented S gene mutations associated with systemic infection or FIP; a further 15% contained a novel S gene mutation and 40% had no mutations at all in the region sequenced. Sensitivity and specificity was not calculated but it is obvious that the sensitivity of the ”traditional“ mutation analysis in this study was poor.
S gene mutation analysis on effusion and other fluid samples
S gene mutation analysis has also been performed on effusions (Felten et al., 2017a, 2017d; Longstaff et al., 2017; Stranieri et al., 2018) with variable sensitivity of 40.0-88.8%. A study (Barker et al., 2017) that evaluated 51 fluid samples (primarily effusions but also included CSF and aqueous humour) from 57 cats with FIP and 47 fluid samples from 45 cats without FIP calculated that S gene mutation analysis, in addition to the detection of FCoV alone by RT-qPCR, did not increase specificity (it stayed at 97.9%) for the diagnosis of FIP, but markedly decreased sensitivity from 78.4% to 60%. Another study (Felten et al., 2017a) that carried out the same calculations on effusion samples, described an increase in specificity from 87.5% to 95.8% for S gene mutation analysis over FCoV RT-PCR alone whilst sensitivity decreased from 97.1% to 68.6%. However, only effusions from three cats without FIP were FCoV RT-PCR-positive and in only one of these, S gene mutation analysis was successful (where mutated virus detected), so the improvement in specificity was not based on detecting non-mutated FCoV. Another study (Hellemans et al., 2020) documented a range of S gene mutations in the majority of effusion samples from cats with FIP.
Another study (Sangl et al., 2020) evaluating aqueous humour samples by FCoV RT-PCR with subsequent mutation analysis concluded that mutation analysis was not helpful for the diagnosis of FIP; in this study, of 11 aqueous humour samples that were FCoV RT-PCR positive in cats with FIP, only four yielded results for mutation analysis.
One study (Felten et al., 2021) that evaluated CSF samples by FCoV RT-PCR with subsequent mutation analysis also concluded that mutation analysis was not helpful for the diagnosis of FIP; in this study, of nine CSF samples that were FCoV RT-PCR positive in cats with FIP, only three yielded results for mutation analysis (all three were positive for the presence of S gene mutations). In this study the sensitivity of mutation analysis in cats with FIP was only 10% (rising to only 16.7% when only cats with neurological FIP were considered); specificity of mutation analysis could not be calculated as none of the cats without FIP yielded positive FCoV RT-PCR results upon which to subsequently perform mutation analysis.
These data suggest that detection of S gene mutations alone cannot be regarded as being confirmative for FIP. If performed it is important to interpret S gene mutation results in association with other factors (signalment, other test results) to determine how likely FIP is in the cat being tested. Generally spike gene analysis appears to be of little benefit over RT-PCR (Barker and Tasker, 2020a).
Indirect detection of the infectious agent
FCoV antibody testing
Antibody testing on blood samples
Serum FCoV antibody tests are usually enzyme-linked immunosorbent assays (ELISA), indirect immunofluorescence antibody (IFA) tests or rapid immunomigration tests (Addie et al., 2015). The porcine coronavirus TGEV or FCoV can be used in these tests as antigen substrates, both being able to detect serum FCoV antibodies; indeed, using TGEV as a substrate in one study (Kummrow et al., 2005) showed higher sensitivity in the detection of serum FCoV antibodies than using FCoV as substrate. In most tests, antibody titres are determined in multiples of serum dilutions. A positive FCoV antibody test indicates that the cat has had contact with FCoV (by natural infection or vaccination) and has developed antibodies; this typically occurs around 10-28 days following natural infection (Meli et al., 2004; Vogel et al., 2010). Although cats with FIP tend to have higher FCoV antibody titres than cats without FIP, there is much overlap, with no difference between median FCoV antibody titres in healthy and suspected FIP cases, so the value in an individual cat to distinguish cats with FIP is very limited (Bell et al., 2006). It has been suggested that a negative serum FCoV antibody result in a suspected FIP case that doesn’t have an effusion is more useful to rule out a diagnosis of FIP than in a cat with effusion (Addie et al., 2009, 2015; Sparkes et al., 1994). However, negative results were reported in three of seven cats with non-effusive neurological FIP (Negrin et al., 2007), although in that study the method of FCoV antibody testing was not described. It is important that the FCoV antibody assay used has adequate sensitivity; otherwise, false negative results can occur (Addie et al., 2015). FCoV antibody tests which begin with a dilution of the sample to 1 in 100, or 1 in 400, are commonly insensitive, missing titres lower than the starting dilution (i.e. those <100 or <400). Only tests which have a starting dilution of 1:25 or less are recommended. Opinions on the usefulness of antibody testing in cats suspected to have FIP vary, but there is no ‘FIP antibody test’; all that can be measured is antibody against FCoV.
Antibody testing on effusions
FCoV antibody tests can also be performed on effusion samples. However, in one study, some cats with FIP (although the diagnosis was not confirmed in all cases) had unexpectedly low FCoV antibody titres in their effusions (Meli et al., 2013) and an inverse correlation between FCoV RNA load, measured by RT-qPCR, and FCoV antibodies was found in some samples, suggesting that the FCoV can bind antibodies rendering them unavailable as a ligand in the antibody test (Meli et al., 2013). False negative results for FCoV antibodies on effusions can be particularly a problem with rapid immunomigration/immunochromatography tests (Meli et al., 2013; Addie et al., 2015). However, other studies (Lorusso et al., 2017; Hellemans et al., 2020) have found no evidence of an inverse correlation between FCoV RNA loads and antibody titres in effusions from cases with suspected FIP and both of these studies concluded that a combination of both FCoV RT-PCR and antibody testing would be more helpful to support a diagnosis of FIP compared to either test alone (Lorusso et al., 2017; Hellemans et al., 2020).
Antibody testing on CSF samples
FCoV antibody testing has been performed on CSF samples in cats with FIP with varied results. One study (Foley et al., 1998) reported it to be useful in diagnosing FIP, with comparison of serum and CSF FCoV titres suggesting intrathecal FCoV antibody production, although no controls were included in this study. Another study (Boettcher et al., 2007) found a significant correlation between serum and CSF FCoV antibody titres, suggesting that any CSF FCoV antibodies detected were derived from blood, and thus their detection was not additionally useful for the diagnosis of FIP. Another study suggested that a CSF FCoV antibody titre of >640 might be useful for the diagnosis of FIP (Soma et al., 2018), although the diagnosis of FIP was not histopathologically confirmed in the cats in this study.
Approach to treatment of FIP
Treatment (or euthanasia) of cats with suspected FIP should only be considered after every effort has been made to obtain a definitive diagnosis, since the wrong treatment can be detrimental for a cat. Situations have been described in which a cat with toxoplasmosis was misdiagnosed as possibly having FIP and was treated with glucocorticoids with fatal consequences (Cohen et al., 2016).
Historically, no effective causal treatment was available for FIP, so every cat with confirmed FIP died or was euthanized. Occasionally, cats have survived for several months or years after diagnosis (Ishida et al., 2004; Ritz et al., 2007; Pedersen, 2014; Hugo and Heading, 2015; Legendre et al., 2017) receiving treatment, for example glucocorticoid or non-steroidal anti-inflammatories, but it is not clear whether prolonged survival was due to the treatment. There are some rare reports of “recovered” cats in the field, but in these cats, a definitive diagnosis has not usually been obtained or reported. Currently, no licensed drug is available that is effective in curing FIP but newer antivirals (discussed below, but not currently licensed) show much promise for effective treatment of FIP and are becoming increasingly used.
Prognosis of FIP
The prognosis for a cat with FIP without effective antiviral treatment is extremely poor. In a prospective study including 43 cats with FIP with effusion, the median survival time after definitive diagnosis was eight days (Ritz et al., 2007). Another study of cats with FIP reported a median survival time of 21 days after presentation for cats with effusions and 38 days for cats without effusions (Tsai et al., 2011). Indeed, some cats, mainly those without effusions, can live for several months up to years (Ishida et al., 2004; Ritz et al., 2007; Hartmann and Ritz, 2008; Pedersen, 2014; Hugo and Heading, 2015; Legendre et al., 2017; Pedersen et al., 2018; Murphy et al., 2018; Tsai et al., 2011). The disease progression between onset of clinical signs and death is variable but is shorter in younger cats and cats with effusions than in older cats and cats without effusion (Pedersen et al., 2014). One cat with FIP lived 200 days after a definitive diagnosis (Ritz et al., 2007; Hartmann and Ritz, 2008), another survived 787 days (Hugo and Heading, 2015). One Birman cat that had never developed effusion died of FIP at six years of age; based on its history, the cat likely had a “mild form” of FIP for many years (Pedersen et al., 2014).
Some parameters can predict survival time; poor general condition, low platelet count, low lymphocyte count, low haematocrit, high bilirubin concentration, low sodium concentration, low potassium concentration, high AST activity, and a large volume of effusion indicate a poor prognosis (Ritz et al., 2007; Tsai et al., 2011). Seizures can also be considered a poor prognostic sign, since they occur significantly more frequently in animals with marked extension of inflammatory lesions in the forebrain (Timmann et al., 2008).
Epidemiological considerations in the management of cats following a diagnosis of FIP
Does a cat with FIP pose a threat to other cats in its household?
Often the question arises whether it is dangerous to bring a cat with FIP back into a household with other cats. In-contact cats have likely been exposed to the same FCoV isolate that originally infected the cat that has FIP. Still, the key question remains whether mutated virus associated with the switch from enteric infection to systemic infection and the development of FIP could be transmitted from cat to cat. In order to answer this question, some facts concerning FCoV epidemiology need to be considered.
According to different studies, 35 to 79% of cats with FIP shed FCoV in their faeces (Addie et al.,1996; Chang et al., 2010; Porter et al., 2014; Barker et al., 2017). However, it appears that some cats, once they have developed FIP, stop shedding virus in their faeces. One study (Chang et al., 2010) on 27 cats with asymptomatic FCoV infection and 28 cats with FIP from the same households revealed that 11 of 17 cats with FIP had no detectable intestinal FCoV and seemingly cleared their primary FCoV infection (Chang et al., 2010). In those cats suffering from FIP with detectable intestinal FCoV, sequence analysis focusing on the FCoV 3c gene revealed that in all but one cat, the virus was different to that associated with FIP lesions, and thus seemed to have been acquired by FCoV superinfection from other cats in the household (Chang et al., 2010). The authors concluded that if a cat with FIP restarts shedding, this is likely due to a new FCoV superinfection and not the original FCoV that resulted in FIP. However, in this study (Chang et al., 2010), the one cat with FIP that was shedding a FCoV strain in its faeces similar to the FCoV strain found in its ascitic fluid was believed to have arisen due to leakage of systemic virus into the intestines due to, for example, an intestinal granuloma. In another study it was reported that faecal FCoV from cats with FIP can carry the same S gene mutations as FCoV found systemically (Barker et al., 2017), and one study found that the full genomic RNA sequences of field FCoV strains isolated at post-mortem examination from the jejunum and the liver of a cat with FIP revealed 100% nucleotide identity between the enteric (jejunum) and non-enteric (liver) derived viral RNA sequences, suggesting that FIP-associated FCoV can be shed under some circumstances (Dye and Siddell, 2007). However, even if FIP-associated FCoV is shed in the faeces of cats with FIP, it may not cause FIP following transmission to another cat as one study demonstrated that faeces of cats with FIP did not cause FIP in another cat (Pedersen et al., 2012). The current understanding is that horizontal transmission of FIP, via a FIP-associated FCoV strain, is not believed to occur very frequently, if at all. Although there is no clear evidence that the disease FIP is transmitted from cat to cat under natural circumstances, FIP can be induced experimentally, such as by inoculation of a FIP-associated FCoV intraperitoneally (Kim et al., 2016), a route which bypasses the natural faecal-oral transmission pathway.
Therefore, based on the current knowledge, it is likely to be relatively safe to take a cat with FIP back into a household with cats that have already been in contact with it, as these cats are likely to already be FCoV-infected. It is, however, not recommended that the cat with FIP has contact with any ‘naïve’ FCoV-uninfected cat, because if the cat with FIP is shedding FCoV, it could infect any naïve cats with FCoV.
In households where a cat with FIP has been euthanised, with no remaining cats in the household, it is recommended that the owner waits for two months before obtaining new cats, as it has been suggested that FCoV preserves its infectivity for days to a few weeks, depending on environmental conditions (Scott, 1988), such as in desiccated faeces.
Management of cats with FIP in the veterinary practice
Cats with FIP in a veterinary practice or hospital should be handled and housed like any other cat, as any cat is a potential source of FCoV infection, and routine hygiene measures should be taken. Thus, there is no benefit in isolating the cat with FIP and it is not necessary to keep cats with FIP in infectious disease isolation wards.
Agents used in the treatment of FIP
Supportive, including anti-inflammatory and effusion drainage, treatments for FIP
Table 2 outlines agents that can be used in the supportive treatment of FIP.
FIP is an immune-mediated disease; thus, symptomatic treatment is aimed at controlling the immune response to FCoV and consists of high doses of immune-suppressive or anti-inflammatory drugs that slow down disease progression, such as prednisolone (initially 2-4 mg/kg q24h PO), that might be tapered down slowly if the cat responds to treatment. Although used in many published cases and in the field, the effect of glucocorticoids never has been substantiated in controlled studies. However, two separate double-blind controlled studies that evaluated feline interferon-omega (Ritz et al., 2007) and propentofylline (Fischer et al., 2011) as treatments for FIP gave all of the cats (both those in the treatment and the control groups) glucocorticoid treatment; the cats given the additional drugs did not survive any longer than those given glucocorticoids only and, indeed, those on glucocorticoids only survived for a median of eight days, confirming a poor outcome with this treatment alone in these cats (Izes et al., 2020c). In one study, cats without effusion treated with both systemic glucocorticoids and the immunomodulator polyprenyl immunostimulant (PPI) had poorer survival than those treated with PPI alone (Legendre et al., 2017); however, a definitive diagnosis of FIP was not established in all cats in this study.
If an effusion is present, some cats benefit from daily removal of the effusion (particularly if pleural effusions are resulting in dyspnoea) and injection of dexamethasone into the abdominal or thoracic cavity (1 mg/kg q24h until effusion is no longer produced, for up to seven days – if effusion is present in both cavities, the dose per cavity should be divided) (Hartmann and Ritz, 2008). Cyclophosphamide (2-4 mg/kg four times per week PO) alone or in combination with glucocorticoids has been sometimes used but there are no data on its efficacy. If indicated, cats should also be treated with broad-spectrum antibiotics (e.g., as long as the effusion is being removed) and supportive therapy (e.g., fluids) (Hartmann and Ritz, 2008).
A thromboxane synthetase inhibitor (ozagrel hydrochloride) that inhibits platelet aggregation and cytokine release was used in two cats with some improvement of clinical signs (Watari et al., 1998), but this was not a controlled study.
A placebo-controlled study in a small number of cats (three treated, three placebo) with experimentally induced FIP found a possible beneficial effect of treatment with antibodies acting against feline tumour necrosis factor alpha (TNF-alpha) (Doki et al., 2016). Progression to FIP was prevented in two of the three cats treated with these antibodies, whereas all three cats developed FIP in the placebo group. TNF-alpha is thought to be involved in FCoV replication in macrophages (Takano et al., 2007a) and contributes to development of clinical signs in cats with FIP. An uncontrolled very small study was published that used anti-human-TNF-alpha antibody treatment alongside itraconazole (Doki et al., 2020b); only three of the 10 cats inoculated in this experimental study developed FIP, and two of these three cats improved with treatment. No field studies have been conducted so far.
Pentoxyfylline or propentofylline has been applied to cats with FIP because they can down-regulate pro-inflammatory cytokines which in turn are thought to increase vasculitis. However, in a placebo-controlled, double-blind study in cats with confirmed FIP, there was no significant difference in survival time, quality of life, or any clinical or laboratory parameter in cats treated with propentofylline versus cats receiving placebo (all cats received glucocorticoids in this study) (Fischer et al., 2011).
Table 2: Drugs that have been suggested for use in cats with FIP: IMMUNOSUPPRESSANTS AND SYMPTOMATIC DRUGS
|Prednisolone/dexa-methasone||Acts as immunosuppressant. No controlled studies available. Some cats benefit from treatment and survive for several months (Ritz et al., 2007). Does not cure FIP.||Currently supportive treatment of choice, especially in cats with effusion (if effusion is present, high dose dexamethasone injection in the body cavity can be considered).|
|Azathioprine||Aims to immunosuppress (and to lower the prednisolone/dexamethasone dose). Toxic in cats. No published studies available.||Not recommended.|
|Chlorambucil||Aims to immunosuppress (and to lower the prednisolone/dexamethasone dose). No published studies.||Might be considered in combination with prednisolone/dexamethasone.|
|Cyclophosphamide||Aims to immunosuppress (and to lower the prednisolone/dexamethasone dose). No published studies.||Might be considered in combination with prednisolone/dexamethasone.|
|Anti-TNF-alpha antibody||Blocks TNF-alpha that is involved in Blocks TNF-alpha that is involved in exacerbating clinical signs of FIP (Takano et al., 2007a, 2007b, 2009). Some efficacy in a placebo-controlled study including few cats (three treated, three placebo) with experimentally induced FIP (Doki et al., 2016). Used in an uncontrolled very small study of cats with experimentally induced FIP alongside itraconazole treatment (Doki et al., 2020b) in which two of three cats with FIP improved.||Placebo-controlled field studies required.|
|Ozagrel hydrochloride||Inhibits thromboxane synthesis leading to reduction of platelet aggregation and cytokine release. Only used in two cats with some improvement of clinical signs (Watari et al., 1998).||Not recommended until further studies available.|
|Pentoxyfylline/Propentofylline||Aims at treating vasculitis. One placebo-controlled double-blind study on propentofylline showed no efficacy (all cats were given glucocorticoids) (Fischer et al., 2011).||Not recommended.|
Antiviral and immunomodulating treatments for FIP
Table 3 outlines antiviral and immunomodulatory agents that have been used for the treatment of FIP.
Currently, no licensed drug has proved effective in curing FIP, although specific antiviral compounds are now showing intense promise. For many drugs, evaluation of data is hampered by the lack of well-controlled clinical trials in which new treatments are compared against a standard care or placebo and the fact that the presence of FIP was not always confirmed in these studies before treatment was initiated, which makes an assessment of the outcome impossible (Hartmann and Ritz, 2008). Some very promising drugs have been developed and evaluated for the treatment of FIP but are not yet commercially available, such as nucleoside analogues and proteinase inhibitors (Sharun et al., 2021). In addition, there are also some promising experimental approaches, including inhibition of the binding of FCoV spike protein to receptors on the host cell membrane that mediates fusion of the viral envelope with host cell membranes (Kim et al., 2013; Liu et al., 2013), circular triple helix-forming oligonucleotide RNA targeting viral RNA (Choong et al., 2014), cholesterol synthesis and transport inhibitors (including itraconazole) inducing cholesterol accumulation in cells and thereby inhibiting FCoV replication (Takano et al., 2017; Takano et al., 2019; Doki et al., 2020a), or small interfering RNAs (siRNA) leading to RNA interference and thus, inhibition of virus replication (McDonagh et al., 2011; McDonagh et al., 2015), but most of these compounds are still in an investigational stage. Some drugs are effective in vitro, but are too toxic for cats, such as ribavirin (Weiss and Oostrom-Ram, 1989; Weiss et al., 1993a, 1993b) or the small molecule chloroquine (Takano et al., 2013; McDonagh et al., 2014)). More hydroxychloroquine, in in vitro studies (Takano et al., 2020), has been suggested as a less toxic alternative to chloroquine. The mechanism of action of the anti-viral properties of the small molecule mefloquine is not known (McDonagh et al., 2014; Yu et al., 2020) but its hepatic metabolism has been studied in vitro (Izes et al., 2020a) as well as its pharmacokinetics in healthy cats (Yu et al., 2020) and its plasma protein binding properties in the plasma of healthy cats and cats with FIP (Izes et al., 2020b); further studies are needed on its pharmacokinetics in cats with FIP. Other drugs have only been investigated in vitro, but in vivo efficacy is unknown, such as vidarabine (Barlough and Scott, 1990) which inhibits polymerases; nelfinavir, a commercially available protease inhibitor of human immunodeficiency virus; and Galanthus nivalis agglutinin (GNA), a carbohydrate-binding agent that binds to FCoV-glycosylated envelope glycoproteins, thereby inhibiting viral attachment to the host cell (van der Meer et al., 2007; Hsieh et al., 2010).
Cyclosporine A can act as an antiviral drug as it binds to cellular cyclophilins thereby inhibiting calcineurin, which is required by many viruses for replication (Tanaka et al., 2012, 2013). Cyclosporine A inhibits FCoV replication in vitro (Tanaka et al., 2012). Cyclosporine A was also associated with a reduction in pleural fluid volume and a decrease in viral load in one cat with FIP, but the cat succumbed to FIP 264 days after treatment initiation (Tanaka et al., 2015). Thus, cyclosporine A might be an option in combination with other therapeutic agents. So far however, well-controlled clinical studies are missing.
Curcumin, a derivative of turmeric, has anti-inflammatory and antiviral properties. Curcumin-encapsulated chitosan nanoparticles (Cur-CS), created to increase the bioavailability of curcumin, were evaluated in vitro and found to decrease the immune-related proteins produced during infection of cell cultures with a FIP-associated virus as well as to inhibit viral replication (Ng et al., 2020). The same study confirmed the enhanced bioavailability of Cur-CS over curcumin in pharmacokinetic analysis in healthy cats. However, another in vitro study failed to find any inhibitory effect of curcumin on FCoV proliferation (McDonagh et al., 2014). Thus, further studies on this agent are required.
Interferons are frequently used in cats with FIP. Human interferon-alpha was effective against a FIP-associated FCoV strain in vitro, but in a placebo-controlled treatment study including 74 specific pathogen-free cats in which FIP was induced experimentally, neither the prophylactic nor the therapeutic administration of high doses (104 or 106 IU/kg) interferon-alpha, feline interferon-beta (103 IU/kg), the immunomodulator Propionibacterium acnes (0.4 mg/cat or 4 mg/cat), or a combination, significantly reduced mortality in treated versus untreated cats (Weiss et al., 1990). However, in the cats treated with 106 IU/kg interferon-alpha in combination with Propionibacterium acnes, the mean survival time was prolonged, but only by three weeks (Weiss et al., 1990). As an explanation for the limited efficacy of interferon-alpha, it has been suggested that ORF-7-encoded accessory protein 7a of FIP-associated strains can act as type-I interferon antagonists and counteract the interferon-alpha-induced antiviral response (Dedeurwaerder et al., 2014). Feline interferon-omega, which is licensed in many European countries, inhibits FCoV replication in vitro (Mochizuki et al., 1994). Preliminary positive results were obtained in one uncontrolled trial, but FIP was not confirmed in the cases that survived (Ishida et al., 2004). In a randomized placebo-controlled double-blind treatment trial in 37 cats with confirmed FIP, feline interferon-omega and immunosuppressive levels of glucocorticoids was not more effective than glucocorticoids alone (Ritz et al., 2007). A published in vitro study evaluating the combination of hydroxychloroquine with interferon-omega (Takano et al., 2020) found that the addition of interferon-omega increased antiviral action for type I FCoV replication, suggesting consideration of combinations of treatment. Oral feline interferon-omega was used in a case of FIP that successfully responded to treatment (Addie et al., 2020b), but the interferon-omega was given subsequent to 56 days of antiviral treatment with oral adenosine nucleoside analogue, so the additional efficacy of the interferon-omega is not clear.
Very promising new drugs include protease inhibitors that prevent viral replication by selectively binding to viral proteases and blocking proteolytic cleavage of protein precursors needed for the production of infectious viral particles. Inhibitors that target the 3C-like protease with broad-spectrum activity against human and animal coronaviruses have been created (Kim et al., 2013). One 3C-like protease inhibitor, GC376, showed strong activity against FCoV in vitro (Kim et al., 2016) and was effective in treating FIP in an experimental setting; of eight cats with experimentally induced FIP, six remained healthy for an eight month follow up period (Kim et al., 2016), although one of these six cats succumbed to neurological FIP subsequently (Pedersen et al., 2018). In a field trial, a cohort of 20 client-owned cats were treated with GC376 at 15 mg/kg SC q12h; this was a higher dose than that used in the experimental study (Kim et al., 2016) due to treatment failure in the first cat enrolled in the trial. Nineteen of 20 treated cats regained health within two weeks of treatment. However, disease signs recurred one to seven weeks after primary treatment. Relapses no longer responsive to treatment occurred in 12/19 cats within one to seven weeks of initial or repeat treatments. Most of these relapsed cats developed neurological FIP. At the time of writing, 7/20 cats were in disease remission (Pedersen et al., 2018); most of these were cats that had presented at a young age with effusion. Cats presenting with neurological signs had been excluded from the study as GC376 does not appear to penetrate the CNS. Some side effects occurred and included injection site reactions and retarded development or abnormal eruption of permanent teeth (Pedersen et al., 2018). No untreated controls were used in this study and FIP was not confirmed in all cats with histopathology and/or immunostaining of FCoV antigen, which hampers the interpretation of data. Considering the studies on this drug, however, protease inhibitors might be a promising new approach especially if combined with other antiviral drugs, but more field trials are necessary.
Another promising treatment approach is the use of nucleoside analogues that act as an alternative substrate for viral RNA synthesis, resulting in RNA chain termination during viral RNA transcription via inhibition of RNA dependent RNA polymerase. One such nucleoside analogue, the compound GS-441524, was shown to be non-toxic in vitro and effectively inhibited replication of FIP-associated FCoV strains and field isolates in two different cell culture systems. In 10 young cats with experimentally induced FIP, GS-441524 (applied SQ q 12 h) caused a rapid reversal of clinical signs and return to a clinically healthy status within two weeks of treatment in all 10 cats. Two of the 10 treated cats had recurrences of clinical signs at four weeks and six weeks post treatment, respectively. These two cats were treated a second time with GS-441524 for two weeks and they responded again, identically to the response seen with the first treatment. All 10 cats remained clinically healthy until the time of publication (> eight months post infection) (Murphy et al., 2018). No signs of toxicity were noted besides a transient "stinging" injection reaction in some cats, such as unusual posturing, licking at the injection site and/or vocalizations, directly after compound administration (Murphy et al., 2018). GS-441524 treatment has also been evaluated in a field study of 31 cats with FIP (Pedersen et al., 2019). Cats were diagnosed with FIP based on signalment, history, clinical examination, prior test results, repeat testing and/or effusion analysis, and FCoV RT-PCR performed on effusions in some cats. Tissue IHC for FCoV antigen that confirmed FIP was only performed on five cats that later underwent post-mortem examination. Cats with neurological or ocular signs were discouraged from the trial due to concerns from the experimental trial of poor penetration of GS-441524 into the brain and/or eye (Murphy et al., 2018). Of the 31 cats with FIP recruited into this study (Pedersen et al., 2019), five had no evidence of effusion. Cats ranged from 3-73 months of age (mean 14 months). The cats were started with a primary treatment course of GS-441524 at a dosage of 2 mg/kg SQ q 24 h for at least 12 weeks (with more than 12 weeks of treatment given if serum protein levels remained elevated). The dosage was increased to 4 mg/kg SQ q 24 h for later treatments in the trial when cats showed a relapse or when a treatment course of longer than 12 weeks was deemed necessary. The study did not include a control group treated with a placebo or standard care protocol. Five of the 31 cats died or were euthanized within 26 days of the first treatment. The remaining 26 cats completed 12 or more weeks of GS-441524 treatment and showed rapid clinical improvement within two weeks. Of these 26 cats, 18 remained healthy, while eight others showed FIP relapses (six cats non-neurological FIP and two cats neurological FIP) at a mean of 23 days following treatment. Three of the eight cats with relapses were treated again with GS-441524 at 2 mg/kg SQ q 24 h; one of these three cats relapsed with neurological FIP and was euthanized whilst the two remaining cats responded well but relapsed with FIP again and were treated again with GS-441524 but at a higher dosage of 4 mg/kg SQ q 24 h. Of the original 31 cats, 25 were classified as long-time survivors after successful treatment, but one of these cats was subsequently euthanized due to presumably unrelated heart disease, while 24 remain healthy at the time of publication (Pedersen et al., 2019). Subsequently, a case series describing GS-441524 treatment (at a higher dose of 5-10 mg/kg SQ q 24 h for at least 12 weeks) in four cats with neurological and ocular signs of FIP was published (Dickinson et al., 2020) documenting very promising results. Three of the four cats were alive and off treatment at the time of publication, 354-528 days after treatment had started; two cats had received 5 mg/kg SQ q 24 h and one cat an escalating dose to 10 mg/kg SQ q 24 h. The remaining cat was euthanased 216 days after starting treatment; this cat had not shown complete resolution of signs on treatment (5 mg/kg SQ q 24 h) and rapid clinical regression occurred when treatment was stopped. Additionally, local skin reactions and discomfort around the SQ injections were cited as a reason for euthanasia.
The studies described above have all administered the nucleoside analogues subcutaneously. Further advice regarding how GS-441524 can be used in the treatment of FIP is continually evolving (Pedersen, 2021b), including recommendations on dose, duration, monitoring and possible side effects of treatment. It appears that younger cats without neurological or ocular signs are those that tend to do most favourably with subcutaneous treatment recommended for 12 weeks; an overall success rate of over 80% for GS-441524 has been quoted by those most experienced in it use (Pedersen, 2021b). Minor renal side effects are reported as well as vasculitis type reactions (different to the injection site reactions that can also occur) that can respond to short-term low dose of steroids (Pedersen, 2021b). Neurological and ocular cases need additional considerations with regards to treatment dose and length (Pedersen, 2021a).
There has been one report of the successful treatment of FIP with an oral preparation of a nucleoside analogue; this was in a cat with ocular signs in the absence of effusions (Addie et al., 2020b). The oral novel nucleoside analogue used is called ‘Mutian’ (constituent is patent-pending  so identity of agent not available although it is suggested to be GS-441524 (Pedersen, 2020a)) and was administered PO for 50 days, initially at a dosage of 8 mg/kg. Within a couple of weeks of starting treatment, the cat had shown a marked improvement in weight, ocular signs and various haematological (e.g. normalization of haematocrit) and biochemical (e.g. marked reductions in AGP and globulin measurements and an increase in the A:G ratio) measurements. The cat was also given anti-inflammatory prednisolone for the first 6 days, and feline interferon-omega PO, subsequent to the Mutian treatment finishing, which was ongoing at the time of publication. The cat appeared to have been cured of FIP. No side effects of treatment were noted although the cat did have increased symmetric dimethylarginine (SDMA) concentrations during treatment (but baseline pre-treatment levels were not measured) which decreased following discontinuation of the Mutian. This cat was also given SAMe supplementation alongside Mutian to protect the liver.
Mutian has been used to eliminate FCoV shedding in cats, and further details on this can be found below in the section ‘Elimination of FCoV shedding’.
Further considerations for use of protease inhibitors & nucleoside analogues
Efficacy in cats with naturally occurring FIP appears greater with GS-441524 (Pedersen et al., 2019; Dickinson et al., 2020) than with GC376 (Pedersen et al., 2018), as only six of 20 cats treated with GC376 remain in remission (Pedersen, unpublished data, 2018; quoted in Pedersen et al., 2019) compared to 25 of 31 cats treated with GS-441524 (Pedersen et al., 2019), and three of the four cats with neurological and ocular signs treated with the higher dose of GS-441524 also went into remission (Dickinson et al., 2020). Disease relapses not responding to retreatment were far more common with GC376 compared to GS-441524, and most of the relapses seen in the GC376 trial were neurological in nature, in contrast to the GS-441524 trial. Both treatments caused similar injection site reactions and appeared to be relatively safe, although GC376 interfered with the development of permanent teeth when given to younger kittens. Although the results of the field studies appear to favour GS-441524 treatment, some of the difference might have been influenced by how the two drugs were administered as the efficacy of GC376 might have been better if all 20 cats had been treated without interruption for 12 weeks, rather than with progressively longer periods starting at just two weeks at the start of the trial in the first five cats. This was done because the 10 experimentally infected cats treated with GS-441524 (Murphy et al., 2018) were also initially given only a two-week treatment course, although two cats required a second treatment. Thus, another GC376 field treatment trial would be warranted using a longer treatment with a higher dosage and a larger number of cats before a final comparison can be made. It would also be important to evaluate both types of drugs in combination. Neither GC376 nor GS-441524 are currently commercially available nor licensed for use in animals, although owners are sourcing agents via the internet and administering these agents themselves. Veterinarians whose clients are using these drugs might need to contact their professional regulatory bodies for guidance on their legal position in dealing with such cases. Provision of supportive care to the cats undergoing treatment and their owners in these circumstances is generally recommended.
Drug resistance is relatively common for anti-viral agents, especially with prolonged drug exposure, high viral mutation rates and sometimes genetics can play a part. In the GC376 treatment study (Pedersen et al., 2018), the 3C-like protease gene sequences of a number of cats were compared from the time of presentation to after GC376 treatment in samples obtained at post-mortem examination after euthanasia for persistent or relapse of FIP; only one cat showed a change in its 3C-like protease gene sequence. This cat had shown a relapse with FIP-caused effusion 30 weeks after starting treatment (which comprised two treatment courses totaling 16 weeks). The three gene changes/mutations found in that cat were then studied for resistance to GC376 (Perera et al., 2019); only one of the mutations conferred a change, a small reduction, in susceptibility to GC376. In the study evaluating GS-441524 treatment (Pedersen et al., 2019), only one cat in that study was thought to have shown evidence of drug resistance, although sequencing studies were not performed. Thus, drug resistance does not appear to be common. It was suggested at a FIP conference that only a small percentage of cats (~ 3%) were resistant to GS-441524 (Pedersen N, personal communication). However, resistance is a real threat and resistance to GS-5734 (Remdesivir), that is now being used in humans, has been associated with amino acid mutations in RNA polymerase and proofreading exonuclease in tissue culture propagated coronaviruses (Agostini et al., 2018). Thus, it may be important to evaluate different types of anti-viral drugs (e.g. GC376, GS-441524) in combination, as done for HIV infection and hepatitis C in humans (Pedersen et al., 2019). Concerns regarding antiviral resistance are behind the reluctance to use antivirals in healthy FCoV infected cats, as described below in the section ‘Elimination of FCoV shedding’.
Remdesivir, GS-5734, a prodrug of GS-441524, has been suggested as a treatment for respiratory coronavirus diseases in humans, most notably with COVID-19 due to SARS-CoV-2 (Beigel et al., 2020; Williamson et al., 2020) and has been considered for coronavirus treatment in cats (Izes et al., 2020c). However one large study failed to show efficacy of remdesivir in human COVID-19 (Dyer, 2020). Remdesivir is altered by infected cells to yield the active ingredient GS-441524. The safety and efficacy of remdesivir for FIP in cats has not been established (Pedersen, 2020b) although anecdotal evidence from field case descriptions suggests that injectable remdesivir is effective for the treatment of FIP (Spanner, 2020).
Immunomodulators are also commonly used in cats with FIP. The idea behind immunomodulator treatment is that these products might stimulate the immune response toward a cell-mediated response or to reduce an overactive Th2 response. An imbalance in T cell versus B cell response has been suggested to attribute to the development of FIP; however, this hypothesis has been questioned (Pedersen et al., 2014). Non-specific stimulation of the immune system might in fact even be contraindicated, since the clinical signs develop and progress because of an immune-mediated response. Therefore, treatment with these drugs is not recommended as long as there is a lack of documented efficacy in well-controlled studies (Hartmann and Ritz, 2008; Hartmann, 2018). Some old case reports suggest some effect through immunomodulator treatment, such as tylosine, promodulin, acemannan, or “para-immunity inducers”, but FIP was not confirmed in these studies (Colgrove and Parker, 1971; Robison et al., 1971; Ford, 1986; Bolcskei and Bilkei, 1995a, 1995b; Hartmann and Ritz, 2008).
Polyprenyl immunostimulant is a drug that has shown promise for immunomodulation. PPI is a commercially available oral agent that is given three times a week and that is considered to act by upregulating Th-1 cytokines. In a case series of three cats with FIP without effusion (confirmed by histopathology in one of the three cats only), PPI was associated with prolonged survival (Legendre and Bartges, 2009). In a field study, treatment with PPI was evaluated in 60 cats that were suspected to have FIP without effusion by primary care and specialist veterinarians, but again confirmation of FIP was not established in all cats and no untreated controls were included (Legendre et al., 2017). Of the 60 treated cats, 16 survived over 100 days; of these, eight survived over 200 days, including four who survived over 300 days. Veterinarians of treated cats that survived over 30 days reported improvements in clinical signs and behaviour. The survival times were significantly longer in cats that were not treated with systemic glucocorticoids concurrently, although topical ophthalmic glucocorticoids did not affect survival (Legendre et al., 2017). Thus, use of PPI might hold some promise for treatment of cats with FIP without effusion, although the number of cats responding to treatment in that study was low overall and FIP was not always confirmed. Controlled studies in cats with confirmed FIP would be necessary to evaluate efficacy.
Table 3: Drugs that have been suggested for use in cats with FIP: ANTIVIRALS AND IMMUNOMODULATORS
|GC376||Inhibits 3C-like protease. Promising results in vitro and in one in vivo experimental study, especially in cats with effusion (Kim et al., 2016), and one field study, although FIP was not confirmed in all cases (Pedersen et al., 2018).||Not commercially available. Further controlled field studies required.|
|GS-441524||Acts as nucleoside analogue that terminates the RNA chain of viral RNA-dependent RNA polymerase. Very promising results in vitro, in one in vivo experimental study (Murphy et al., 2018), and in two field studies, although FIP was not confirmed in all cases (Pedersen et al., 2019; Dickinson et al., 2020).||Not commercially available. Further controlled field studies required.|
|Ribavirin||Inhibits FCoV replication in vitro, but very toxic in cats (Weiss and Oostrom-Ram, 1989; Weiss et al., 1993a, 1993b).||Not recommended.|
|Vidarabine||Inhibits FCoV replication in vitro, but in vivo efficacy unknown (Takano et al., 2013). Toxic to cats if given systemically.||Not recommended.|
|Nelfinavir||Acts as protease inhibitor that showed synergistic effects against FCoV with Galanthus nivalis agglutinin in vitro (Hsieh et al., 2010). No in vivo data available.||Not recommended until in vivo studies available.|
|Galanthus nivalis agglutinin (GNA)||Binds to FCoV-glycosylated envelope glycoproteins, thereby inhibiting viral attachment to the host cell. Showed synergistic effects against FCoV with nelfinavir in vitro (Hsieh et al., 2010). No in vivo data available.||Not recommended until in vivo studies available.|
|Cyclosporine A and non-immunosuppressive derivatives (e.g., alosporivir)||Inhibits cyclophilins and thereby blocks replication of FCoV in vitro (Tanaka et al., 2012, 2013). Reduced viral load in one cat (Tanaka et al., 2015). Can lead to immunosuppression, depending on the cyclosporine A derivative.||Further field studies needed.|
|Indomethacin||Acts as cyclopentenone cyclooxygenase (COX) metabolite with activity against several RNA viruses, including canine coronavirus (Amici et al., 2006). No data on efficacy against FCoV in vitro or in cats with FIP available.||Not recommended until further studies available. Safety in cats unknown (e. g., general risk of non-steroidal anti-inflammatories in cats).|
|Chloroquine||Inhibits FCoV replication in vitro and has anti-inflammatory effects in vivo (Takano et al., 2013). Can increase liver enzyme activities. Effective as a small molecule inhibitor of FCoV replication in vitro (McDonagh et al., 2014).||Not recommended until further studies available.|
|Hydroxychloroquine||Inhibits type I and II FCoV replication in vitro, with less evidence of cytotoxicity than chloroquine (Takano et al., 2020); addition of interferon-omega increased antiviral action against type I FCoV replication.||Not recommended until further studies available.|
|Mefloquine||Effectively inhibits replication in vitro as a small molecule inhibitor of FCoV (McDonagh et al., 2014). Hepatic metabolism studied in vitro (Izes et al., 2020a), and its pharmokinetics in healthy cats (Yu et al., 2020). Its plasma protein binding properties have been studied with blood from cats with and without FIP, and a simple high performance liquid chromatography assay developed to measure mefloquine (Izes et al., 2020b). Causes vomiting if not given with food but generally appeared safe in healthy cats.||Field studies required.|
|Itraconazole||Inhibits cholesterol transport in type I FCoVs in vitro (Takano et al., 2019) and thus, inhibits FCoV replication. Used in an uncontrolled very small study of cats with experimentally induced FIP alongside anti-human-TNF-alpha antibody treatment (Doki et al., 2020b) in which two of three cats with FIP improved.||Further controlled field studies needed.|
|Feline interferon-omega||Inhibits FCoV replication in vitro and reduced FCoV shedding in 9/11 cats (without FIP) in a shelter (Gil et al., 2013), but was not effective in one placebo-controlled study as treatment in cats with FIP with effusion that were concurrently treated with glucocorticoids (Ritz et al., 2007).||Lack of efficacy in one placebo-controlled study, but further studies in cats without effusions and with lower concurrent glucocorticoid doses would be useful.|
|Curcumin||Decreased the immune-related proteins produced during infection of cell cultures with FIP-associated FCoV and inhibited viral replication in vitro as curcumin-encapsulated chitosan nanoparticles (Ng et al., 2020). Enhanced bioavailability as curcumin-encapsulated chitosan nanoparticles over curcumin in pharmacokinetic analysis in healthy cats. Not effective as a small molecule inhibitor of FCoV replication in vitro (McDonagh et al., 2014).||Further studies needed.|
|Human interferon-alpha |
– SC high dose
|Inhibits FCoV replication in vitro, but SC high dose treatment was not effective in an experimental trial (Weiss et al., 1990). Leads to development of antibodies to human interferon-alpha within 3-7 weeks in cats (Zeidner et al., 1990).||Not recommended.|
– PO low dose
|Acts only as immunostimulant and not as an antiviral agent if given orally. No trials published.||Not recommended until further studies available.|
|Polyprenyl immunostimulant (PPI)||Acts as immunomodulator with some efficacy in case reports and one uncontrolled field trial in cats without effusion, but FIP was not definitively diagnosed in all cases (Legendre and Bartges, 2009; Legendre et al., 2017). Median survival of cats treated with glucocorticoids in addition was lower than median survival with PPI alone.||Available in USA and in Europe by importation. Can be considered in cats with FIP without effusion. Placebo-controlled clinical studies in cats with confirmed FIP required.|
Efficacy of FIP vaccines
At present there is one intranasal vaccine commercially available in the USA and in some European countries. It contains a temperature-sensitive mutant of the type II FCoV strain DF2; type I coronaviruses are, however, more prevalent in the field in most countries (Addie and Jarrett, 2001; Addie et al., 2003; Kummrow et al., 2005). The vaccine aims to induce local mucosal immune responses through the induction of IgA and cell-mediated immunity. However, it also induces development of systemic antibodies against FCoV, although usually with low titres. The efficacy of this vaccine is in question. Results from experimental studies have been inconsistent, with preventable fractions between 0 and 75% (Gerber et al., 1990; Hoskins et al., 1995a; Hoskins et al., 1995b, McArdle et al., 1995; Gerber et al., 1995; Scott et al., 1995a, Scott et al., 1995b). Results from field studies have been equally inconsistent (Fehr et al., 1995, 1997; Postorino Reeves, 1995). No difference in the development of FIP between the vaccinated and placebo groups was found during the first 150 days after vaccination when the vaccine was used in Persian breeding colonies (Fehr et al., 1995). However, after 150 days, significantly fewer cases of FIP occurred in the vaccinated cats compared to the placebo group (Fehr et al., 1995) and retrospectively virus was found in the blood samples of the cats who developed FIP. In another trial, a preventable fraction of 75% was found when the vaccine was tested in a large cat shelter in the USA (Postorino Reeves, 1995), although the published study description was very short making it difficult to interpret the study fully. In this study, all kittens were antibody-negative prior to vaccination. The conclusion is that the vaccine is likely not effective in antibody-positive cats that have already been exposed to FCoV. The ADE of infection that was a feature of some experimental vaccine trials (McArdle et al., 1995; Scott et al., 1995a; Scott et al., 1995b) , where more vaccinated than control cats developed FIP, has not been observed in field studies, suggesting that the vaccine can be considered safe (Postorino Reeves, 1995; Fehr et al., 1995, 1997).
Use of the FIP vaccine
The ABCD considers the FIP vaccine to be non-core. It cannot be used in FCoV antibody-positive cats, which severely limits its use as many cats are FCoV antibody-positive. FCoV antibody-negative kittens could potentially benefit from vaccination, particularly if they subsequently enter an FCoV-endemic environment and thus would be at risk of developing FIP. The fact that in multi-cat environments most kittens are already infected at the age of 16 weeks further limits the usefulness of the vaccine (Addie and Jarrett, 1992; Lutz et al., 2002; Pedersen et al., 2008).
If vaccination is to be given, the 1st dose should not be given before 16 weeks of age with a 2nd dose being given three weeks after the 1st dose.
If primary vaccination has been performed, annual boosters might be considered. Although studies on the duration of immunity are lacking, it is thought to be short-lived (Addie et al., 2003).
Control of FCoV and FIP in specific situations
FIP is especially a problem of cats kept in larger groups, particularly in breeding catteries and rescue situations. Breeding catteries are high-risk environments for FIP. In Europe, it is likely that the vast majority of breeding catteries have endemic FCoV infection (Felten et al., 2020). Very rarely an unusually high number of cats (>10%; Barker et al., 2013) develop FIP within a multi-cat environment. Such “mini-outbreaks” (referred to as epizootics) are very occasionally reported (Graham et al., 2012; Wang et al., 2013; Barker et al., 2013) and more have been observed by the authors of these guidelines (unpublished data). Several factors might contribute to these “mini-outbreaks”. These factors include FCoV that have a high chance of becoming FIP-associated FCoV (thus, need a low number of mutations to become FIP-associated), high viral replication and thus viral loads in the environment and spread of these FCoV within a highly susceptible cat population.
General approach to FCoV & FIP control
Reducing FCoV transmission
Since FCoV is transmitted predominantly via the faecal–oral route, hygiene is the mainstay of FIP control in any multi-cat environment. FCoV infection is maintained in a household by continual cycles of infection and re-infection (Addie et al., 2003; Foley et al., 2003), the source of infection being faeces in the litter tray. Rarely is FIP a problem among cats leading an indoor–outdoor lifestyle or in stray cats that bury their faeces outside unless these cats originate from multi-cat environments (Riemer et al., 2016).
The goal in every cat household must be to reduce the FCoV infection pressure and risk of transmission. This can be achieved by keeping not more than three well-adapted cats per room (and keeping such cat groups stable), observing strict hygiene, and providing outdoor access if possible. If outside access is not possible, enough litter boxes should be provided (one more than the number of cats). Litter trays should be positioned in different rooms away from food and water bowls. They should have faeces removed at least twice a day, and litter tray utensils should be cleaned daily. Litter trays should be completely emptied at least weekly and cleaned using detergent. A study (Addie et al., 2020a) suggested that a Fuller’s earth-based litter that tracked minimally was associated with a reduced viral load in a multi-cat household compared to another Fuller’s earth litter; this effect was believed to be due to a binding effect of the clay in the Fuller’s earth as well as the non-tracking property of the litter to help reduce spread. Further larger studies are required. Although FCoV is only rarely shed in saliva, food and water bowls should be cleaned daily using detergent or in a dishwasher at a cycle of at least 60°C, because of the risk of fomite contamination.
Managing FCoV shedders
In some breeding catteries, attempts to at least control FCoV spread by segregation of cats has been made. Faecal shedding in cats occurs over several months or sometimes lifelong, especially in multi-cat households (Addie and Jarrett, 2001; Addie et al., 2003; Pedersen et al., 2008). Identification of cats that are persistently shedding a high FCoV load, and their separation from low shedders and faecal RT-PCR-negative cats, has been suggested for reducing transmission rates (Addie and Jarrett, 1995; Kass and Dent, 1995). Persistent high FCoV load shedders can be detected using quantitative RT-PCR on faecal samples or rectal swabs, which can be collected by the cat owner. Repeated individual RT-PCR of four faecal samples or swabs collected one week apart has been recommended to correctly identify non-shedders. The recommended frequency and interval of faecal sample collection for the identification of shedding cats in multicat households by RT-PCR has been suggested as at least three faecal samples collected at between one week and one-month intervals (Klein-Richers et al., 2020). The use of FCoV antibody testing on blood samples at a single time point, instead of repeated faecal sampling for RT-qPCR, to identify FCoV shedders was not found to be useful in another study (Felten et al., 2020), despite the finding of a positive correlation between antibody titres and the likelihood and frequency of faecal FCoV shedding and viral load (Felten et al., 2020). This was because, despite this correlation, the study identified antibody-positive cats that did not have any FCoV RNA in any of their four sequentially collected faecal samples (15 of 82 cats) and antibody-negative cats that had FCoV RNA in all of their four sequentially collected faecal samples (2 of the 82 cats).
The laboratory performing the RT-PCR from faecal swabs should provide the FCoV loads and/or an interpretation of the results as to whether or not a cat is considered a persistent high FCoV load shedder. When persistent high load shedders are identified within a multi-cat environment, access to their litter trays by non-shedders should be strictly avoided. Ideally three categories of cats are formed to separate from each other: the persistent high shedders, low shedders and non-shedders. However, if not feasible and for practical reasons, at least separation of the high shedders from the low/non-shedders can be recommended. In addition, the recommendations on how to reduce the FCoV infection pressure listed above should be strictly enforced, and FCoV-infected cats should not be exposed to stressful situations. The screening for persistent high FCoV load shedders gives a temporary picture and results can change over time. Cats can be retested six to nine months later to evaluate whether the situation has changed.
Elimination of FCoV shedding
One published study (Addie et al., 2020c) has described the use of an oral novel nucleoside analogue ‘Mutian X’ (constituent is patent-pending  so identity of agent not available although it is suggested to be GS-441524 (Pedersen, 2020a)) to eliminate FCoV shedding (identified by faecal RT-qPCR) in 29 cats within four households with endemic infection. A second study also describes the use of oral ‘Mutian’ to eliminate FCoV infection in four cats in a household that lived with a cat that was treated successfully for FIP with oral Mutian (Addie et al., 2020b). Both of these studies describe households in which the owners were wanting to eliminate FCoV shedding due to previous FIP cases in their households, including to prevent re-infection of an FIP recovered cat by other cats. In both studies a 4-day course of 4 mg/kg orally q 24 h was effective in eliminating FCoV shedding; follow up periods to confirm duration of elimination were variable in the studies. In the larger study (Addie et al., 2020c), two cats that completed the study in a household of four cats were still negative 155 and 157 days after stopping Mutian X, whilst the other cats from other households that had follow up periods were negative between three to 51 days after stopping Mutian X. The study was not placebo-controlled, so spontaneous elimination of FCoV by the treated cats cannot be ruled out, but the abrupt fall in FCoV shedding seen when Mutian X treatment was started was consistent with the effect being due to the treatment. The rationale behind using Mutian X to eliminate FCoV shedding is to establish households that are free of FCoV to remove the risk of FIP arising. One potential problem with this approach, however, is that because FCoV is so common in cat populations, and can be carried on fomites, it is likely to be hard to maintain a FCoV-negative status long term and it is highly unlikely that treatment leads to more permanent immunity than natural infection (Pedersen, 2020a; Pedersen, 2021a). Additionally, it is of note that many advise that the use of antivirals should be preserved only for the treatment of cats with FIP, in view of the potential for resistance to develop (Pedersen, 2021a), and even though Mutian X is not licensed for treatment of FIP, it is known to be one of the agents that is used to treat the disease in the field (Pedersen, 2020a). More studies are required on induction of viral resistance, and currently, for these reasons, ABCD does not recommend that this agent is used for control of FCoV infection in cats without FIP.
Breeding catteries are those households in which the reduction of FCoV infection pressure is of particular importance. A study of 37 breeding catteries in Germany, that performed RT-PCR on faecal samples collected from cats in the catteries, did not find any to be free of FCoV (Klein-Richers et al., 2020), showing how highly prevalent FCoV is in such environments. In this study, in which all households had ≥ 5 cats, only having cats of < 1 year of age was associated with an increased risk of FCoV shedding; management and husbandry measures (e.g. thoroughness of cleaning, number of litter trays, cleaning and disinfection frequency) was not associated with prevalence of faecal shedding (Klein-Richers et al., 2020). Special measures in kittens can be considered. FIP usually occurs after the kittens have left the breeder and are in a new household (Cave et al., 2002). It has been suggested that most kittens are considered protected from FCoV infection by maternally-derived antibodies (MDA) until they are five to six weeks of age. In some studies, FCoV transmission has been prevented by isolating pregnant queens two weeks before birth and then moving their kittens away to a clean environment away from other cats when they are five to six weeks old and maintaining them there until they go to a new home (Addie and Jarrett, 1992, 1995). For this method to succeed, the breeder is required to follow strict quarantine hygiene methods. However, the procedure failed in another study in which kittens were found to shed FCoV already as early as at the age of two weeks (Lutz et al., 2002), questioning the protection through MDA. In addition, specialist veterinary behaviourists often advise against early weaning due to a risk of socialisation problems in these kittens (Philip and Seitz, 1959; Guyot et al., 1980; Bateson, 1981).
There are PCR tests commercially available which purport to detect cats that are resistant to FIP (discussed briefly in the section above on Pathogenesis and Immunity). These tests are currently not recommended as a bases for breeding decisions. Positive selective breeding for “resistance to FIP” in a colony of laboratory cats was shown to decrease the survival of the offspring after intraperitoneal infection with FIP-associated FCoV (Pedersen et al., 2016). The diminished resistance to FIP in these cats was associated with decreased genomic heterozygosity.
Rescue facilities, shelters and boarding catteries
Preventing FCoV infection in rescue facilities, shelters, and boarding catteries is extremely difficult. In catteries and shelters with more than six cats, FCoV infection is virtually always present (Pedersen, 2009). Incoming cats should be kept in quarantine for a minimum of three weeks. After entry into a shelter, shedding of FCoV increases dramatically within one week amongst cats that were already infected at entry, and more than one half of initially negative cats were shedding FCoV a week later (Pedersen et al., 2004). Strict hygiene precautions, like movement control, hygiene protocols for care workers, cleaning and disinfection, must be enforced to reduce virus contamination and viral spread. Special care should be given to sterilising litter trays between use in different cats, having litter trays and scoops dedicated to each cat pen, and avoiding fomite transmission on cleaning utensils such as brushes. Ideally, cats should be kept in small groups of three or less cats per room (Addie et al., 2009) (see above) and with limited exchange of animals. Stress reduction is of particular importance, as stress may lead to increased virus production and a risk of development of FIP. New catteries should be designed with infectious disease control and stress reduction as priorities (Möstl et al., 2013; Wagner et al., 2018a, 2018b). More information on control of infectious diseases in shelters can be found in the ABCD guidelines on Infectious diseases in shelter situations and their management.
Management of apparently healthy FCoV-infected cats
A FIP vaccine is not useful in FCoV-infected cats. The question has been raised whether FCoV-infected cats should receive other vaccinations, since vaccination was identified as a stressor preceding onset of FIP in one study (Riemer et al., 2016). However, studies are lacking to support that FCoV-infected cats should be vaccinated less often than non-infected cats. Therefore, until the contrary has been demonstrated, healthy FCoV-positive cats should receive vaccination similarly to non-infected cats.
Any treatment inducing immunosuppression might increase the risk of FIP development in FCoV-infected cats (Addie et al., 2015). However, cats might have diseases that require immunosuppressive treatment in the presence of FCoV infection.
Maintaining a FCoV-negative status
Once a household or a geographic area has achieved a FCoV-negative status, every effort should be made to keep it FCoV-free. Rectal swabs for RT-PCR, taken four times, a week apart, and a serum or plasma antibody test can help to prevent introduction of infected cats into a FCoV-free home or geographical area (Addie et al., 2012). The veterinarians of the Falkland (Malvinas) Islands instituted a policy that only FCoV antibody-negative cats could be imported to the Islands, protecting the islands’ cats from FIP by preventing the introduction of FCoV (Addie et al., 2012). However, as outlined above in the section on ‘Measures for reduction of FCoV infection pressure and risk of FCoV transmission in multi-cat environments’, there might be limitations to antibody testing as method of control as some antibody-negative cats may shed FCoV in their faeces (Felten et al., 2020) and testing faeces by RT-PCR is the prefered way to identify shedders.
ABCD Europe gratefully acknowledges the support of Boehringer Ingelheim (the founding sponsor of the ABCD) and Virbac.
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